Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed  you- class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of  you,  one of the original  you -class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The  you  gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that  you  mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type  you  gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.you-you,youyouyouyou

Introduction The coordination of growth, proliferation, and differentiation during development requires transmission of information in the form of extracellular signals. Hedgehog signaling is of fundamental importance in the development of a wide variety of tissues and organ systems. Much of the initial functional analysis of Hedgehog signaling focused on the patterning of  Drosophila  larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development (reviewed in [ 1 ]). Misregulation of Hedgehog signaling has been implicated in several diseases and developmental abnormalities, including basal cell carcinoma [ 2 , 3 , 4 ], medulloblastoma [ 5 , 6 , 7 ], pancreatic cancer [ 8 ], and holoprosencephaly [ 9 , 10 ]. After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In  Drosophila , diffusion of lipid-modified Hedgehog proteins is dependent on the action of  tout velu,  a gene involved in the synthesis of heparan sulfate proteoglycans [ 11 , 12 ]. The diffusion of Hedgehog is also attenuated via sequestration by its receptor, Patched [ 13 ]. In vertebrates, Hedgehog proteins may be further regulated by binding to the  growth-arrest specific  gene product Gas1 [ 14 ], and Hedgehog-interacting protein Hip1, which is itself induced by Hedgehog signaling [ 15 ]. Moreover, the ability of Hedgehog proteins to diffuse over significant distances in the developing vertebrate limb bud appears to depend on the cholesterol modification of the Hedgehog protein; this modification may facilitate the assembly of Hedgehog proteins into a multimeric structure, perhaps conferring increased stability or mobility [ 16 , 17 ]. Genetic and biochemical evidence suggests that the low-density receptor-related protein Megalin may also play a role in Hedgehog signaling in vertebrates, perhaps by binding to Hedgehog proteins and facilitating their endocytosis [ 18 , 19 ]. Hedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. In zebrafish embryos at 24 h post fertilization (hpf), skeletal muscle can be subdivided into two distinct classes based on morphological characteristics and gene expression. Slow muscle fibers are mononucleate, express characteristic slow muscle forms of myosin heavy chain, and show strong nuclear expression of the transcription factor  prox1 . In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of  prox1  expression [ 28 ]. Cell labeling experiments have demonstrated that slow muscle fibers derive from the adaxial cells that lie immediately adjacent to the notochord [ 21 ]. As development progresses, a subset of these developing slow muscle cells migrates laterally through the myotome to form the superficial slow fibers [ 21 , 27 , 28 ]. Slow muscle fibers that remain near the midline—the muscle pioneers—express high levels of Engrailed and organize the somites into their distinctive chevron shape [ 22 , 28 , 29 , 30 ]. The remaining muscle cells in the interior of the myotome form multinucleate fast muscle fibers [ 21 , 28 ]. Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations [ 25 , 26 , 31 , 32 , 33 ]. Conversely, slow muscle is expanded at the expense of fast muscle in embryos with increased Hedgehog pathway activity [ 20 , 23 , 24 , 34 ]. Moreover, addition of Hedgehog protein to cultured zebrafish myoblasts induces expression of slow-muscle-specific forms of myosin heavy chain [ 35 ]. Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish [ 32 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ]. Many of these Hedgehog pathway mutants share characteristic defects, the most obvious of which is abnormal somite morphology resulting from disrupted slow muscle specification and the lack of horizontal myoseptum [ 22 ]. These mutants are thus termed “ you -class” mutants because of their U-shaped somites. Five of the seven  you -class mutations have been cloned, and four of these genes,  syu/shh, yot/gli2, smu/smoh,  and  con/disp1,  encode members of the Hedgehog signaling pathway [ 32 , 33 , 36 , 37 , 39 ]. The exception is  ubo/prdm1,  which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ 27 , 42 ]. Careful analysis reveals differences between the  ubo  and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in  ubo  mutants [ 22 , 43 ]. Examination of Hedgehog-induced gene expression also reveals a clear distinction between Hedgehog pathway mutations and  ubo:  Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target  ptc1,  whereas  ptc1  expression is normal in  ubo  mutants, indicating that they can receive Hedgehog signals [ 22 , 25 , 26 , 32 , 33 , 36 , 39 ]. Previous phenotypic characterization of mutants for the eponymous  you -class gene,  you,  has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ 22 , 43 ]. Moreover, expression of Hedgehog target genes, including  ptc1  and adaxial  myod,  is reduced in  you  mutants [ 25 ]. These results suggest that the  you  gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the  you  gene has remained unknown. We report the positional cloning of the  you  gene and show that it encodes a novel extracellular EGF-CUB (epidermal growth factor–complement Uegf Bmp1) protein required for Hedgehog signaling. Functional studies provide evidence that  you  is essential for the transport or stability of Hedgehog signals in the extracellular environment.
Introduction
The coordination of growth, proliferation, and differentiation during development requires transmission of information in the form of extracellular signals. Hedgehog signaling is of fundamental importance in the development of a wide variety of tissues and organ systems. Much of the initial functional analysis of Hedgehog signaling focused on the patterning of  Drosophila  larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development (reviewed in [ 1 ]). Misregulation of Hedgehog signaling has been implicated in several diseases and developmental abnormalities, including basal cell carcinoma [ 2 , 3 , 4 ], medulloblastoma [ 5 , 6 , 7 ], pancreatic cancer [ 8 ], and holoprosencephaly [ 9 , 10 ].After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In  Drosophila , diffusion of lipid-modified Hedgehog proteins is dependent on the action of  tout velu,  a gene involved in the synthesis of heparan sulfate proteoglycans [ 11 , 12 ]. The diffusion of Hedgehog is also attenuated via sequestration by its receptor, Patched [ 13 ]. In vertebrates, Hedgehog proteins may be further regulated by binding to the  growth-arrest specific  gene product Gas1 [ 14 ], and Hedgehog-interacting protein Hip1, which is itself induced by Hedgehog signaling [ 15 ]. Moreover, the ability of Hedgehog proteins to diffuse over significant distances in the developing vertebrate limb bud appears to depend on the cholesterol modification of the Hedgehog protein; this modification may facilitate the assembly of Hedgehog proteins into a multimeric structure, perhaps conferring increased stability or mobility [ 16 , 17 ]. Genetic and biochemical evidence suggests that the low-density receptor-related protein Megalin may also play a role in Hedgehog signaling in vertebrates, perhaps by binding to Hedgehog proteins and facilitating their endocytosis [ 18 , 19 ].tout velu,growth-arrest specificHedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. In zebrafish embryos at 24 h post fertilization (hpf), skeletal muscle can be subdivided into two distinct classes based on morphological characteristics and gene expression. Slow muscle fibers are mononucleate, express characteristic slow muscle forms of myosin heavy chain, and show strong nuclear expression of the transcription factor  prox1 . In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of  prox1  expression [ 28 ]. Cell labeling experiments have demonstrated that slow muscle fibers derive from the adaxial cells that lie immediately adjacent to the notochord [ 21 ]. As development progresses, a subset of these developing slow muscle cells migrates laterally through the myotome to form the superficial slow fibers [ 21 , 27 , 28 ]. Slow muscle fibers that remain near the midline—the muscle pioneers—express high levels of Engrailed and organize the somites into their distinctive chevron shape [ 22 , 28 , 29 , 30 ]. The remaining muscle cells in the interior of the myotome form multinucleate fast muscle fibers [ 21 , 28 ].prox1prox1Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations [ 25 , 26 , 31 , 32 , 33 ]. Conversely, slow muscle is expanded at the expense of fast muscle in embryos with increased Hedgehog pathway activity [ 20 , 23 , 24 , 34 ]. Moreover, addition of Hedgehog protein to cultured zebrafish myoblasts induces expression of slow-muscle-specific forms of myosin heavy chain [ 35 ].Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish [ 32 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ]. Many of these Hedgehog pathway mutants share characteristic defects, the most obvious of which is abnormal somite morphology resulting from disrupted slow muscle specification and the lack of horizontal myoseptum [ 22 ]. These mutants are thus termed “ you -class” mutants because of their U-shaped somites. Five of the seven  you -class mutations have been cloned, and four of these genes,  syu/shh, yot/gli2, smu/smoh,  and  con/disp1,  encode members of the Hedgehog signaling pathway [ 32 , 33 , 36 , 37 , 39 ]. The exception is  ubo/prdm1,  which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ 27 , 42 ]. Careful analysis reveals differences between the  ubo  and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in  ubo  mutants [ 22 , 43 ]. Examination of Hedgehog-induced gene expression also reveals a clear distinction between Hedgehog pathway mutations and  ubo:  Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target  ptc1,  whereas  ptc1  expression is normal in  ubo  mutants, indicating that they can receive Hedgehog signals [ 22 , 25 , 26 , 32 , 33 , 36 , 39 ].youyousyu/shh, yot/gli2, smu/smoh,con/disp1,ubo/prdm1,ubouboubo:ptc1,ptc1uboPrevious phenotypic characterization of mutants for the eponymous  you -class gene,  you,  has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ 22 , 43 ]. Moreover, expression of Hedgehog target genes, including  ptc1  and adaxial  myod,  is reduced in  you  mutants [ 25 ]. These results suggest that the  you  gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the  you  gene has remained unknown. We report the positional cloning of the  you  gene and show that it encodes a novel extracellular EGF-CUB (epidermal growth factor–complement Uegf Bmp1) protein required for Hedgehog signaling. Functional studies provide evidence that  you  is essential for the transport or stability of Hedgehog signals in the extracellular environment.youyou,ptc1myod,youyouyouyouyouResults
 you  Function Is Required for Hedgehog Signaling In wild-type zebrafish embryos, the dorsal and ventral portions of each myotome converge at a point where the horizontal myoseptum forms, giving the somites their characteristic chevron shape ( Figure 1 A). In contrast,  you  mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the  you  class ( Figure 1 B; [ 22 ]). The formation of the horizontal myoseptum in zebrafish depends on the proper development of slow muscle, a process that is defective in  you- class mutants [ 22 , 31 ]. Hedgehog activity in the context of slow muscle cell specification can be assayed by analyzing Engrailed expression in the muscle pioneers and adaxial  myod  expression during somitogenesis. Wild-type embryos at 24 hpf exhibit strong Engrailed staining in characteristic elongate nuclei of 2–6 muscle pioneers—slow muscle cells that develop along the prospective horizontal myoseptum—per myotomal segment ( Figure 1 C). In addition, weaker Engrailed expression can be observed in the typically more rounded nuclei of mulitnucleate fast muscle fibers, which are situated farther from the horizontal myoseptum ( Figure 1 C; [ 28 , 30 ]).  you  mutant embryos completely lacked the strong Engrailed expression in the muscle pioneers ( Figure 1 D; [ 22 ]), but very weak labeling was sometimes observed in a small number of cells. When weakly expressing cells were present, they were confined to the nine most anterior somites. In wild-type embryos,  myod  is expressed during somitogenesis in the adaxial cells of both somitic and presomitic mesoderm, and laterally along the posterior borders of developing somites ( Figure 1 E).  you  mutant embryos retained the lateral expression of  myod,  but the adaxial expression of  myod  was absent in the trunk and reduced in the tail bud ( Figure 1 F; [ 25 ]). In addition, expression of  ptc1— both a member of the Hedgehog pathway and a sensitive transcriptional readout of Hedgehog signaling—was reduced in the adaxial cells of  you  mutants at 22 somites and other stages ( Figure 1 G and  1 H; data not shown; [ 25 ]). Figure 1
 you  Mutants Exhibit Hedgehog-Associated Defects in Slow Muscle and Ventral Spinal Cord (A and B) Lateral views of live zebrafish at 22 hpf. Wild-type embryos (A) have chevron-shaped somites and a clearly visible floor plate (arrowhead), while  you  mutants (B) exhibit U-shaped somites and an indistinct floor plate (arrowhead). (C and D) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Wild-type embryos (C) show strong Engrailed expression in muscle pioneers (arrow), and weaker expression in multinucleate medial fast fibers (arrowheads). Engrailed expression in  you  mutants (D) is mostly absent, though very weak expression can occasionally be observed (arrowhead). (E and F) Dorsal views of posterior trunk and tail bud in 12-somite embryos. Wild-type embryos (E) exhibit adaxial  myod  expression throughout the somitic (arrowhead) and presomitic (arrow) mesoderm, while  you  mutants (F) lack expression in the somitic (arrowhead) and in parts of the presomitic (arrow) mesoderm. (G and H) Lateral view of somites 9–15 in whole-mount embryos at 22 hpf. Wild-type embryos (G) exhibit strong expression of  ptc1,  while  you  mutants (H) show weaker levels of  ptc1  expression. (I and J) Lateral view of spinal cord in the posterior trunk of whole-mount embryos at 24 hpf. Wild-type embryos (I) show expression of  nkx2.2  in the ventral spinal cord, while in  you  mutants (J) this expression is strongly reduced. (K–N) Dorsal views of whole-mount embryos at bud stage (10 hpf). Expression in  you  mutant embryos of both  ehh  (L) and  shh  (N) is similar to that observed wild-type embryos (K and M). Anterior is to the left in all images. Genotypes of all embryos were determined by PCR after photography. In addition to these disruptions in developing somites,  you  mutant embryos showed defects in patterning of the central nervous system.  nkx2.2,  a Hedgehog-induced marker of ventral cell types in the spinal cord [ 44 ], was absent in the trunk and tail of  you  mutants ( Figure 1 I and  1 J). Moreover, our analysis and prior work has revealed that  you  embryos show delayed and weakened blood circulation in the dorsal aorta (data not shown; [ 45 ]). These results and previous phenotypic analyses support the conclusion that  you  gene function is required for Hedgehog signaling in development of slow muscle, ventral spinal cord, and the dorsal aorta. In zebrafish, three  hedgehog  genes —ehh, shh, and twhh —are expressed at the midline in early embryonic stages. To determine whether  you  is required for  hedgehog  gene transcription, we analyzed the expression of  hedgehog  genes in wild-type and  you  mutant embryos. Expression of all three  hedgehog  genes appeared normal in  you  embryos at bud stage (10 hpf;  Figure 1 K– 1 N; data not shown). Positional Cloning of  you
 As the first step toward identifying the  you  gene, we mapped the  you  mutation to a 1-cM (12 recombinants among 1,156 meioses) region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270 ( Figure 2 A). By comparing the position of the  you  mutation to zebrafish genetic maps ([ 46 , 47 ]; unpublished data), we excluded as candidate genes more than 60 zebrafish orthologs of Hedgehog pathway genes and genes known to interact with the Hedgehog pathway. We therefore adopted a positional cloning strategy to identify the gene disrupted by the  you  mutation. Using a marker linked with Z15270 on a contig from the Sanger Institute whole-genome shotgun assembly, we screened a pooled bacterial artificial chromosome (BAC) library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked  you  ( Figure 2 A). Figure 2 Positional Cloning of the  you  Locus (A) Genetic and physical map of the  you  region on LG 7, showing the initial flanking SSLPs and the BACs used in the chromosome walk. The number of recombinants in 1,156 meioses is shown for the SSLP markers and for mapped BAC end sequences. (B) Diagram of BAC zC93A15, with expressed sequence tag markers BM184987 and AI722938 shown flanking the  you  locus. Both BM184987 and AI722938 showed one recombinant out of 6,514 meioses, and were genetically localized on opposite sides of  you . (C) The genomic region of the  you  locus is depicted by horizontal black bars. Gaps in these bars represent fragments of genomic sequence that were not obtained in the sequencing analysis. Exons are depicted by blue rectangles, and the number of recombinants in 6,514 meioses is shown below each mapped exon. Single nucleotide polymorphisms in four exons always segregated with the  you  locus, and one of these exons harbored a single nucleotide lesion predicted to change a glutamine codon to a stop codon and truncate the open reading frame. To reduce the critical interval that contained  you,  we improved the resolution of our map by increasing our mapping panel to 6,514 meioses. By scoring sequences identified from BAC zC93A15 in key recombinants from the mapping panel, we localized the  you  gene to a portion of this BAC between polymorphisms identified in expressed sequence tag markers BM184987 and AI722938 ( Figure 2 B). Further sequence analysis and mapping identified other exons of the same gene as AI722938, some of which were on the opposite side of the mutation from the original AI722938 marker ( Figure 2 C). We isolated a full-length cDNA clone for this gene and used this new sequence information to identify polymorphisms in other exons. In all, high-resolution mapping identified four exons that failed to recombine with  you  and confirmed that the two ends of the gene flanked the  you ty97  mutation. Sequence analysis of the four non-recombining exons in wild-type and  you ty97  mutant genomic DNA identified a nonsense mutation that truncates the predicted protein approximately two-thirds of the way through the open reading frame. These findings, together with others described below, indicate that this gene is disrupted by the  you  mutation.
 you  Is Orthologous to  Scube2
 The protein encoded by  you  comprises 1,010 amino acids, and comparison of the predicted  you  amino acid sequence against the protein database indicated that the  you  protein is highly similar to a family of proteins founded by mouse SCUBE1 (Signal sequence, CUB domain, EGF-related; [ 48 ]). Pairwise sequence comparisons between  you  and SCUBE family members revealed that the You protein most closely matched SCUBE2 in mouse (65% identity) and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes  SCUBE2, LMO1, STK33,  and  ST5  exhibited conserved synteny with orthologous genes in both mouse and zebrafish ([ 49 ]; unpublished data). SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB [ 50 ]. In all identified SCUBE family members, the N-terminal signal sequence is followed by nine EGF repeats, a spacer region, and a single C-terminal CUB domain [ 48 , 51 , 52 , 53 ].  Figure 3 A shows an alignment comparing the predicted  you  amino acid sequence with selected SCUBE proteins in mouse and human. Similarity between the You protein and mouse SCUBE2 was particularly high in the CUB domain (89% identity), the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in  Figure 3 A. Conservation in amino acid sequence was notably higher in this region (66% identical) than in the remainder of the spacer domain (33% identical). This six-cysteine repeat motif does not match the general structure of EGF repeats. The functional significance of this motif is not presently known, but it does lie within a region of SCUBE1 that is required in cell culture for secretion and cell surface expression [ 52 ]. Figure 3 SCUBE Protein Alignment and Truncation of the You Protein In (A) and (C), the signal peptide is labeled in blue, the nine EGF domains are labeled in red, and the CUB domain is labeled in green. (A) Alignment of the predicted You amino acid sequence with SCUBE proteins in mouse and human. Identical amino acids are shaded, and similar amino acids are boxed. Conserved cysteines in these domains and elsewhere in the alignment are indicated by filled circles. A conserved six-cysteine repeat motif N-terminal to the CUB domain is labeled in yellow. The location of the glutamine residue at amino acid 644 in the zebrafish protein, which is changed to a stop codon in  you ty97  mutants, is boxed in bold. (B) Sequence traces from homozygous wild-type and  you ty97  embryos. In  you ty97  mutants, a C to T transition is predicted to change a glutamine codon (CAA) to a stop codon (TAA) and truncate the open reading frame. (C) Model of You protein domain structure. The You protein in  you ty97  mutants is predicted to be truncated prior to the six-cysteine repeat motifs, the CUB domain, and the conserved C-terminus. In  you ty97  mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon ( Figure 3 A and  3 B). The predicted mutant protein is truncated immediately prior to the six-cysteine repeat motif, so that these repeats, the CUB domain, and the highly conserved C-terminus are lacking ( Figure 3 C). Morpholino Phenocopy and RNA Rescue of  you
 To confirm that this zebrafish EGF-CUB gene is disrupted in  you  mutants, we performed morpholino oligonucleotide (MO) injection experiments to phenocopy defects seen in  you,  and mRNA injection experiments to rescue the  you  phenotype in mutants ( Figure 4 ). All wild-type embryos injected with a MO targeting the translational start site showed reduced expression of  myod  ( n  = 112) in the adaxial cells ( Figure 4 B), and an absence of strong Engrailed expression ( n  = 70) in the muscle pioneers ( Figure 4 D). Embryos injected with a mismatch control MO did not exhibit these defects in either  myod  ( n  = 65) or Engrailed ( n  = 48) expression ( Figure 4 A and  4 C). Figure 4 MO-Induced Phenocopy of  you  Defects and Rescue of the  you  Phenotype with mRNA Injection (A, B, E, and F) Dorsal view of the posterior trunk and tail bud of 12-somite embryos. (C, D, G, and H) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Anterior is to the left in all images. Injection at the 1–4-cell stage of 420 pg of a MO targeting the translational start site of the  you  mRNA (ATG MO) resulted in decreased adaxial expression of  myod  in the somitic (arrowhead) and presomitic (arrow) mesoderm of wild-type embryos (B). Injection of an equivalent amount of a mismatch control (mismatch MO) did not produce these defects (A). Similarly, wild-type embryos injected with 420 pg of the mismatch MO (C) exhibited strong Engrailed expression in muscle pioneers (arrow) and weaker expression in medial fast fibers (arrowheads). In contrast, Engrailed expression was strongly reduced in wild-type embryos injected with 420 pg of the ATG MO (D), though very weak expression was still observed (arrowhead). Genotypically  you  mutant embryos (E) showed rescued expression of adaxial  myod  in somitic (arrowhead) and presomitic (arrow) mesoderm when injected with 50 pg of synthetic  you  mRNA at the 1–4-cell stage, while mutants injected with 50 pg of a frameshift mutant form of  you  mRNA (F) did not exhibit rescue of adaxial  myod  expression. At 24 hpf, genotypically  you  mutant embryos injected at the 1–4-cell stage with 50 pg of  you  mRNA (G) showed rescue of strong Engrailed expression in the muscle pioneers (arrow) and weaker expression in the medial fast fibers (arrowheads). Mutant embryos injected with 50 pg of the mutant mRNA (H) did not show rescued Engrailed expression, though very weak Engrailed expression (arrowhead) was observed in some cases. Engrailed expression at the MHB was normal in all analyzed embryos (data not shown). Genotypes of embryos shown in (E–H) were determined by PCR after photography. When injected with 50 pg of synthetic wild-type  you  mRNA, 98.7% ( n  = 665) of embryos from  you/+  intercrosses showed expression of  myod  in adaxial cells at 12 somites ( Figure 4 E). Genotyping of 571 embryos with wild-type  myod  expression from these intercrosses showed that 137 (24%) were homozygous mutant for  you . In contrast, 23.6% ( n  = 127) of embryos from  you/+  intercrosses injected with a mutant form of  you  mRNA lacked expression of  myod  in adaxial cells ( Figure 4 F); 20 of the mutants were genotyped and confirmed to be  you  mutant homozygotes. Similarly, all embryos ( n  = 62) from a  you/+  intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers ( Figure 4 G; genotyping of 32 phenotypically wild-type embryos showed that seven were homozygous  you ty97 ), whereas 24% ( n  = 33) of embryos injected with control mRNA lacked Engrailed expression ( Figure 4 H; eight phenotypic mutants were confirmed as homozygous for the  you  mutation). In addition, injection of  you  MOs resulted in loss of  nkx2.2  expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of  you  mRNA was sufficient to rescue  nkx2.2  expression in  you  mutants (data not shown).
 you  Expression
 you  transcripts appear to be maternally deposited in zebrafish embryos ( Figure 5 A) and are distributed widely in the embryo through early gastrulation stages ( Figure 5 B). During late gastrulation, the distribution of  you  transcripts in the embryo began to be restricted, and at bud stage (10 hpf)  you  was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes ( Figure 5 C). During somitogenesis,  you  expression continued to be refined, such that six-somite embryos exhibited expression in the eye field, in stripes in the midbrain and the midbrain–hindbrain boundary (MHB), in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis ( Figure 5 D). At 24 hpf ( Figure 5 E and  5 F),  you  transcripts were localized to the border of the ventral telencephalon and the dorsal diencephalon and to the ventral tectum, and were strongly expressed in the MHB, the hindbrain, and along the length of the embryo in the dorsal spinal cord. In addition,  you  expression was observed in the ventral tail and posterior to the yolk extension at the developing urogenital opening ( Figure 5 E). In the following 24 h of development, dorsal spinal cord expression continued, and  you  transcripts persisted in a complex and dynamic pattern in the brain. At 48 hpf ( Figure 5 G and  5 H),  you  expression was particularly strong in the cerebellum and in the hindbrain along the rhombic lip. Figure 5 Expression of  you  Examined by In Situ Hybridization (A and B) Maternal  you  transcripts were evident in cleavage-stage embryos (A) (16-cell; 1.5 hpf), and  you  mRNA was widely expressed into the gastrula period (B) (shield stage; 6 hpf). In addition,  you  mRNA was detectable by RT-PCR at 2 hpf, prior to the zebrafish midblastula transition. (C) Toward the end of gastrulation,  you  transcripts began to be restricted, so that at the bud stage (10 hpf),  you  expression was evident in the eye field (white arrowhead), in the developing midbrain and hindbrain (black arrowheads), and in posterior paraxial stripes (arrow). (D) During early somitogenesis (12 hpf),  you  expression was observed in the eye field (white arrowhead), in stripes in the midbrain and the MHB (black arrowheads), in a complex pattern in the hindbrain (white arrow), and in paraxial stripes along the developing trunk and tail bud (black arrow). (E and F) At 24 hpf,  you  transcripts were observed dorsal to the hypothalamus (black arrow), at the boundary between the telencephalon and the diencephalon (white arrow), in the ventral tectum (white arrowhead), in the region of the presumptive cerebellum (asterisk), and dorsally along the length of the spinal cord. Additional expression of  you  at this stage and later was observed in the ventral tail and at the urogenital opening (arrowheads; data not shown). (G and H) At 48 hpf,  you  transcripts were highly expressed in the cerebellum (black arrow), and were also present in the rhombic lip (white arrowhead), and continuing along the length of the anterior–posterior axis in the dorsal spinal cord (black arrowhead; data not shown). Orientation of images: (A) lateral view; (B) lateral view, dorsal to the right; (C, D, F, and G) dorsal views, anterior to the left; (E and H) lateral views, anterior to the left. Permissive Role of  you  Upstream of the Hedgehog Cellular Response To explore the possibility that the  you  gene may induce a gain-of-function phenotype when overexpressed, we injected wild-type embryos with an amount (50 pg) of synthetic  you  mRNA that was sufficient to rescue the phenotypic defects observed in  you  mutants ( Figure 6 ). When compared to embryos injected with a mutant form of  you  mRNA ( myod, n  = 81,  Figure 6 A; Engrailed,  n  = 25,  Figure 6 E;  nkx2.2, n  = 17,  Figure 6 I), overexpression of  you  in wild-type embryos did not result in obvious ectopic expression of  myod  ( n  = 394;  Figure 6 B) ,  Engrailed ( n  = 25;  Figure 6 F), or  nkx2.2  ( n  = 17;  Figure 6 J). This result suggests that  you  functions as a permissive factor in Hedgehog signaling, rather than as a potent activator of the Hedgehog pathway. When 50 pg of synthetic mRNA encoding a potent Hedgehog pathway activator  (shh)  was injected into embryos from a  you/+  intercross, ectopic expression of  myod  ( n  = 53;  Figure 6 C and  6 D), Engrailed ( n  = 78;  Figure 6 G and  6 H), and  nkx2.2  ( n  = 78;  Figure 6 K and  6 L) was induced in all embryos. Genotyping a subset of these embryos indicated that both genotypically wild-type and  you  mutant embryos showed ectopic expression of each of these markers  (myod:  41 wild-type, 12  you;  Engrailed: 34 wild-type, 8  you; nkx2.2:  63 wild-type, 15  you).  Because downstream targets of the Hedgehog pathway were rescued or upregulated in  shh- injected  you  mutants, components of the Hedgehog pathway downstream of  shh  are most likely functional in  you  embryos. These results are consistent with  you  acting upstream of or parallel with  shh  in the Hedgehog pathway. Figure 6 Early Overexpression of  you  in Wild-type Embryos and Rescue of  you  Defects by  shh  mRNA Injection (A–D) Dorsal views of the posterior trunk and tail bud of whole-mount embryos at 12 somites (15 hpf). (E–F) Lateral views of somites 2–7 in 24-hpf embryos. (G–H) Lateral views of somites 8–13 in 24-hpf embryos. (I–L) Lateral views of 24-hpf embryos. Anterior is to the left in all images. When 50 pg of  you  mRNA was injected into wild-type embryos at the 1–4-cell stage, no obvious expansion of  myod  (B), Engrailed (F), or  nkx2.2  (J) expression was observed when compared either with wild-type embryos injected with equivalent amounts of mutant mRNA (A, E, and I) or with uninjected embryos (see  Figure 1 ). Muscle pioneers were counted in a subset of the embryos; there were 4.0 ± 0.8 Engrailed-expressing muscle pioneers per somite in embryos injected with the control mRNA ( n  = 3 embryos, 33 somites) and 4.6 ± 1.1 muscle pioneers per somite in embryos injected with synthetic  you  mRNA ( n  = 8 embryos, 88 somites). Injection of 50 pg of  shh  mRNA into embryos at the 1–4-cell stage resulted in expansion of  myod,  Engrailed, and  nkx2.2  expression in both wild-type (C, G, and K) and  you  mutant (D, H, and L) embryos. s hh  injection rescued adaxial expression of  myod  (D), muscle pioneer expression of Engrailed (H), and ventral spinal cord expression of  nkx2.2  (L) in genotypically mutant  you  embryos (compare with  Figure 1 ). Genotypes of all embryos were determined by PCR after photography. Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down  patched  activity with MOs ( Figure 7 ). Expression of  myod  in adaxial cells was rescued or expanded in all  you  mutant embryos that were injected with MOs targeting  ptc1  ( Figure 7 E and  7 G;  n  = 8 mutants) or a combination of MOs targeting both  ptc1  and  ptc2  ( Figure 7 I and  7 K;  n  = 13 mutants).  you  mutant embryos injected with a  ptc1  mismatch control MO did not exhibit rescued  myod  expression ( Figure 7 A and  7 C;  n  = 8 mutants). In similar experiments, injection of  patched  MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of  you  mutant embryos ( Figure 7 B,  7 D,  7 F,  7 H,  7 J, and  7 L;  ptc1  MO,  n  = 11 mutants;  ptc1  +  ptc2  MO,  n  = 11 mutants). Figure 7 Knockdown of  patched  Function Rescues Slow Muscle Defects in  you
 After injection of 420 pg of a mismatch control  ptc1  MO, adaxial expression of  myod  (A) and Engrailed (B) was normal in wild-type embryos, but absent in  you  mutant embryos (C and D). When injected with 420 pg of a MO targeting  ptc1,  however,  myod  expression in mutants (E) was rescued to levels comparable to wild-type embryos (G). Engrailed expression was slightly expanded in both wild-type (F) and mutant (H) embryos injected with 420 pg of  ptc1  MOs. Both adaxial  myod  expression and Engrailed expression was slightly expanded in wild-type (I and J) and  you  mutant embryos (K and L) injected with MOs targeting both  ptc1  and  ptc2  (420 pg each). Embryos assayed for  myod  expression are shown in flat mount at the 12-somite stage, and somites 5–9 of Engrailed-expressing embryos are shown in lateral view at 24 hpf. Anterior is to the left in all panels. Genotypes of all embryos were determined by PCR after photography.
 you  Acts Non-Autonomously in Muscle Pioneer Differentiation To determine whether a cell must be wild-type for  you  function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts ( Figure 8 ). Cells derived from  you  mutant embryos were able to differentiate as muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei of mononucleate cells at the proper position in the somite ( Figure 8 C– 8 E;  n  = 13 embryos). Similarly, cells from embryos in which  you  function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown). Figure 8
 you  Acts Non-Autonomously in Muscle Pioneers and Is Not Required in Cells Producing Hedgehog Signals (A) Donor embryos were labeled at the 1–4-cell stage with Oregon Green dextran. (B) Cells from donor embryos were transplanted into unlabeled hosts during late blastula and early gastrula stages. (C–E) Images from a chimera made by transplanting cells from labeled mutant donors (green in C and E) into unlabeled wild-type hosts. At 24 hpf, muscle pioneer cells in chimeric embryos were labeled with anti-Engrailed antibody (red in D and E). When transplanted into wild-type embryos, mutant cells were able to differentiate as muscle pioneers, as shown by co-labeling with the anti-Engrailed antibody (E, yellow arrows). (F–K) Images from chimeras made by transplanting cells from labeled wild-type donors (green in F, H, I, and K) into unlabeled mutant hosts. Expression of Engrailed (red in G, H, J, and K) in some mutant muscle pioneers (one marked by red arrows in G, H, J, and K) was rescued in a subset of embryos (see also  Table 1 ). Donor cells in the embryo shown in (F–H) contributed solely to muscle and to non-floor-plate identities within the neural tube. Moreover, in a subset of chimeras, cells derived from wild-type donors differentiated as muscle pioneer cells (yellow arrows in J and K), simultaneously showing both the characteristic strong nuclear Engrailed expression and the typical flattened and mononucleate morphology of this cell type. The somite labeled with the arrows in J and K contains two muscle pioneers, one derived from the wild-type donor (yellow arrow) and another derived from the mutant host (red arrow). Donor cells in the embryo shown in (I–K) contributed primarily to muscle and to non-floor-plate identities within the neural tube; in addition, a group of seven floor plate cells derived from the wild-type donor was present in the tail of this embryo (not shown). Table 1 Chimeras with Wild-Type Donor Cells in  you  Mutant Hosts Rescue of muscle pioneers in wild-type→ you  mutant chimeras was evidenced by the presence of mutant (i.e., host) cells with strong Engrailed expression in elongate nuclei at the proper position in 1–8 somites. Wild-type contribution indicates structures that included cells from labeled wild-type donors CNS, non-floor-plate cells in neural tube; FP, floor plate Muscle Pioneer Differentiation Does Not Require  you  Function in Axial Hedgehog-Producing Cells To determine which cell types must be wild-type for  you  function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into  you  mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in  you  mutants ( Table 1 ;  Figure 8 F– 8 K). In addition, wild-type cells differentiated as muscle pioneers in two of the ten chimeras with rescued mutant muscle pioneers. In these cases ( Figure 8 I– 8 K), the muscle pioneer identity of cells strongly expressing Engrailed was further confirmed by presence of the lineage tracer dye, which showed that these cells had the characteristic flattened and mononucleate morphology of muscle pioneers. In all ten embryos with rescued Engrailed expression, wild-type cells were present in the muscle and in non-floor-plate regions within the neural tube. In five of the chimeric embryos with rescued Engrailed expression, the floor plate and notochord were derived entirely from  you  mutant cells, indicating that  you  function is not required in axial Hedgehog-producing cells. Collectively, the transplantation experiments indicate that  you  function is not required in either the signaling or responding cells, and instead suggest that  you  is essential for the transport or stability of Hedgehog signals.
Results
you  Function Is Required for Hedgehog Signaling In wild-type zebrafish embryos, the dorsal and ventral portions of each myotome converge at a point where the horizontal myoseptum forms, giving the somites their characteristic chevron shape ( Figure 1 A). In contrast,  you  mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the  you  class ( Figure 1 B; [ 22 ]). The formation of the horizontal myoseptum in zebrafish depends on the proper development of slow muscle, a process that is defective in  you- class mutants [ 22 , 31 ]. Hedgehog activity in the context of slow muscle cell specification can be assayed by analyzing Engrailed expression in the muscle pioneers and adaxial  myod  expression during somitogenesis. Wild-type embryos at 24 hpf exhibit strong Engrailed staining in characteristic elongate nuclei of 2–6 muscle pioneers—slow muscle cells that develop along the prospective horizontal myoseptum—per myotomal segment ( Figure 1 C). In addition, weaker Engrailed expression can be observed in the typically more rounded nuclei of mulitnucleate fast muscle fibers, which are situated farther from the horizontal myoseptum ( Figure 1 C; [ 28 , 30 ]).  you  mutant embryos completely lacked the strong Engrailed expression in the muscle pioneers ( Figure 1 D; [ 22 ]), but very weak labeling was sometimes observed in a small number of cells. When weakly expressing cells were present, they were confined to the nine most anterior somites. In wild-type embryos,  myod  is expressed during somitogenesis in the adaxial cells of both somitic and presomitic mesoderm, and laterally along the posterior borders of developing somites ( Figure 1 E).  you  mutant embryos retained the lateral expression of  myod,  but the adaxial expression of  myod  was absent in the trunk and reduced in the tail bud ( Figure 1 F; [ 25 ]). In addition, expression of  ptc1— both a member of the Hedgehog pathway and a sensitive transcriptional readout of Hedgehog signaling—was reduced in the adaxial cells of  you  mutants at 22 somites and other stages ( Figure 1 G and  1 H; data not shown; [ 25 ]). Figure 1
 you  Mutants Exhibit Hedgehog-Associated Defects in Slow Muscle and Ventral Spinal Cord (A and B) Lateral views of live zebrafish at 22 hpf. Wild-type embryos (A) have chevron-shaped somites and a clearly visible floor plate (arrowhead), while  you  mutants (B) exhibit U-shaped somites and an indistinct floor plate (arrowhead). (C and D) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Wild-type embryos (C) show strong Engrailed expression in muscle pioneers (arrow), and weaker expression in multinucleate medial fast fibers (arrowheads). Engrailed expression in  you  mutants (D) is mostly absent, though very weak expression can occasionally be observed (arrowhead). (E and F) Dorsal views of posterior trunk and tail bud in 12-somite embryos. Wild-type embryos (E) exhibit adaxial  myod  expression throughout the somitic (arrowhead) and presomitic (arrow) mesoderm, while  you  mutants (F) lack expression in the somitic (arrowhead) and in parts of the presomitic (arrow) mesoderm. (G and H) Lateral view of somites 9–15 in whole-mount embryos at 22 hpf. Wild-type embryos (G) exhibit strong expression of  ptc1,  while  you  mutants (H) show weaker levels of  ptc1  expression. (I and J) Lateral view of spinal cord in the posterior trunk of whole-mount embryos at 24 hpf. Wild-type embryos (I) show expression of  nkx2.2  in the ventral spinal cord, while in  you  mutants (J) this expression is strongly reduced. (K–N) Dorsal views of whole-mount embryos at bud stage (10 hpf). Expression in  you  mutant embryos of both  ehh  (L) and  shh  (N) is similar to that observed wild-type embryos (K and M). Anterior is to the left in all images. Genotypes of all embryos were determined by PCR after photography. In addition to these disruptions in developing somites,  you  mutant embryos showed defects in patterning of the central nervous system.  nkx2.2,  a Hedgehog-induced marker of ventral cell types in the spinal cord [ 44 ], was absent in the trunk and tail of  you  mutants ( Figure 1 I and  1 J). Moreover, our analysis and prior work has revealed that  you  embryos show delayed and weakened blood circulation in the dorsal aorta (data not shown; [ 45 ]). These results and previous phenotypic analyses support the conclusion that  you  gene function is required for Hedgehog signaling in development of slow muscle, ventral spinal cord, and the dorsal aorta. In zebrafish, three  hedgehog  genes —ehh, shh, and twhh —are expressed at the midline in early embryonic stages. To determine whether  you  is required for  hedgehog  gene transcription, we analyzed the expression of  hedgehog  genes in wild-type and  you  mutant embryos. Expression of all three  hedgehog  genes appeared normal in  you  embryos at bud stage (10 hpf;  Figure 1 K– 1 N; data not shown).
you  Function Is Required for Hedgehog Signaling
youIn wild-type zebrafish embryos, the dorsal and ventral portions of each myotome converge at a point where the horizontal myoseptum forms, giving the somites their characteristic chevron shape ( Figure 1 A). In contrast,  you  mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the  you  class ( Figure 1 B; [ 22 ]). The formation of the horizontal myoseptum in zebrafish depends on the proper development of slow muscle, a process that is defective in  you- class mutants [ 22 , 31 ]. Hedgehog activity in the context of slow muscle cell specification can be assayed by analyzing Engrailed expression in the muscle pioneers and adaxial  myod  expression during somitogenesis. Wild-type embryos at 24 hpf exhibit strong Engrailed staining in characteristic elongate nuclei of 2–6 muscle pioneers—slow muscle cells that develop along the prospective horizontal myoseptum—per myotomal segment ( Figure 1 C). In addition, weaker Engrailed expression can be observed in the typically more rounded nuclei of mulitnucleate fast muscle fibers, which are situated farther from the horizontal myoseptum ( Figure 1 C; [ 28 , 30 ]).  you  mutant embryos completely lacked the strong Engrailed expression in the muscle pioneers ( Figure 1 D; [ 22 ]), but very weak labeling was sometimes observed in a small number of cells. When weakly expressing cells were present, they were confined to the nine most anterior somites. In wild-type embryos,  myod  is expressed during somitogenesis in the adaxial cells of both somitic and presomitic mesoderm, and laterally along the posterior borders of developing somites ( Figure 1 E).  you  mutant embryos retained the lateral expression of  myod,  but the adaxial expression of  myod  was absent in the trunk and reduced in the tail bud ( Figure 1 F; [ 25 ]). In addition, expression of  ptc1— both a member of the Hedgehog pathway and a sensitive transcriptional readout of Hedgehog signaling—was reduced in the adaxial cells of  you  mutants at 22 somites and other stages ( Figure 1 G and  1 H; data not shown; [ 25 ]).youyouyou-myodyoumyodyoumyod,myodptc1—youIn addition to these disruptions in developing somites,  you  mutant embryos showed defects in patterning of the central nervous system.  nkx2.2,  a Hedgehog-induced marker of ventral cell types in the spinal cord [ 44 ], was absent in the trunk and tail of  you  mutants ( Figure 1 I and  1 J). Moreover, our analysis and prior work has revealed that  you  embryos show delayed and weakened blood circulation in the dorsal aorta (data not shown; [ 45 ]). These results and previous phenotypic analyses support the conclusion that  you  gene function is required for Hedgehog signaling in development of slow muscle, ventral spinal cord, and the dorsal aorta.younkx2.2,youyouyouIn zebrafish, three  hedgehog  genes —ehh, shh, and twhh —are expressed at the midline in early embryonic stages. To determine whether  you  is required for  hedgehog  gene transcription, we analyzed the expression of  hedgehog  genes in wild-type and  you  mutant embryos. Expression of all three  hedgehog  genes appeared normal in  you  embryos at bud stage (10 hpf;  Figure 1 K– 1 N; data not shown).hedgehog—ehh, shh, and twhhyouhedgehoghedgehogyouhedgehogyouPositional Cloning of  you
 As the first step toward identifying the  you  gene, we mapped the  you  mutation to a 1-cM (12 recombinants among 1,156 meioses) region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270 ( Figure 2 A). By comparing the position of the  you  mutation to zebrafish genetic maps ([ 46 , 47 ]; unpublished data), we excluded as candidate genes more than 60 zebrafish orthologs of Hedgehog pathway genes and genes known to interact with the Hedgehog pathway. We therefore adopted a positional cloning strategy to identify the gene disrupted by the  you  mutation. Using a marker linked with Z15270 on a contig from the Sanger Institute whole-genome shotgun assembly, we screened a pooled bacterial artificial chromosome (BAC) library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked  you  ( Figure 2 A). Figure 2 Positional Cloning of the  you  Locus (A) Genetic and physical map of the  you  region on LG 7, showing the initial flanking SSLPs and the BACs used in the chromosome walk. The number of recombinants in 1,156 meioses is shown for the SSLP markers and for mapped BAC end sequences. (B) Diagram of BAC zC93A15, with expressed sequence tag markers BM184987 and AI722938 shown flanking the  you  locus. Both BM184987 and AI722938 showed one recombinant out of 6,514 meioses, and were genetically localized on opposite sides of  you . (C) The genomic region of the  you  locus is depicted by horizontal black bars. Gaps in these bars represent fragments of genomic sequence that were not obtained in the sequencing analysis. Exons are depicted by blue rectangles, and the number of recombinants in 6,514 meioses is shown below each mapped exon. Single nucleotide polymorphisms in four exons always segregated with the  you  locus, and one of these exons harbored a single nucleotide lesion predicted to change a glutamine codon to a stop codon and truncate the open reading frame. To reduce the critical interval that contained  you,  we improved the resolution of our map by increasing our mapping panel to 6,514 meioses. By scoring sequences identified from BAC zC93A15 in key recombinants from the mapping panel, we localized the  you  gene to a portion of this BAC between polymorphisms identified in expressed sequence tag markers BM184987 and AI722938 ( Figure 2 B). Further sequence analysis and mapping identified other exons of the same gene as AI722938, some of which were on the opposite side of the mutation from the original AI722938 marker ( Figure 2 C). We isolated a full-length cDNA clone for this gene and used this new sequence information to identify polymorphisms in other exons. In all, high-resolution mapping identified four exons that failed to recombine with  you  and confirmed that the two ends of the gene flanked the  you ty97  mutation. Sequence analysis of the four non-recombining exons in wild-type and  you ty97  mutant genomic DNA identified a nonsense mutation that truncates the predicted protein approximately two-thirds of the way through the open reading frame. These findings, together with others described below, indicate that this gene is disrupted by the  you  mutation.
Positional Cloning of  you
youAs the first step toward identifying the  you  gene, we mapped the  you  mutation to a 1-cM (12 recombinants among 1,156 meioses) region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270 ( Figure 2 A). By comparing the position of the  you  mutation to zebrafish genetic maps ([ 46 , 47 ]; unpublished data), we excluded as candidate genes more than 60 zebrafish orthologs of Hedgehog pathway genes and genes known to interact with the Hedgehog pathway. We therefore adopted a positional cloning strategy to identify the gene disrupted by the  you  mutation. Using a marker linked with Z15270 on a contig from the Sanger Institute whole-genome shotgun assembly, we screened a pooled bacterial artificial chromosome (BAC) library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked  you  ( Figure 2 A).youyouyouyouyouTo reduce the critical interval that contained  you,  we improved the resolution of our map by increasing our mapping panel to 6,514 meioses. By scoring sequences identified from BAC zC93A15 in key recombinants from the mapping panel, we localized the  you  gene to a portion of this BAC between polymorphisms identified in expressed sequence tag markers BM184987 and AI722938 ( Figure 2 B). Further sequence analysis and mapping identified other exons of the same gene as AI722938, some of which were on the opposite side of the mutation from the original AI722938 marker ( Figure 2 C). We isolated a full-length cDNA clone for this gene and used this new sequence information to identify polymorphisms in other exons. In all, high-resolution mapping identified four exons that failed to recombine with  you  and confirmed that the two ends of the gene flanked the  you ty97  mutation. Sequence analysis of the four non-recombining exons in wild-type and  you ty97  mutant genomic DNA identified a nonsense mutation that truncates the predicted protein approximately two-thirds of the way through the open reading frame. These findings, together with others described below, indicate that this gene is disrupted by the  you  mutation.you,youyouyou ty97you ty97youyou  Is Orthologous to  Scube2
 The protein encoded by  you  comprises 1,010 amino acids, and comparison of the predicted  you  amino acid sequence against the protein database indicated that the  you  protein is highly similar to a family of proteins founded by mouse SCUBE1 (Signal sequence, CUB domain, EGF-related; [ 48 ]). Pairwise sequence comparisons between  you  and SCUBE family members revealed that the You protein most closely matched SCUBE2 in mouse (65% identity) and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes  SCUBE2, LMO1, STK33,  and  ST5  exhibited conserved synteny with orthologous genes in both mouse and zebrafish ([ 49 ]; unpublished data). SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB [ 50 ]. In all identified SCUBE family members, the N-terminal signal sequence is followed by nine EGF repeats, a spacer region, and a single C-terminal CUB domain [ 48 , 51 , 52 , 53 ].  Figure 3 A shows an alignment comparing the predicted  you  amino acid sequence with selected SCUBE proteins in mouse and human. Similarity between the You protein and mouse SCUBE2 was particularly high in the CUB domain (89% identity), the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in  Figure 3 A. Conservation in amino acid sequence was notably higher in this region (66% identical) than in the remainder of the spacer domain (33% identical). This six-cysteine repeat motif does not match the general structure of EGF repeats. The functional significance of this motif is not presently known, but it does lie within a region of SCUBE1 that is required in cell culture for secretion and cell surface expression [ 52 ]. Figure 3 SCUBE Protein Alignment and Truncation of the You Protein In (A) and (C), the signal peptide is labeled in blue, the nine EGF domains are labeled in red, and the CUB domain is labeled in green. (A) Alignment of the predicted You amino acid sequence with SCUBE proteins in mouse and human. Identical amino acids are shaded, and similar amino acids are boxed. Conserved cysteines in these domains and elsewhere in the alignment are indicated by filled circles. A conserved six-cysteine repeat motif N-terminal to the CUB domain is labeled in yellow. The location of the glutamine residue at amino acid 644 in the zebrafish protein, which is changed to a stop codon in  you ty97  mutants, is boxed in bold. (B) Sequence traces from homozygous wild-type and  you ty97  embryos. In  you ty97  mutants, a C to T transition is predicted to change a glutamine codon (CAA) to a stop codon (TAA) and truncate the open reading frame. (C) Model of You protein domain structure. The You protein in  you ty97  mutants is predicted to be truncated prior to the six-cysteine repeat motifs, the CUB domain, and the conserved C-terminus. In  you ty97  mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon ( Figure 3 A and  3 B). The predicted mutant protein is truncated immediately prior to the six-cysteine repeat motif, so that these repeats, the CUB domain, and the highly conserved C-terminus are lacking ( Figure 3 C).
you  Is Orthologous to  Scube2
youScube2The protein encoded by  you  comprises 1,010 amino acids, and comparison of the predicted  you  amino acid sequence against the protein database indicated that the  you  protein is highly similar to a family of proteins founded by mouse SCUBE1 (Signal sequence, CUB domain, EGF-related; [ 48 ]). Pairwise sequence comparisons between  you  and SCUBE family members revealed that the You protein most closely matched SCUBE2 in mouse (65% identity) and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes  SCUBE2, LMO1, STK33,  and  ST5  exhibited conserved synteny with orthologous genes in both mouse and zebrafish ([ 49 ]; unpublished data).youyouyouyouSCUBE2, LMO1, STK33,ST5SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB [ 50 ]. In all identified SCUBE family members, the N-terminal signal sequence is followed by nine EGF repeats, a spacer region, and a single C-terminal CUB domain [ 48 , 51 , 52 , 53 ].  Figure 3 A shows an alignment comparing the predicted  you  amino acid sequence with selected SCUBE proteins in mouse and human. Similarity between the You protein and mouse SCUBE2 was particularly high in the CUB domain (89% identity), the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in  Figure 3 A. Conservation in amino acid sequence was notably higher in this region (66% identical) than in the remainder of the spacer domain (33% identical). This six-cysteine repeat motif does not match the general structure of EGF repeats. The functional significance of this motif is not presently known, but it does lie within a region of SCUBE1 that is required in cell culture for secretion and cell surface expression [ 52 ].youIn  you ty97  mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon ( Figure 3 A and  3 B). The predicted mutant protein is truncated immediately prior to the six-cysteine repeat motif, so that these repeats, the CUB domain, and the highly conserved C-terminus are lacking ( Figure 3 C).you ty97Morpholino Phenocopy and RNA Rescue of  you
 To confirm that this zebrafish EGF-CUB gene is disrupted in  you  mutants, we performed morpholino oligonucleotide (MO) injection experiments to phenocopy defects seen in  you,  and mRNA injection experiments to rescue the  you  phenotype in mutants ( Figure 4 ). All wild-type embryos injected with a MO targeting the translational start site showed reduced expression of  myod  ( n  = 112) in the adaxial cells ( Figure 4 B), and an absence of strong Engrailed expression ( n  = 70) in the muscle pioneers ( Figure 4 D). Embryos injected with a mismatch control MO did not exhibit these defects in either  myod  ( n  = 65) or Engrailed ( n  = 48) expression ( Figure 4 A and  4 C). Figure 4 MO-Induced Phenocopy of  you  Defects and Rescue of the  you  Phenotype with mRNA Injection (A, B, E, and F) Dorsal view of the posterior trunk and tail bud of 12-somite embryos. (C, D, G, and H) Lateral views of somites 8–13 in whole-mount embryos at 24 hpf. Anterior is to the left in all images. Injection at the 1–4-cell stage of 420 pg of a MO targeting the translational start site of the  you  mRNA (ATG MO) resulted in decreased adaxial expression of  myod  in the somitic (arrowhead) and presomitic (arrow) mesoderm of wild-type embryos (B). Injection of an equivalent amount of a mismatch control (mismatch MO) did not produce these defects (A). Similarly, wild-type embryos injected with 420 pg of the mismatch MO (C) exhibited strong Engrailed expression in muscle pioneers (arrow) and weaker expression in medial fast fibers (arrowheads). In contrast, Engrailed expression was strongly reduced in wild-type embryos injected with 420 pg of the ATG MO (D), though very weak expression was still observed (arrowhead). Genotypically  you  mutant embryos (E) showed rescued expression of adaxial  myod  in somitic (arrowhead) and presomitic (arrow) mesoderm when injected with 50 pg of synthetic  you  mRNA at the 1–4-cell stage, while mutants injected with 50 pg of a frameshift mutant form of  you  mRNA (F) did not exhibit rescue of adaxial  myod  expression. At 24 hpf, genotypically  you  mutant embryos injected at the 1–4-cell stage with 50 pg of  you  mRNA (G) showed rescue of strong Engrailed expression in the muscle pioneers (arrow) and weaker expression in the medial fast fibers (arrowheads). Mutant embryos injected with 50 pg of the mutant mRNA (H) did not show rescued Engrailed expression, though very weak Engrailed expression (arrowhead) was observed in some cases. Engrailed expression at the MHB was normal in all analyzed embryos (data not shown). Genotypes of embryos shown in (E–H) were determined by PCR after photography. When injected with 50 pg of synthetic wild-type  you  mRNA, 98.7% ( n  = 665) of embryos from  you/+  intercrosses showed expression of  myod  in adaxial cells at 12 somites ( Figure 4 E). Genotyping of 571 embryos with wild-type  myod  expression from these intercrosses showed that 137 (24%) were homozygous mutant for  you . In contrast, 23.6% ( n  = 127) of embryos from  you/+  intercrosses injected with a mutant form of  you  mRNA lacked expression of  myod  in adaxial cells ( Figure 4 F); 20 of the mutants were genotyped and confirmed to be  you  mutant homozygotes. Similarly, all embryos ( n  = 62) from a  you/+  intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers ( Figure 4 G; genotyping of 32 phenotypically wild-type embryos showed that seven were homozygous  you ty97 ), whereas 24% ( n  = 33) of embryos injected with control mRNA lacked Engrailed expression ( Figure 4 H; eight phenotypic mutants were confirmed as homozygous for the  you  mutation). In addition, injection of  you  MOs resulted in loss of  nkx2.2  expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of  you  mRNA was sufficient to rescue  nkx2.2  expression in  you  mutants (data not shown).
Morpholino Phenocopy and RNA Rescue of  you
youTo confirm that this zebrafish EGF-CUB gene is disrupted in  you  mutants, we performed morpholino oligonucleotide (MO) injection experiments to phenocopy defects seen in  you,  and mRNA injection experiments to rescue the  you  phenotype in mutants ( Figure 4 ). All wild-type embryos injected with a MO targeting the translational start site showed reduced expression of  myod  ( n  = 112) in the adaxial cells ( Figure 4 B), and an absence of strong Engrailed expression ( n  = 70) in the muscle pioneers ( Figure 4 D). Embryos injected with a mismatch control MO did not exhibit these defects in either  myod  ( n  = 65) or Engrailed ( n  = 48) expression ( Figure 4 A and  4 C).youyou,youmyodnnmyodnnWhen injected with 50 pg of synthetic wild-type  you  mRNA, 98.7% ( n  = 665) of embryos from  you/+  intercrosses showed expression of  myod  in adaxial cells at 12 somites ( Figure 4 E). Genotyping of 571 embryos with wild-type  myod  expression from these intercrosses showed that 137 (24%) were homozygous mutant for  you . In contrast, 23.6% ( n  = 127) of embryos from  you/+  intercrosses injected with a mutant form of  you  mRNA lacked expression of  myod  in adaxial cells ( Figure 4 F); 20 of the mutants were genotyped and confirmed to be  you  mutant homozygotes. Similarly, all embryos ( n  = 62) from a  you/+  intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers ( Figure 4 G; genotyping of 32 phenotypically wild-type embryos showed that seven were homozygous  you ty97 ), whereas 24% ( n  = 33) of embryos injected with control mRNA lacked Engrailed expression ( Figure 4 H; eight phenotypic mutants were confirmed as homozygous for the  you  mutation). In addition, injection of  you  MOs resulted in loss of  nkx2.2  expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of  you  mRNA was sufficient to rescue  nkx2.2  expression in  you  mutants (data not shown).younyou/+myodmyodyounyou/+youmyodyounyou/+you ty97nyouyounkx2.2younkx2.2youyou  Expression
 you  transcripts appear to be maternally deposited in zebrafish embryos ( Figure 5 A) and are distributed widely in the embryo through early gastrulation stages ( Figure 5 B). During late gastrulation, the distribution of  you  transcripts in the embryo began to be restricted, and at bud stage (10 hpf)  you  was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes ( Figure 5 C). During somitogenesis,  you  expression continued to be refined, such that six-somite embryos exhibited expression in the eye field, in stripes in the midbrain and the midbrain–hindbrain boundary (MHB), in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis ( Figure 5 D). At 24 hpf ( Figure 5 E and  5 F),  you  transcripts were localized to the border of the ventral telencephalon and the dorsal diencephalon and to the ventral tectum, and were strongly expressed in the MHB, the hindbrain, and along the length of the embryo in the dorsal spinal cord. In addition,  you  expression was observed in the ventral tail and posterior to the yolk extension at the developing urogenital opening ( Figure 5 E). In the following 24 h of development, dorsal spinal cord expression continued, and  you  transcripts persisted in a complex and dynamic pattern in the brain. At 48 hpf ( Figure 5 G and  5 H),  you  expression was particularly strong in the cerebellum and in the hindbrain along the rhombic lip. Figure 5 Expression of  you  Examined by In Situ Hybridization (A and B) Maternal  you  transcripts were evident in cleavage-stage embryos (A) (16-cell; 1.5 hpf), and  you  mRNA was widely expressed into the gastrula period (B) (shield stage; 6 hpf). In addition,  you  mRNA was detectable by RT-PCR at 2 hpf, prior to the zebrafish midblastula transition. (C) Toward the end of gastrulation,  you  transcripts began to be restricted, so that at the bud stage (10 hpf),  you  expression was evident in the eye field (white arrowhead), in the developing midbrain and hindbrain (black arrowheads), and in posterior paraxial stripes (arrow). (D) During early somitogenesis (12 hpf),  you  expression was observed in the eye field (white arrowhead), in stripes in the midbrain and the MHB (black arrowheads), in a complex pattern in the hindbrain (white arrow), and in paraxial stripes along the developing trunk and tail bud (black arrow). (E and F) At 24 hpf,  you  transcripts were observed dorsal to the hypothalamus (black arrow), at the boundary between the telencephalon and the diencephalon (white arrow), in the ventral tectum (white arrowhead), in the region of the presumptive cerebellum (asterisk), and dorsally along the length of the spinal cord. Additional expression of  you  at this stage and later was observed in the ventral tail and at the urogenital opening (arrowheads; data not shown). (G and H) At 48 hpf,  you  transcripts were highly expressed in the cerebellum (black arrow), and were also present in the rhombic lip (white arrowhead), and continuing along the length of the anterior–posterior axis in the dorsal spinal cord (black arrowhead; data not shown). Orientation of images: (A) lateral view; (B) lateral view, dorsal to the right; (C, D, F, and G) dorsal views, anterior to the left; (E and H) lateral views, anterior to the left.
you  Expression
youyou  transcripts appear to be maternally deposited in zebrafish embryos ( Figure 5 A) and are distributed widely in the embryo through early gastrulation stages ( Figure 5 B). During late gastrulation, the distribution of  you  transcripts in the embryo began to be restricted, and at bud stage (10 hpf)  you  was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes ( Figure 5 C). During somitogenesis,  you  expression continued to be refined, such that six-somite embryos exhibited expression in the eye field, in stripes in the midbrain and the midbrain–hindbrain boundary (MHB), in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis ( Figure 5 D). At 24 hpf ( Figure 5 E and  5 F),  you  transcripts were localized to the border of the ventral telencephalon and the dorsal diencephalon and to the ventral tectum, and were strongly expressed in the MHB, the hindbrain, and along the length of the embryo in the dorsal spinal cord. In addition,  you  expression was observed in the ventral tail and posterior to the yolk extension at the developing urogenital opening ( Figure 5 E). In the following 24 h of development, dorsal spinal cord expression continued, and  you  transcripts persisted in a complex and dynamic pattern in the brain. At 48 hpf ( Figure 5 G and  5 H),  you  expression was particularly strong in the cerebellum and in the hindbrain along the rhombic lip.youyouyouyouyouyouyouyouPermissive Role of  you  Upstream of the Hedgehog Cellular Response To explore the possibility that the  you  gene may induce a gain-of-function phenotype when overexpressed, we injected wild-type embryos with an amount (50 pg) of synthetic  you  mRNA that was sufficient to rescue the phenotypic defects observed in  you  mutants ( Figure 6 ). When compared to embryos injected with a mutant form of  you  mRNA ( myod, n  = 81,  Figure 6 A; Engrailed,  n  = 25,  Figure 6 E;  nkx2.2, n  = 17,  Figure 6 I), overexpression of  you  in wild-type embryos did not result in obvious ectopic expression of  myod  ( n  = 394;  Figure 6 B) ,  Engrailed ( n  = 25;  Figure 6 F), or  nkx2.2  ( n  = 17;  Figure 6 J). This result suggests that  you  functions as a permissive factor in Hedgehog signaling, rather than as a potent activator of the Hedgehog pathway. When 50 pg of synthetic mRNA encoding a potent Hedgehog pathway activator  (shh)  was injected into embryos from a  you/+  intercross, ectopic expression of  myod  ( n  = 53;  Figure 6 C and  6 D), Engrailed ( n  = 78;  Figure 6 G and  6 H), and  nkx2.2  ( n  = 78;  Figure 6 K and  6 L) was induced in all embryos. Genotyping a subset of these embryos indicated that both genotypically wild-type and  you  mutant embryos showed ectopic expression of each of these markers  (myod:  41 wild-type, 12  you;  Engrailed: 34 wild-type, 8  you; nkx2.2:  63 wild-type, 15  you).  Because downstream targets of the Hedgehog pathway were rescued or upregulated in  shh- injected  you  mutants, components of the Hedgehog pathway downstream of  shh  are most likely functional in  you  embryos. These results are consistent with  you  acting upstream of or parallel with  shh  in the Hedgehog pathway. Figure 6 Early Overexpression of  you  in Wild-type Embryos and Rescue of  you  Defects by  shh  mRNA Injection (A–D) Dorsal views of the posterior trunk and tail bud of whole-mount embryos at 12 somites (15 hpf). (E–F) Lateral views of somites 2–7 in 24-hpf embryos. (G–H) Lateral views of somites 8–13 in 24-hpf embryos. (I–L) Lateral views of 24-hpf embryos. Anterior is to the left in all images. When 50 pg of  you  mRNA was injected into wild-type embryos at the 1–4-cell stage, no obvious expansion of  myod  (B), Engrailed (F), or  nkx2.2  (J) expression was observed when compared either with wild-type embryos injected with equivalent amounts of mutant mRNA (A, E, and I) or with uninjected embryos (see  Figure 1 ). Muscle pioneers were counted in a subset of the embryos; there were 4.0 ± 0.8 Engrailed-expressing muscle pioneers per somite in embryos injected with the control mRNA ( n  = 3 embryos, 33 somites) and 4.6 ± 1.1 muscle pioneers per somite in embryos injected with synthetic  you  mRNA ( n  = 8 embryos, 88 somites). Injection of 50 pg of  shh  mRNA into embryos at the 1–4-cell stage resulted in expansion of  myod,  Engrailed, and  nkx2.2  expression in both wild-type (C, G, and K) and  you  mutant (D, H, and L) embryos. s hh  injection rescued adaxial expression of  myod  (D), muscle pioneer expression of Engrailed (H), and ventral spinal cord expression of  nkx2.2  (L) in genotypically mutant  you  embryos (compare with  Figure 1 ). Genotypes of all embryos were determined by PCR after photography. Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down  patched  activity with MOs ( Figure 7 ). Expression of  myod  in adaxial cells was rescued or expanded in all  you  mutant embryos that were injected with MOs targeting  ptc1  ( Figure 7 E and  7 G;  n  = 8 mutants) or a combination of MOs targeting both  ptc1  and  ptc2  ( Figure 7 I and  7 K;  n  = 13 mutants).  you  mutant embryos injected with a  ptc1  mismatch control MO did not exhibit rescued  myod  expression ( Figure 7 A and  7 C;  n  = 8 mutants). In similar experiments, injection of  patched  MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of  you  mutant embryos ( Figure 7 B,  7 D,  7 F,  7 H,  7 J, and  7 L;  ptc1  MO,  n  = 11 mutants;  ptc1  +  ptc2  MO,  n  = 11 mutants). Figure 7 Knockdown of  patched  Function Rescues Slow Muscle Defects in  you
 After injection of 420 pg of a mismatch control  ptc1  MO, adaxial expression of  myod  (A) and Engrailed (B) was normal in wild-type embryos, but absent in  you  mutant embryos (C and D). When injected with 420 pg of a MO targeting  ptc1,  however,  myod  expression in mutants (E) was rescued to levels comparable to wild-type embryos (G). Engrailed expression was slightly expanded in both wild-type (F) and mutant (H) embryos injected with 420 pg of  ptc1  MOs. Both adaxial  myod  expression and Engrailed expression was slightly expanded in wild-type (I and J) and  you  mutant embryos (K and L) injected with MOs targeting both  ptc1  and  ptc2  (420 pg each). Embryos assayed for  myod  expression are shown in flat mount at the 12-somite stage, and somites 5–9 of Engrailed-expressing embryos are shown in lateral view at 24 hpf. Anterior is to the left in all panels. Genotypes of all embryos were determined by PCR after photography.
Permissive Role of  you  Upstream of the Hedgehog Cellular Response
youTo explore the possibility that the  you  gene may induce a gain-of-function phenotype when overexpressed, we injected wild-type embryos with an amount (50 pg) of synthetic  you  mRNA that was sufficient to rescue the phenotypic defects observed in  you  mutants ( Figure 6 ). When compared to embryos injected with a mutant form of  you  mRNA ( myod, n  = 81,  Figure 6 A; Engrailed,  n  = 25,  Figure 6 E;  nkx2.2, n  = 17,  Figure 6 I), overexpression of  you  in wild-type embryos did not result in obvious ectopic expression of  myod  ( n  = 394;  Figure 6 B) ,  Engrailed ( n  = 25;  Figure 6 F), or  nkx2.2  ( n  = 17;  Figure 6 J). This result suggests that  you  functions as a permissive factor in Hedgehog signaling, rather than as a potent activator of the Hedgehog pathway. When 50 pg of synthetic mRNA encoding a potent Hedgehog pathway activator  (shh)  was injected into embryos from a  you/+  intercross, ectopic expression of  myod  ( n  = 53;  Figure 6 C and  6 D), Engrailed ( n  = 78;  Figure 6 G and  6 H), and  nkx2.2  ( n  = 78;  Figure 6 K and  6 L) was induced in all embryos. Genotyping a subset of these embryos indicated that both genotypically wild-type and  you  mutant embryos showed ectopic expression of each of these markers  (myod:  41 wild-type, 12  you;  Engrailed: 34 wild-type, 8  you; nkx2.2:  63 wild-type, 15  you).  Because downstream targets of the Hedgehog pathway were rescued or upregulated in  shh- injected  you  mutants, components of the Hedgehog pathway downstream of  shh  are most likely functional in  you  embryos. These results are consistent with  you  acting upstream of or parallel with  shh  in the Hedgehog pathway.youyouyouyoumyod, nnnkx2.2, nyoumyodn,nnkx2.2nyou(shh)you/+myodnnnkx2.2nyou(myod:you;you; nkx2.2:you).shh-youshhyouyoushhAdditional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down  patched  activity with MOs ( Figure 7 ). Expression of  myod  in adaxial cells was rescued or expanded in all  you  mutant embryos that were injected with MOs targeting  ptc1  ( Figure 7 E and  7 G;  n  = 8 mutants) or a combination of MOs targeting both  ptc1  and  ptc2  ( Figure 7 I and  7 K;  n  = 13 mutants).  you  mutant embryos injected with a  ptc1  mismatch control MO did not exhibit rescued  myod  expression ( Figure 7 A and  7 C;  n  = 8 mutants). In similar experiments, injection of  patched  MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of  you  mutant embryos ( Figure 7 B,  7 D,  7 F,  7 H,  7 J, and  7 L;  ptc1  MO,  n  = 11 mutants;  ptc1  +  ptc2  MO,  n  = 11 mutants).patchedmyodyouptc1nptc1ptc2nyouptc1myodnpatchedyouptc1nptc1ptc2nyou  Acts Non-Autonomously in Muscle Pioneer Differentiation To determine whether a cell must be wild-type for  you  function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts ( Figure 8 ). Cells derived from  you  mutant embryos were able to differentiate as muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei of mononucleate cells at the proper position in the somite ( Figure 8 C– 8 E;  n  = 13 embryos). Similarly, cells from embryos in which  you  function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown). Figure 8
 you  Acts Non-Autonomously in Muscle Pioneers and Is Not Required in Cells Producing Hedgehog Signals (A) Donor embryos were labeled at the 1–4-cell stage with Oregon Green dextran. (B) Cells from donor embryos were transplanted into unlabeled hosts during late blastula and early gastrula stages. (C–E) Images from a chimera made by transplanting cells from labeled mutant donors (green in C and E) into unlabeled wild-type hosts. At 24 hpf, muscle pioneer cells in chimeric embryos were labeled with anti-Engrailed antibody (red in D and E). When transplanted into wild-type embryos, mutant cells were able to differentiate as muscle pioneers, as shown by co-labeling with the anti-Engrailed antibody (E, yellow arrows). (F–K) Images from chimeras made by transplanting cells from labeled wild-type donors (green in F, H, I, and K) into unlabeled mutant hosts. Expression of Engrailed (red in G, H, J, and K) in some mutant muscle pioneers (one marked by red arrows in G, H, J, and K) was rescued in a subset of embryos (see also  Table 1 ). Donor cells in the embryo shown in (F–H) contributed solely to muscle and to non-floor-plate identities within the neural tube. Moreover, in a subset of chimeras, cells derived from wild-type donors differentiated as muscle pioneer cells (yellow arrows in J and K), simultaneously showing both the characteristic strong nuclear Engrailed expression and the typical flattened and mononucleate morphology of this cell type. The somite labeled with the arrows in J and K contains two muscle pioneers, one derived from the wild-type donor (yellow arrow) and another derived from the mutant host (red arrow). Donor cells in the embryo shown in (I–K) contributed primarily to muscle and to non-floor-plate identities within the neural tube; in addition, a group of seven floor plate cells derived from the wild-type donor was present in the tail of this embryo (not shown). Table 1 Chimeras with Wild-Type Donor Cells in  you  Mutant Hosts Rescue of muscle pioneers in wild-type→ you  mutant chimeras was evidenced by the presence of mutant (i.e., host) cells with strong Engrailed expression in elongate nuclei at the proper position in 1–8 somites. Wild-type contribution indicates structures that included cells from labeled wild-type donors CNS, non-floor-plate cells in neural tube; FP, floor plate
you  Acts Non-Autonomously in Muscle Pioneer Differentiation
youTo determine whether a cell must be wild-type for  you  function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts ( Figure 8 ). Cells derived from  you  mutant embryos were able to differentiate as muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei of mononucleate cells at the proper position in the somite ( Figure 8 C– 8 E;  n  = 13 embryos). Similarly, cells from embryos in which  you  function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown).youyounyouChimeras with Wild-Type Donor Cells in  you  Mutant Hosts
youRescue of muscle pioneers in wild-type→ you  mutant chimeras was evidenced by the presence of mutant (i.e., host) cells with strong Engrailed expression in elongate nuclei at the proper position in 1–8 somites. Wild-type contribution indicates structures that included cells from labeled wild-type donorsyouCNS, non-floor-plate cells in neural tube; FP, floor plateMuscle Pioneer Differentiation Does Not Require  you  Function in Axial Hedgehog-Producing Cells To determine which cell types must be wild-type for  you  function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into  you  mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in  you  mutants ( Table 1 ;  Figure 8 F– 8 K). In addition, wild-type cells differentiated as muscle pioneers in two of the ten chimeras with rescued mutant muscle pioneers. In these cases ( Figure 8 I– 8 K), the muscle pioneer identity of cells strongly expressing Engrailed was further confirmed by presence of the lineage tracer dye, which showed that these cells had the characteristic flattened and mononucleate morphology of muscle pioneers. In all ten embryos with rescued Engrailed expression, wild-type cells were present in the muscle and in non-floor-plate regions within the neural tube. In five of the chimeric embryos with rescued Engrailed expression, the floor plate and notochord were derived entirely from  you  mutant cells, indicating that  you  function is not required in axial Hedgehog-producing cells. Collectively, the transplantation experiments indicate that  you  function is not required in either the signaling or responding cells, and instead suggest that  you  is essential for the transport or stability of Hedgehog signals.
Muscle Pioneer Differentiation Does Not Require  you  Function in Axial Hedgehog-Producing Cells
youTo determine which cell types must be wild-type for  you  function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into  you  mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in  you  mutants ( Table 1 ;  Figure 8 F– 8 K). In addition, wild-type cells differentiated as muscle pioneers in two of the ten chimeras with rescued mutant muscle pioneers. In these cases ( Figure 8 I– 8 K), the muscle pioneer identity of cells strongly expressing Engrailed was further confirmed by presence of the lineage tracer dye, which showed that these cells had the characteristic flattened and mononucleate morphology of muscle pioneers. In all ten embryos with rescued Engrailed expression, wild-type cells were present in the muscle and in non-floor-plate regions within the neural tube. In five of the chimeric embryos with rescued Engrailed expression, the floor plate and notochord were derived entirely from  you  mutant cells, indicating that  you  function is not required in axial Hedgehog-producing cells. Collectively, the transplantation experiments indicate that  you  function is not required in either the signaling or responding cells, and instead suggest that  you  is essential for the transport or stability of Hedgehog signals.youyouyouyouyouyouyouDiscussion Using a positional cloning approach, we have shown that the  you  gene encodes a novel EGF-CUB protein essential for Hedgehog signaling in zebrafish. High-resolution mapping indicates that the  you  locus is tightly linked to a zebrafish homolog of mouse  Scube2. you ty97  mutants harbor a nonsense lesion that truncates the open reading frame upstream of the CUB domain and other highly conserved sequences. MO-mediated knockdown of the gene phenocopies defects observed in  you  mutants, and injection of wild-type mRNA into mutant embryos rescues the  you  phenotype. Taken together, these experiments provide compelling evidence that the  you  mutation disrupts this EGF-CUB gene. Biochemical studies of SCUBE family members have shown that they are extracellular, membrane-associated glycoproteins [ 52 , 53 ], but no previous work implicates these proteins in the Hedgehog signaling pathway. Although the Hedgehog pathway has been extensively studied in flies and mammals, several factors have likely obscured the connection between this  Scube  gene and the Hedgehog pathway. Prior to this study, no loss of function analysis had been performed on any  Scube  family gene; there is no known  Scube  gene in the fly, and mouse mutants have not been reported. Also, because overexpression of synthetic  you  mRNA does not significantly hyperactivate Hedgehog signaling,  Scube  gene function in the Hedgehog pathway would not be apparent in gain-of-function screens to identify pathway components.
 you  mutant embryos exhibit phenotypic defects characteristic of reduced Hedgehog signaling in zebrafish, indicating that the  you  gene is a positively acting component of the Hedgehog pathway. Development of slow muscle is disrupted, as shown by lack of adaxial  myod  expression during somitogenesis and the absence of Engrailed-expressing muscle pioneer cells. Moreover,  you  mutants lack  nkx2.2  expression in the ventral spinal cord, showing that specification of ventral neural fates is disrupted in this region of  you  embryos. Additionally, expression of the Hedgehog target gene  ptc1  is reduced in  you  mutants. Analysis of the mutant phenotype, therefore, demonstrates that the  you  gene is essential for Hedgehog signaling in development of slow muscle and ventral spinal cord fates. Current evidence indicates that EGF-CUB proteins, including You, have extracellular functions. The  you  gene product and other SCUBE proteins contain a signal peptide sequence targeting the protein for secretion, as well as EGF and CUB domains characteristic of extracellular proteins [ 48 , 50 , 51 , 52 , 53 ]. The You homolog SCUBE1 is a glycosylated peripheral membrane protein when expressed in 293T cells, and is also present at low levels in the culture medium [ 52 ]. EGF and CUB domains are found together in a small but diverse group of extracellularly acting proteins, including the complement subunits C1s and C1r, the metalloproteinase Tolloid, the sea urchin extracellular matrix protein Fibropellin, the serum glycoprotein Attractin, and the scavenger receptor Cubilin. CUB domains have been implicated in mediating protein–protein interactions and may confer specificity to ligand binding; for example, specific CUB domains in Cubilin have been implicated in facilitating the binding and subsequent endocytosis of specific ligands (reviewed in [ 54 ]). Although  you  is essential for Hedgehog signaling, our analysis indicates that  you  mutant cells are able to produce and respond to Hedgehog signals.  hedgehog  gene expression in the embryonic midline is normal in  you  mutants, indicating that You acts downstream of  hedgehog  gene transcription (see  Figure 1 ). Further evidence that  you  function is not required in cells generating Hedgehog signals derives from the analysis of chimeric embryos: muscle pioneers can differentiate in chimeras in which the notochord and floor plate are formed entirely from mutant cells (see  Figure 8 ;  Table 1 ). Conversely,  you  mutants can respond to Hedgehog pathway activation, mediated by either  shh  overexpression or disruption of  patched,  demonstrating that the defect in the mutants lies upstream of cellular response mechanism (see  Figures 6  and  7 ). In addition,  you  mutant cells can respond to Hedgehog and differentiate as muscle pioneers when transplanted into a wild-type host (see  Figure 8 ), indicating that  you  gene function is not required in cells responding to Hedgehog signals. The analysis of chimeras also demonstrates that the presence of wild-type cells in the paraxial mesoderm and neural plate is sufficient to allow  you  mutant cells to respond to Hedgehog signals produced from  you  mutant notochord and floor plate. Thus, our epistasis and transplantation experiments provide evidence that You functions in the extracellular environment in the field of responding cells, likely acting to transport or stabilize Hedgehog signals. It is possible that You interacts with components of the extracellular matrix, some of which are known to regulate the action of Hedgehog signals in other systems [ 11 , 12 , 55 , 56 ]. Another possible model is that You activates the Hedgehog pathway indirectly, by inhibiting an extracellular pathway antagonist such as Hip1 or Gas1. Our results, however, do not support such a model. Overexpression of high levels of synthetic  you  mRNA (up to ten times the amount required to rescue  you  mutants) in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial  myod,  Engrailed in muscle pioneers, or  nkx2.2  in the ventral spinal cord (see  Figure 6 ; data not shown). The finding that overexpression of  you  does not significantly hyperactivate Hedgehog targets argues against simple models in which  you  functions to counteract an endogenous repressor of the Hedgehog pathway. An interesting aspect of the  you  mutant phenotype is that it encompasses only a subset of defects seen in other zebrafish Hedgehog pathway mutants. Whereas mutants for  syu/shh, yot/gli2, smu/smoh,  and  con/disp1  have prominent midline abnormalities in the head, such as ipsilateral retinotectal projections, reduction of anterior pituitary, and defects in medial neurocranial cartilage, these phenotypes are not evident in  you  mutants [ 26 , 32 , 33 , 37 , 43 , 57 , 58 , 59 ]. Also, development of pectoral fins, which is disrupted in  syu, con,  and  smu  [ 26 , 60 ], appears normal in  you  embryos.  you  mutants, therefore, show characteristic Hedgehog signaling defects in slow muscle specification, patterning of ventral spinal cord, and the development of the dorsal aorta, but  you  is apparently not required for Hedgehog signaling in some other regions of the zebrafish embryo. Because the primary cell types disrupted in  you  mutants all develop in close proximity to the notochord, it is possible that  you  gene function may be required for the transport or stability of Hedgehog signals in the vicinity of the developing notochord but not some other regions. The notochord is a defining feature of chordates, and a notochord-associated function would explain why no  you  counterpart is required for Hedgehog signaling in the fly. It is not clear, however, why Hedgehog signaling near the notochord would require a special extracellular mediator. Another possibility is that maternal  you  function could mask earlier requirements in zygotic  you  mutants; future work with maternal-zygotic  you  mutants is needed to address this possibility. A third explanation of the requirement for  you  in only a subset of Hedgehog-regulated processes is that additional factors with redundant functions may substitute for  you  in other regions of the embryo. Intriguingly, expression of another  Scube  gene in mouse— Scube1— is observed in many embryonic tissues known to require Hedgehog signaling for their proper development, including the ventral forebrain, limb bud, somites, and developing gonad [ 48 ]. These results suggest that an additional zebrafish  Scube  gene may also play a role in the development of other areas of the embryo where Hedgehog signaling is active. Moreover, interactions between SCUBE proteins may be important for Hedgehog signaling; biochemical analysis suggests that SCUBE1 and SCUBE2 proteins can interact to form both homodimers and heterodimers [ 52 ]. In addition to its role in promoting Hedgehog signaling in the developing muscle pioneers, ventral neural tube, and dorsal aorta, the expression pattern of  you  suggests that the gene may act in other cell types and perhaps in other pathways. During gastrulation, when Hedgehog signaling is required for specification of muscle pioneers [ 28 ],  you  is widely expressed. In 24-hpf embryos, however,  you  is expressed strongly in specific regions in the forebrain, midbrain, and hindbrain, and dorsally along the length of the spinal cord. Some of these expression domains overlap with regions of Hedgehog signaling, whereas others do not. One region where Hedgehog activity and  you  expression intersect at later embryonic stages is the cerebellum, where Hedgehog signaling plays a well-defined role in the proliferation of granule cell precursors in mammals [ 61 , 62 ]. In the trunk and tail, however,  you  expression in the dorsal spinal cord corresponds neither with known sources of Hedgehog signals nor with cells that require Hedgehog signaling for their proper development. This result suggests that  you  may function in other pathways later in development. Future studies will define the role of  you  in the Hedgehog pathway and address the possibility that  you  and other  Scube  family genes also function in other signaling pathways.
Discussion
Using a positional cloning approach, we have shown that the  you  gene encodes a novel EGF-CUB protein essential for Hedgehog signaling in zebrafish. High-resolution mapping indicates that the  you  locus is tightly linked to a zebrafish homolog of mouse  Scube2. you ty97  mutants harbor a nonsense lesion that truncates the open reading frame upstream of the CUB domain and other highly conserved sequences. MO-mediated knockdown of the gene phenocopies defects observed in  you  mutants, and injection of wild-type mRNA into mutant embryos rescues the  you  phenotype. Taken together, these experiments provide compelling evidence that the  you  mutation disrupts this EGF-CUB gene.youyouScube2. you ty97youyouyouBiochemical studies of SCUBE family members have shown that they are extracellular, membrane-associated glycoproteins [ 52 , 53 ], but no previous work implicates these proteins in the Hedgehog signaling pathway. Although the Hedgehog pathway has been extensively studied in flies and mammals, several factors have likely obscured the connection between this  Scube  gene and the Hedgehog pathway. Prior to this study, no loss of function analysis had been performed on any  Scube  family gene; there is no known  Scube  gene in the fly, and mouse mutants have not been reported. Also, because overexpression of synthetic  you  mRNA does not significantly hyperactivate Hedgehog signaling,  Scube  gene function in the Hedgehog pathway would not be apparent in gain-of-function screens to identify pathway components.ScubeScubeScubeyouScubeyou  mutant embryos exhibit phenotypic defects characteristic of reduced Hedgehog signaling in zebrafish, indicating that the  you  gene is a positively acting component of the Hedgehog pathway. Development of slow muscle is disrupted, as shown by lack of adaxial  myod  expression during somitogenesis and the absence of Engrailed-expressing muscle pioneer cells. Moreover,  you  mutants lack  nkx2.2  expression in the ventral spinal cord, showing that specification of ventral neural fates is disrupted in this region of  you  embryos. Additionally, expression of the Hedgehog target gene  ptc1  is reduced in  you  mutants. Analysis of the mutant phenotype, therefore, demonstrates that the  you  gene is essential for Hedgehog signaling in development of slow muscle and ventral spinal cord fates.youyoumyodyounkx2.2youptc1youyouCurrent evidence indicates that EGF-CUB proteins, including You, have extracellular functions. The  you  gene product and other SCUBE proteins contain a signal peptide sequence targeting the protein for secretion, as well as EGF and CUB domains characteristic of extracellular proteins [ 48 , 50 , 51 , 52 , 53 ]. The You homolog SCUBE1 is a glycosylated peripheral membrane protein when expressed in 293T cells, and is also present at low levels in the culture medium [ 52 ]. EGF and CUB domains are found together in a small but diverse group of extracellularly acting proteins, including the complement subunits C1s and C1r, the metalloproteinase Tolloid, the sea urchin extracellular matrix protein Fibropellin, the serum glycoprotein Attractin, and the scavenger receptor Cubilin. CUB domains have been implicated in mediating protein–protein interactions and may confer specificity to ligand binding; for example, specific CUB domains in Cubilin have been implicated in facilitating the binding and subsequent endocytosis of specific ligands (reviewed in [ 54 ]).youAlthough  you  is essential for Hedgehog signaling, our analysis indicates that  you  mutant cells are able to produce and respond to Hedgehog signals.  hedgehog  gene expression in the embryonic midline is normal in  you  mutants, indicating that You acts downstream of  hedgehog  gene transcription (see  Figure 1 ). Further evidence that  you  function is not required in cells generating Hedgehog signals derives from the analysis of chimeric embryos: muscle pioneers can differentiate in chimeras in which the notochord and floor plate are formed entirely from mutant cells (see  Figure 8 ;  Table 1 ). Conversely,  you  mutants can respond to Hedgehog pathway activation, mediated by either  shh  overexpression or disruption of  patched,  demonstrating that the defect in the mutants lies upstream of cellular response mechanism (see  Figures 6  and  7 ). In addition,  you  mutant cells can respond to Hedgehog and differentiate as muscle pioneers when transplanted into a wild-type host (see  Figure 8 ), indicating that  you  gene function is not required in cells responding to Hedgehog signals. The analysis of chimeras also demonstrates that the presence of wild-type cells in the paraxial mesoderm and neural plate is sufficient to allow  you  mutant cells to respond to Hedgehog signals produced from  you  mutant notochord and floor plate. Thus, our epistasis and transplantation experiments provide evidence that You functions in the extracellular environment in the field of responding cells, likely acting to transport or stabilize Hedgehog signals. It is possible that You interacts with components of the extracellular matrix, some of which are known to regulate the action of Hedgehog signals in other systems [ 11 , 12 , 55 , 56 ].youyouhedgehogyouhedgehogyouyoushhpatched,youyouyouyouAnother possible model is that You activates the Hedgehog pathway indirectly, by inhibiting an extracellular pathway antagonist such as Hip1 or Gas1. Our results, however, do not support such a model. Overexpression of high levels of synthetic  you  mRNA (up to ten times the amount required to rescue  you  mutants) in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial  myod,  Engrailed in muscle pioneers, or  nkx2.2  in the ventral spinal cord (see  Figure 6 ; data not shown). The finding that overexpression of  you  does not significantly hyperactivate Hedgehog targets argues against simple models in which  you  functions to counteract an endogenous repressor of the Hedgehog pathway.youyoumyod,nkx2.2youyouAn interesting aspect of the  you  mutant phenotype is that it encompasses only a subset of defects seen in other zebrafish Hedgehog pathway mutants. Whereas mutants for  syu/shh, yot/gli2, smu/smoh,  and  con/disp1  have prominent midline abnormalities in the head, such as ipsilateral retinotectal projections, reduction of anterior pituitary, and defects in medial neurocranial cartilage, these phenotypes are not evident in  you  mutants [ 26 , 32 , 33 , 37 , 43 , 57 , 58 , 59 ]. Also, development of pectoral fins, which is disrupted in  syu, con,  and  smu  [ 26 , 60 ], appears normal in  you  embryos.  you  mutants, therefore, show characteristic Hedgehog signaling defects in slow muscle specification, patterning of ventral spinal cord, and the development of the dorsal aorta, but  you  is apparently not required for Hedgehog signaling in some other regions of the zebrafish embryo. Because the primary cell types disrupted in  you  mutants all develop in close proximity to the notochord, it is possible that  you  gene function may be required for the transport or stability of Hedgehog signals in the vicinity of the developing notochord but not some other regions. The notochord is a defining feature of chordates, and a notochord-associated function would explain why no  you  counterpart is required for Hedgehog signaling in the fly. It is not clear, however, why Hedgehog signaling near the notochord would require a special extracellular mediator. Another possibility is that maternal  you  function could mask earlier requirements in zygotic  you  mutants; future work with maternal-zygotic  you  mutants is needed to address this possibility. A third explanation of the requirement for  you  in only a subset of Hedgehog-regulated processes is that additional factors with redundant functions may substitute for  you  in other regions of the embryo. Intriguingly, expression of another  Scube  gene in mouse— Scube1— is observed in many embryonic tissues known to require Hedgehog signaling for their proper development, including the ventral forebrain, limb bud, somites, and developing gonad [ 48 ]. These results suggest that an additional zebrafish  Scube  gene may also play a role in the development of other areas of the embryo where Hedgehog signaling is active. Moreover, interactions between SCUBE proteins may be important for Hedgehog signaling; biochemical analysis suggests that SCUBE1 and SCUBE2 proteins can interact to form both homodimers and heterodimers [ 52 ].yousyu/shh, yot/gli2, smu/smoh,con/disp1yousyu, con,smuyouyouyouyouyouyouyouyouyouyouyouScubeScube1—ScubeIn addition to its role in promoting Hedgehog signaling in the developing muscle pioneers, ventral neural tube, and dorsal aorta, the expression pattern of  you  suggests that the gene may act in other cell types and perhaps in other pathways. During gastrulation, when Hedgehog signaling is required for specification of muscle pioneers [ 28 ],  you  is widely expressed. In 24-hpf embryos, however,  you  is expressed strongly in specific regions in the forebrain, midbrain, and hindbrain, and dorsally along the length of the spinal cord. Some of these expression domains overlap with regions of Hedgehog signaling, whereas others do not. One region where Hedgehog activity and  you  expression intersect at later embryonic stages is the cerebellum, where Hedgehog signaling plays a well-defined role in the proliferation of granule cell precursors in mammals [ 61 , 62 ]. In the trunk and tail, however,  you  expression in the dorsal spinal cord corresponds neither with known sources of Hedgehog signals nor with cells that require Hedgehog signaling for their proper development. This result suggests that  you  may function in other pathways later in development. Future studies will define the role of  you  in the Hedgehog pathway and address the possibility that  you  and other  Scube  family genes also function in other signaling pathways.youyouyouyouyouyouyouyouScubeMaterials and Methods Fish strains Zebrafish embryos were maintained at 28.5 °C and were staged according to [ 63 ]. Wild-type embryos were derived from the WIK strain. All phenotypic analysis of  you  mutants was performed with embryos homozygous for the  you ty97  allele [ 22 ]. Genetic mapping The mapping panel was generated by crossing  you ty97 /+ individuals with wild-type fish from the WIK strain.  you ty97  heterozygotes in the F1 generation were intercrossed, and mutant and wild-type embryos in the F2 progeny were collected at 3–4 dpf for mapping. Genomic DNA was prepared from these embryos as described [ 64 ]. Primer sequences for SSLP markers were obtained from the MGH zebrafish database ( http://zebrafish.mgh.harvard.edu ). For initial localization of  you,  bulked segregant analysis (reviewed in [ 65 ]) was performed on DNA pools from 20 mutant and 20 wild-type embryos. Putative zebrafish orthologs of  hedgehog -related genes were identified by reciprocal BLAST analysis and localized to the Heat Shock Panel as previously described [ 47 ]. BAC screening, chromosome walking, and BAC sequencing The CHORI211 BAC library was screened by PCR to identify positive BAC clones ( http://www.rzpd.de ). BAC end sequences were obtained from the Sanger Institute database ( http://trace.ensembl.org , and PCR primers were designed to amplify regions of these sequences. PCR amplicons were sequenced from homozygous wild-type and mutant embryos to identify nucleotide differences that generated restriction enzyme fragment length polymorphisms. These polymorphisms were tested on the mapping panel, and markers showing tighter linkage with  you  were iteratively screened against the BAC library until a contiguous stretch of genomic sequence with ends that flanked  you  was identified. The BAC zC93A15 was subcloned into pBluescript SK+ (Stratagene, La Jolla, California, United States) following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City, California, United States). Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15. Plasmid constructs A full-length  you  clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the  you  protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant  you  clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for  you  were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic  you  mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States). In situ hybridization, antibody labeling, and genotyping Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from  you /+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [ 66 ]. Other probes used were zebrafish  myod  [ 67 ],  ptc1  [ 68 ],  nkx2.2  [ 44 ],  ehh  [ 20 ],  shh  [ 69 ], and monoclonal antibody 4D9 [ 29 ]. Genotyping was performed by scoring a polymorphism in AI722938 (primer 1,
GTGAAAGCAAAAAGCAAGCA; primer 2,
GCACTGCATTATGTTTGTGGA; followed by a Hinf I digest).
 Microinjections Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described [ 70 ]. RNA was diluted in 0.2 M KCl with 5 mg/ml Phenol Red prior to injection. MOs were obtained from Gene Tools (Philomath, Oregon, United States). A MO targeted to the  you  translational initiation site (5′-
GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAg
AGTCg
AAACAcCTg
CCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting  ptc1  and  ptc2  were obtained from [ 28 ].
 Transplantations Cellular transplantations were done according to standard methods [ 22 ]. Embryos derived from  you/+  intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from  you /+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into  you  mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in  you /+ intercrosses.
Materials and Methods
Fish strains Zebrafish embryos were maintained at 28.5 °C and were staged according to [ 63 ]. Wild-type embryos were derived from the WIK strain. All phenotypic analysis of  you  mutants was performed with embryos homozygous for the  you ty97  allele [ 22 ]. Genetic mapping The mapping panel was generated by crossing  you ty97 /+ individuals with wild-type fish from the WIK strain.  you ty97  heterozygotes in the F1 generation were intercrossed, and mutant and wild-type embryos in the F2 progeny were collected at 3–4 dpf for mapping. Genomic DNA was prepared from these embryos as described [ 64 ]. Primer sequences for SSLP markers were obtained from the MGH zebrafish database ( http://zebrafish.mgh.harvard.edu ). For initial localization of  you,  bulked segregant analysis (reviewed in [ 65 ]) was performed on DNA pools from 20 mutant and 20 wild-type embryos. Putative zebrafish orthologs of  hedgehog -related genes were identified by reciprocal BLAST analysis and localized to the Heat Shock Panel as previously described [ 47 ]. BAC screening, chromosome walking, and BAC sequencing The CHORI211 BAC library was screened by PCR to identify positive BAC clones ( http://www.rzpd.de ). BAC end sequences were obtained from the Sanger Institute database ( http://trace.ensembl.org , and PCR primers were designed to amplify regions of these sequences. PCR amplicons were sequenced from homozygous wild-type and mutant embryos to identify nucleotide differences that generated restriction enzyme fragment length polymorphisms. These polymorphisms were tested on the mapping panel, and markers showing tighter linkage with  you  were iteratively screened against the BAC library until a contiguous stretch of genomic sequence with ends that flanked  you  was identified. The BAC zC93A15 was subcloned into pBluescript SK+ (Stratagene, La Jolla, California, United States) following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City, California, United States). Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15. Plasmid constructs A full-length  you  clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the  you  protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant  you  clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for  you  were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic  you  mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States). In situ hybridization, antibody labeling, and genotyping Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from  you /+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [ 66 ]. Other probes used were zebrafish  myod  [ 67 ],  ptc1  [ 68 ],  nkx2.2  [ 44 ],  ehh  [ 20 ],  shh  [ 69 ], and monoclonal antibody 4D9 [ 29 ]. Genotyping was performed by scoring a polymorphism in AI722938 (primer 1,
GTGAAAGCAAAAAGCAAGCA; primer 2,
GCACTGCATTATGTTTGTGGA; followed by a Hinf I digest).
 Microinjections Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described [ 70 ]. RNA was diluted in 0.2 M KCl with 5 mg/ml Phenol Red prior to injection. MOs were obtained from Gene Tools (Philomath, Oregon, United States). A MO targeted to the  you  translational initiation site (5′-
GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAg
AGTCg
AAACAcCTg
CCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting  ptc1  and  ptc2  were obtained from [ 28 ].
 Transplantations Cellular transplantations were done according to standard methods [ 22 ]. Embryos derived from  you/+  intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from  you /+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into  you  mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in  you /+ intercrosses.

Fish strains Zebrafish embryos were maintained at 28.5 °C and were staged according to [ 63 ]. Wild-type embryos were derived from the WIK strain. All phenotypic analysis of  you  mutants was performed with embryos homozygous for the  you ty97  allele [ 22 ].
Fish strains
Zebrafish embryos were maintained at 28.5 °C and were staged according to [ 63 ]. Wild-type embryos were derived from the WIK strain. All phenotypic analysis of  you  mutants was performed with embryos homozygous for the  you ty97  allele [ 22 ].youyou ty97Genetic mapping The mapping panel was generated by crossing  you ty97 /+ individuals with wild-type fish from the WIK strain.  you ty97  heterozygotes in the F1 generation were intercrossed, and mutant and wild-type embryos in the F2 progeny were collected at 3–4 dpf for mapping. Genomic DNA was prepared from these embryos as described [ 64 ]. Primer sequences for SSLP markers were obtained from the MGH zebrafish database ( http://zebrafish.mgh.harvard.edu ). For initial localization of  you,  bulked segregant analysis (reviewed in [ 65 ]) was performed on DNA pools from 20 mutant and 20 wild-type embryos. Putative zebrafish orthologs of  hedgehog -related genes were identified by reciprocal BLAST analysis and localized to the Heat Shock Panel as previously described [ 47 ].
Genetic mapping
The mapping panel was generated by crossing  you ty97 /+ individuals with wild-type fish from the WIK strain.  you ty97  heterozygotes in the F1 generation were intercrossed, and mutant and wild-type embryos in the F2 progeny were collected at 3–4 dpf for mapping. Genomic DNA was prepared from these embryos as described [ 64 ]. Primer sequences for SSLP markers were obtained from the MGH zebrafish database ( http://zebrafish.mgh.harvard.edu ). For initial localization of  you,  bulked segregant analysis (reviewed in [ 65 ]) was performed on DNA pools from 20 mutant and 20 wild-type embryos. Putative zebrafish orthologs of  hedgehog -related genes were identified by reciprocal BLAST analysis and localized to the Heat Shock Panel as previously described [ 47 ].you ty97you ty97you,hedgehogBAC screening, chromosome walking, and BAC sequencing The CHORI211 BAC library was screened by PCR to identify positive BAC clones ( http://www.rzpd.de ). BAC end sequences were obtained from the Sanger Institute database ( http://trace.ensembl.org , and PCR primers were designed to amplify regions of these sequences. PCR amplicons were sequenced from homozygous wild-type and mutant embryos to identify nucleotide differences that generated restriction enzyme fragment length polymorphisms. These polymorphisms were tested on the mapping panel, and markers showing tighter linkage with  you  were iteratively screened against the BAC library until a contiguous stretch of genomic sequence with ends that flanked  you  was identified. The BAC zC93A15 was subcloned into pBluescript SK+ (Stratagene, La Jolla, California, United States) following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City, California, United States). Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15.
BAC screening, chromosome walking, and BAC sequencing
The CHORI211 BAC library was screened by PCR to identify positive BAC clones ( http://www.rzpd.de ). BAC end sequences were obtained from the Sanger Institute database ( http://trace.ensembl.org , and PCR primers were designed to amplify regions of these sequences. PCR amplicons were sequenced from homozygous wild-type and mutant embryos to identify nucleotide differences that generated restriction enzyme fragment length polymorphisms. These polymorphisms were tested on the mapping panel, and markers showing tighter linkage with  you  were iteratively screened against the BAC library until a contiguous stretch of genomic sequence with ends that flanked  you  was identified. The BAC zC93A15 was subcloned into pBluescript SK+ (Stratagene, La Jolla, California, United States) following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City, California, United States). Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15.youyouPlasmid constructs A full-length  you  clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the  you  protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant  you  clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for  you  were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic  you  mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States).
Plasmid constructs
A full-length  you  clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the  you  protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant  you  clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for  you  were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic  you  mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States).youyouyouyouyouIn situ hybridization, antibody labeling, and genotyping Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from  you /+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [ 66 ]. Other probes used were zebrafish  myod  [ 67 ],  ptc1  [ 68 ],  nkx2.2  [ 44 ],  ehh  [ 20 ],  shh  [ 69 ], and monoclonal antibody 4D9 [ 29 ]. Genotyping was performed by scoring a polymorphism in AI722938 (primer 1,
GTGAAAGCAAAAAGCAAGCA; primer 2,
GCACTGCATTATGTTTGTGGA; followed by a Hinf I digest).
In situ hybridization, antibody labeling, and genotyping
Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from  you /+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [ 66 ]. Other probes used were zebrafish  myod  [ 67 ],  ptc1  [ 68 ],  nkx2.2  [ 44 ],  ehh  [ 20 ],  shh  [ 69 ], and monoclonal antibody 4D9 [ 29 ]. Genotyping was performed by scoring a polymorphism in AI722938 (primer 1,
GTGAAAGCAAAAAGCAAGCA; primer 2,
GCACTGCATTATGTTTGTGGA; followed by a Hinf I digest).youmyodptc1nkx2.2ehhshhMicroinjections Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described [ 70 ]. RNA was diluted in 0.2 M KCl with 5 mg/ml Phenol Red prior to injection. MOs were obtained from Gene Tools (Philomath, Oregon, United States). A MO targeted to the  you  translational initiation site (5′-
GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAg
AGTCg
AAACAcCTg
CCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting  ptc1  and  ptc2  were obtained from [ 28 ].
Microinjections
Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described [ 70 ]. RNA was diluted in 0.2 M KCl with 5 mg/ml Phenol Red prior to injection. MOs were obtained from Gene Tools (Philomath, Oregon, United States). A MO targeted to the  you  translational initiation site (5′-
GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAg
AGTCg
AAACAcCTg
CCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting  ptc1  and  ptc2  were obtained from [ 28 ].youptc1ptc2Transplantations Cellular transplantations were done according to standard methods [ 22 ]. Embryos derived from  you/+  intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from  you /+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into  you  mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in  you /+ intercrosses.
Transplantations
Cellular transplantations were done according to standard methods [ 22 ]. Embryos derived from  you/+  intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from  you /+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into  you  mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in  you /+ intercrosses.you/+youyouyouSupporting Information Accession Numbers The  you  cDNA sequence has been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) under accession number AY741664. The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716),  con/disp1  (LocusID 378448), Cubilin (LocusID 8029),  ehh  (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725),  myod  (LocusID 30513) , nkx2.2  (LocusID 30697), Patched (LocusID 35851),  prox1  (LocusID 30679),  ptc1  (LocusID 30181),  ptc2  (LocusID 30189), sea urchin Fibropellin (LocusID 373313),  smu/smoh  (LocusID 30225),  syu/ssh  (LocusID 30269), Tolloid (LocusID 42945),  tout velu  (LocusID 36614),  twhh  (LocusID 30444),  ubo/prdm1  (LocusID 323473),  yot/gli2  (LocusID 30154), human  LMO1  (LocusID 4004) ,  human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human  ST5  (LocusID 6764), human  STK33  (LocusID 65975) ,  mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).
Supporting Information
Accession Numbers The  you  cDNA sequence has been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) under accession number AY741664. The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716),  con/disp1  (LocusID 378448), Cubilin (LocusID 8029),  ehh  (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725),  myod  (LocusID 30513) , nkx2.2  (LocusID 30697), Patched (LocusID 35851),  prox1  (LocusID 30679),  ptc1  (LocusID 30181),  ptc2  (LocusID 30189), sea urchin Fibropellin (LocusID 373313),  smu/smoh  (LocusID 30225),  syu/ssh  (LocusID 30269), Tolloid (LocusID 42945),  tout velu  (LocusID 36614),  twhh  (LocusID 30444),  ubo/prdm1  (LocusID 323473),  yot/gli2  (LocusID 30154), human  LMO1  (LocusID 4004) ,  human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human  ST5  (LocusID 6764), human  STK33  (LocusID 65975) ,  mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).
Accession Numbers
The  you  cDNA sequence has been deposited in GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) under accession number AY741664.youThe LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716),  con/disp1  (LocusID 378448), Cubilin (LocusID 8029),  ehh  (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725),  myod  (LocusID 30513) , nkx2.2  (LocusID 30697), Patched (LocusID 35851),  prox1  (LocusID 30679),  ptc1  (LocusID 30181),  ptc2  (LocusID 30189), sea urchin Fibropellin (LocusID 373313),  smu/smoh  (LocusID 30225),  syu/ssh  (LocusID 30269), Tolloid (LocusID 42945),  tout velu  (LocusID 36614),  twhh  (LocusID 30444),  ubo/prdm1  (LocusID 323473),  yot/gli2  (LocusID 30154), human  LMO1  (LocusID 4004) ,  human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human  ST5  (LocusID 6764), human  STK33  (LocusID 65975) ,  mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).con/disp1ehhmyod, nkx2.2prox1ptc1ptc2smu/smohsyu/sshtout velutwhhubo/prdm1yot/gli2LMO1,ST5STK33,