Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system.Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all  Drosophila genome for mutations that affect tracheal (respiratory) system development. We included steps to bypass complexities from mutant effects on other tissues and steps to exclude mutations in general cell “housekeeping genes.” We isolated mutations in ∼70 genes that identify major steps in the organogenesis program including an integral cell growth control step. Many of the new tracheal genes are “morphogenesis genes” that encode proteins involved in cell structure or intracellular transport. The results suggest that genetic screens can elucidate a full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ.Elucidating the genetic programs that control formation and maintenance of body organs is a central goal of developmental biology, and understanding how these programs go awry in disease has important implications for medicine. Many such organogenesis genes have been identified, but most are early-acting “patterning genes” encoding signaling proteins and gene regulators that control expression of a poorly characterized set of downstream “morphogenesis genes,” which encode proteins that generate the remarkable organ forms and structures of the constituent cells. We screened ∼40% of the fruit fly  Drosophila body axis formation and other early developmental events Drosophila tracheal system organogenesis that included clonal analysis of gene function in the tracheal system, to identify all tracheal genes including those with pleiotropic phenotypes. The results of a screen of the third chromosome, representing ∼40% of the Drosophila genome Elucidating the genetic programs of organ formation and maintenance is a central goal of developmental biology and medicine. Many organogenesis genes have been isolated in systematic genetic screens in model organisms, and many others have been identified by their organ-selective expression patterns and by candidate gene analysis. These approaches have been very successful at discovering the signaling pathways and transcription factors that induce and pattern organs and specify cell fates, but they have been much less successful at identifying the downstream effectors that execute morphogenesis programs, what we call morphogenesis genes Drosophila tracheal (respiratory) system is a branched tubular network that transports oxygen throughout the body The C. elegans excretory system The tracheal system develops from 10 pairs of tracheal sacs that arise by invagination of the embryonic ectoderm trachealess, isolated in the classical screens for embryonic patterning mutants by the complete and selective absence of the tracheal system trachealess, a Drosophila homolog of mammalian FGFRs was isolated and named Breathless because it is selectively expressed in the developing tracheal system and required for branching pointed, blistered/pruned, and escargot required, respectively, for secondary and terminal branching and branch fusion branchless, breathless) and transcription factors The first important tracheal gene identified was Drosophila third chromosome genes, including early essential genes and genes with pleiotropic phenotypes. We sought to identify most or all of the genetically separable steps in tracheal development; to identify new tracheal genes associated with each step; and to provide an estimate of the total number of genes required to build an organ. We were especially interested in identifying tracheal morphogenesis genes. Our approach involved clonal analysis in the tracheal system of all chemically-induced mutations that did not survive late enough in development as homozygotes to assess their tracheal function, and a secondary screen to exclude general “house-keeping” genes. We isolated mutations representing ∼70 genes, 14 of which we identified molecularly, implicating most of the genes as morphogenesis genes and revealing new cell biological pathways in tracheal development. Many of the mutations affect terminal branch morphogenesis, genetically subdividing this poorly understood process into five major morphogenetic steps including an integral cell growth step.We conducted a large-scale screen of chemically-induced mutations to assess the function of nearly all Drosophila genes To identify tracheal morphogenesis genes, we screened the third chromosome for EMS-induced mutations that affect larval tracheal morphology. Approximately 4,300 mutagenized third chromosomes were generated, and balanced lines were established for each. Three-quarters (73%) of the lines were homozygous lethal. Assuming a Poisson distribution, there were ∼1.3 lethal mutations per mutagenized third chromosome and a total of 5600 lethal mutations screened. Because there are ∼3600 essential btl-GAL4 and UAS-GFP transgenes (abbreviated btl>GFP) to label tracheal cells, were scored for tracheal defects by fluorescence microscopy. The balancer chromosome carried a Tub-GAL80 transgene that inhibits Gal4 and blocks expression of UAS-GFP so only homozygous mutant animals expressed GFP, facilitating screening ) with btl>GFP  with surrounding wild type tracheal cells , enhancing the sensitivity of the screen and detection of cell non-autonomous effects in the tracheal system.These pre-pupal lethal lines were analyzed in a second step of the screen, using a genetic mosaic strategy in which we examined clones of homozygous mutant tracheal cells in otherwise heterozygous larvae . We devi btl>GFP . This faOver 600 mutants with highly penetrant and expressive tracheal defects were identified. However, the vast majority were lethal mutations in genes required cell autonomously for tracheal cell growth and survival, and later found in a secondary screen (see below) to be presumptive housekeeping genes and discarded. 133 tracheal mutations were saved , 18 fromMost of the mutations could be placed into one of five phenotypic classes : (1) celComplementation tests allowed assignment of 68 mutations to 24 loci . In addistumps and trachealess) whose mosaic phenotype we determined, and the lower than expected allele frequencies (mean 2.6) obtained for the 28 definitively identified loci, indicate that the screen did not achieve the degree of saturation predicted by a Poisson distribution. Nevertheless, the screen was extensive so we think it is likely it identified mutations in most processes and molecular pathways involved in tracheal tube morphogenesis.It is difficult to estimate the number of mutations we obtained in previously known tracheal genes because the mosaic loss of function phenotype is not known for most tracheal genes, and the number of complementation tests necessary to determine this number directly is prohibitive. However, the apparent absence of mutations in two known tracheal genes  clones for comparison or to exclude partial masking of phenotypes due to perdurance of wild type protein in mutant cells.These mutations eliminated specific tracheal cell types or blocked their differentiation.no terminal cell clones-3L and no terminal cell clones-3R) gave normal numbers of mutant clones in the mosaic screen but the mutant cells rarely if ever included terminal cells  mutants, dorsal branch, fat body, and CNS terminal cells were frequently missing, but terminal cells in other positions were unaffected (steeple (AI87) mutants frequently lacked dorsal branch terminal cells  and missing parts (loose caboose (AB56) mutants had frequent defects in fusion of the most posterior dorsal branches  mutant cells were several times larger than normal (jolly green giant (1149) caused a more subtle increase in cell size, most readily detected in terminal cells, and no obvious alteration in lumen morphology. Both miracle-gro and jolly green giant mutations also cause overgrowth of cells outside the tracheal system, because eyes derived from miracle-gro or jolly green giant clones in the EGUF/hid assay Two complementation groups showed tracheal cell overgrowth phenotypes in the mosaic screen, and the enlarged terminal cells had more branches (see below). n normal , and thetrachealess, resembled clones homozygous mutant for the housekeeping gene glutamyl-prolyl-tRNA synthetase (hid system lotus (312) (etiolated (1736), formed eyes of normal size and shape, demonstrating that they are tracheal-selective growth mutations. Other aspects of the lotus phenotype are detailed below.In roughly 500 lines, homozygous mutant cells in mosaic animals were small or absent, and mutant terminal cells when present were small with few or no branches . Most ofthetase . To distus (312)  and etiosprout (574) caused a general reduction of tracheal cell size, as well as a growth defect in the EGUF/Hid assay. However, it was distinguished from all the general undergrowth mutations described above by the ability of the small mutant terminal cells to branch extensively, like a bonsai plant (ai plant .cincher (773) and corset (897), caused a growth defect selective for dorsal trunk cells: homozygous mutant dorsal trunk cells were less than half normal size, whereas mutant terminal cell clones were unaffected , the canonical terminal branching gene winded alleles  showed severe pruning defects, like blistered null alleles: mutant terminal cells had few branches, and any residual branches were typically thin and wispy and lacked subcellular tubes , comparable to blistered partial loss of function alleles.We identified ∼40 mutants in which terminal cell clones had fewer branches than normal but were distinct from the general cell growth genes described above because mutant dorsal trunk cells were of normal size and morphology. This phenotype is similar to that of ar tubes . Other smiracle-gro alleles described above, which also increased cell size. Although some extra branches observed in miracle-gro terminal cells might simply be terminal branches that are normally present but bigger than normal and hence easier to detect, there clearly were also extra branches not present in wild type, such as those in proximal positions of miracle-gro terminal cells. In many cases, these extra seamless tubes coursed through the soma of the terminal cell, giving it a striking multiple lumen phenotype , thick and severely pruned branches , and excess protrusions , as detailed below.Eight mutations affected terminal cell branch pattern or shape and included mutations that caused selective pruning  mutant terminal cells ramified much less extensively than wild type, but the branching defect appears selective for side branches and possibly other later rounds of terminal branching  the first terminal branches appeared largely normal, although sometimes thickened, but subsequent branches were absent or severely compromised: small, unbranched, and with a narrow bore tube. These mutants demonstrate genetic differences between early and later rounds of terminal branching.ranching . Likewislotus (312), conjoined (356), and oak gall (696) were severely pruned, in the most extreme cases with just a single significant branch, and the remaining branches were unusually thick  mutant terminal cells had variable numbers of cytoplasmic protrusions emanating along the length of terminal branches , convoluted lumens , multiple convoluted lumens , lumen dilation and other irregularities , and large vacuoles , as detailed below.These mutations caused defects in seamless tubes including missing and discontinuous lumens  lacked or had seriously compromised seamless tubes, whereas other aspects of terminal branches appeared normal and lumens of other branches were unaffected. In impatent mutant terminal cells, the lumGFP reporter accumulated in large puncta, up to 1–2 um in diameter, located mostly in the cell body , whereas others form lumens that are difficult to detect by brightfield microscopy because they remain filled with matrix (see below). The three alleles of ell body . These wwhacked mutations  eliminated the distal portions of seamless tubes, with the lumen typically terminating prematurely in an irregularly-shaped local dilatation (moon cheese (1524) and carbuncle (804) caused a variable discontinuous lumen phenotype in which regions along the terminal branch were missing lumen but flanked by regions containing blind-ended and irregularly-shaped lumen (Some of the other mutations in this class displayed a more complex spectrum of defects. atation . moon ched lumen . These mwavy lumens (894) and wobbly lumens (BG13), terminal cell lumens formed but followed a convoluted path through the cytoplasm  defines another locus whose phenotype was remarkably similar to tendrils except that it began to manifest a day or so earlier in development. creeper, braided and ivy had similar but less penetrant phenotypes.whacked had irregularly shaped, and often prematurely terminated tubes. cystic lumens (1243) caused areas of lumen dilation and constriction  mutant cells, the vacuoles were centered over gas-filled lumens, suggesting that transport into the lumenal space is defective (black hole and dark matter (1417) caused vacuole accumulation specifically in tracheal terminal cells, whereas moon cheese (1524) and carbuncle (804) caused vacuole accumulation in all tracheal cells. For moon cheese and carbuncle, a secreted form of GFP (lum-GFP) accumulated to high levels within mutant cells, particularly within the vacuoles, suggesting a defect in trafficking of secreted proteins. moon cheese and carbuncle also caused a variable discontinuous tube defect described above.Other mutations caused large cytoplasmic vesicles that excluded cytoplasmic GFP in mutant tracheal cells. In efective . black hlotus (312), conjoined (356), oak gall (696) interfered variably with the formation of tubes sealed by autocellular junctions  and lumen constriction , as detailed below.These mutations caused defects in multicellular tubes, including lumen dilation , bulgy (636), and balloon (1448) caused subtle outward bulges in the lumen of dorsal trunk tubes , dyspneic , and constricted (960) caused lumenal constrictions at the sites of mutant cells , and the only lumGFP detected in control clones was the rare puncta near branch tips or the junctions between branches (scrub (659), lumGFP was not detected in either matrix-filled lumens or cytoplasmic puncta (data not shown); these mutations might alter lumenal targeting such that lumGFP is secreted from other positions in the cell so does not accumulate intracellularly.We expected that some mutants from the screen that appeared to lack lumens would instead be lumen clearance and gas-filling mutants in which the lumen was present but difficult to detect by brightfield optics because it remained filled with matrix, which has a similar refractive index as the surrounding cytoplasm. To identify such mutants, lines from the screen that appeared under brightfield optics to lack lumens were subjected to a secondary screen using a transgene expressing a fusion protein containing the signal peptide of p23 sthmatic . By contd lumens , or in wd lumens . The onlbranches . In mutaasthmatic), in the tips of seamless tubes , or at the junction between seamless and autocellular tubes , as detailed below.These mutations caused liquid clearance defects in all seamless tubes  had gas-filling defects that typically affected only new or distal portions of subcellular tubes . asthmatic mutations, which caused a strong terminal cell gas-filling defect as noted above, also caused a partially penetrant autocellular tube gas-filling defect (flashflood (AP67) selectively blocked clearance of lateral tracheal trunks, as detected in homozygous third instar larvae.Nine mutants representing four complementation groups showed dramatic defects in liquid clearance/gas-filling of tracheal tubes containing autocellular junctions. Two of the complementation groups, g defect . flashfl were previously implicated in tracheal development. However, new functions were revealed for each by our clonal analysis. Two are allelic to canonical tracheal genes in the branchless/breathless FGF pathway, the breathless FGFR itself pointedshort of breath and dyspneic, which our results implicate in lumen clearance and gas-filling and as cell autonomous promoters of tube expansion, are allelic to krotzkopf verkehrt (kkv) and knickkopf (knk), chitin synthesis pathway genes previously shown to coordinate the behavior of cells in the tracheal epithelium during tube expansion 784lopped is allelic to fatiga that encodes Drosophila Hif1 prolyl hydroxylase, and appears to be required in terminal cells for normal branching. Previous studies with hypomorphic fatiga alleles gave opposite results fatiga) result in stunted tracheal development while later exposure stimulates branching failed fusions is allelic to polychaetoid, which has been implicated in branch fusion and in tracheal cell intercalation, but our mosaic analysis, along with other new data from our lab, suggest another function for polychaetoid in tip cell selection .To begin to define the molecular functions of tracheal genes identified in the screen, we mapped representative mutations and molecularly identified 14 of the genes . Six of vine, moon cheese, and whacked) had not been genetically defined tendrils, which is allelic to rhea and encodes talin vine, which encodes the Drosophila ortholog of CCTgamma, show that talin-dependent integrin adhesion and a component of the TriChaperonin complex are required for maintenance of terminal branches and lumenal organization.The other eight molecularly identified genes had not been previously implicated in tracheal development; indeed, three . The identification of membrane lipid and vesicle trafficking genes in terminal branching supports the idea that outgrowth of cellular processes and lumen formation require targeting of apical and basolateral membrane components at a distance from the cell soma. It will be important to determine the number of trafficking pathways involved, how the pathways are activated at the appropriate times and places, and how the identified t-SNARE, Rab-GAP, and ESCRTI component function in the pathways.The four other genes implicate membrane and vesicle trafficking genes in tracheal development. Such genes have been speculated to function in tube morphogenesis but few have been genetically identified. Thus, all 14 molecularly characterized genes from the screen reveal new cell biological pathways in tracheal development or new functions for established pathways.moon cheese/membrin, burs/TSG101, and whacked/RabGAP; the cell junction and cytoskeletal genes failed fusions/polychaetoid/ZO-1, rhea/tendrils/talin, vine/cctγ; and the chitin synthesis genes short of breath/kkv/chitin synthase and dyspneic/knk. Three others are established patterning genes: the receptor btl/no-terminal cell clones-L, the transcription factor pnt/no terminal cell clones-R, and the transcription factor regulator lopped/fatiga/Hif prolyl hydroxylase. The remaining three are more difficult to categorize because they encode enzymes that likely couple patterning signals to cytoplasmic outgrowth (winded/(CdsA) and cell growth (Tsc1/jolly green giant and miracle-gro), as discussed below. Thus, over three-quarters of the identified genes  appear to be downstream effectors/morphogenesis genes  or genes that couple patterning signals to morphogenesis , supporting our hypothesis that systematic clonal analysis is an effective way of identifying such genes.The identities of the molecularly characterized genes allowed us to assess the success of the screen in identifying morphogenesis effectors. Although it was not possible to unambiguously classify all 14 genes in this way from their sequence alone, eight very likely function as morphogenesis effectors: the vesicle trafficking genes miracle-gro cell overgrowth mutations (Section 2A). In terminal cells, not only was the soma enlarged but there were many ectopic seamless tubes coursing through it  it was systematic and extensive, surveying most third chromosome genes, nearly 40% of the genome; (ii) it included a clonal analysis of mutations using a new cell marking method that allowed facile identification of tracheal functions of pleiotropic mutations such as vesicle trafficking and cytoskeletal genes; and (iii) it employed a secondary screen in another tissue (eye) to exclude mutations in general housekeeping genes. Housekeeping genes are a huge class of genes that would have dominated the results of our clonal screen, as they have in previous clonal screens Many of the mutations we identified, particularly in the clonal analysis, affect terminal branching, a morphogenesis process for which little was known beyond the key signaling pathway and transcription factors that control terminal cell selection. The systematic nature of our screen and the distinct phenotypes of terminal branching mutations we identified provide a comprehensive genetic outline of this morphogenetic process . We propblistered SRF transcription complex and its downstream effector genes The earliest step of cell selection and specification in terminal branching is a well-characterized process that previous genetic studies have shown is controlled by the Branchless FGF pathway (Bnl/Btl/RAS/Pointed) that induces expression of the Blistered/Pruned SRF transcription factor that selects cells at the ends of budding branches for a terminal branching fate breathless FGFR and pointedOur screen identified third chromosome genes previously implicated in the selection process, but revealed an interesting new aspect of the process because of the novel “no terminal cell clones” phenotype. These turned out to be mutations in steeple, missing parts) required for specification of a subset of terminal cells in specific regions or branches. These may encode region-specific enhancers of the Bnl-Btl pathway because sporadic failure of terminal cell formation is seen in animals in which this signaling pathway is partially compromised We also discovered genes  required for phosphoinositide (PI) synthesis, suggesting that a PI-dependent signaling process, presumably like those involved in other receptor tyrosine kinase (RTK) signaling pathways topiary, paltry, truncated) might encode additional signal transduction components or targets of the SRF transcription factor required for polarized cell growth (see below).Although the Branchless pathway and Blistered SRF transcription complex are key regulators of branch budding and outgrowth spikes encodes a negative regulator of bud site selection because small ectopic buds form in mutant terminal cells. One appealing idea is that spikes restricts the normal budding response of terminal cells to the sites of maximal induction by Branchless FGF. TSG101/erupted/burs and denuded regulate branch pattern by promoting lateral and late rounds of terminal branching, perhaps by catalyzing the local disassembly or reorganization of the cytoskeleton within a maturing terminal branch.We propose that other branching genes regulate bud site selection and the pattern of branching. lotus, oak gall, conjoined) affected branch number but also dramatically altered the size and shape of the remaining branches (see below). These were difficult to categorize purely as branching genes or growth regulators, so we place them in a special class at the boundary between those categories because they share features of both. They may function as integrators of branch outgrowth and size control signals.One set of genes , so an appealing model is that these conditions and this signaling pathway inhibit the activity of wobbly lumens and wavy lumens, which themselves function to restrict the length or the position of terminal branch lumens.The striking phenotype of carbuncle and membrin/moon cheese, suggesting that lumens of seamless tubes are made piecemeal and these genes promote their connection. We also identified four genes  required to make functional connections between seamless tubes and the adjacent, architecturally distinct autocellular tubes that form by wrapping. The short lumenal gap in these mutant terminal cells may result from a failure to connect the tubes or a structural defect that prevents clearance of the connection.Lumen continuity requires ichorous and asthmatic are required for clearance and gas filling of most or all terminal branches, and littoral and panting and others are required to clear the tips of terminal branches. Recent studies highlight the importance of secretion into the lumen and subsequent endocytosis of lumenal matrix and liquid during tube expansion and air-filling of large multicellular tracheal tubes For tracheal branches to become functional, the lumenal matrix must be cleared and replaced by gas, the molecular composition of which is unknown. rhea/tendrils/talincctgamma/vine, creeper, braided, and ivy are similar to tendrils, suggesting that they function in the same integrin/talin cell adhesion and cytoskeletal support system. For example, the cctgamma/vine chaperonin may facilitate the folding or assembly of talin or some other component in the support system, an appealing hypothesis given that CCT chaperonins have been shown in other systems to mediate the folding of cytoskeletal proteins These genes maintain the elaborate shape and structure of terminal branches under mechanical stress such as muscle contraction. In the mutant These maintenance genes emphasize the importance of analyzing the onset and evolution of a mutant phenotype when elucidating gene function, because similar phenotypes can arise from early developmental aberrations or later defects in maintenance. Other elaborate cell types and organs likely also require maintenance genes. For example, mutations in mouse Dlg5 perturb delivery of adherens complex proteins to the plasma membrane of brain and kidney epithelial tubes, resulting in cyst formation branchless and the receptor breathless are induced by hypoxia during larval life, and terminal cell outgrowth and branching are stimulated and directed to hypoxic cells by local production of Branchless FGF How are the genetically separable terminal branch morphogenesis processes described above coordinated and controlled in time and space? An important part of this control and coordination almost certainly involves the Branchless FGF pathway. Expression of both the ligand cdsA/winded severely abrogated terminal branching.Because Branchless functions as a chemoattractant, it must also provide a spatial cue that guides terminal branch outgrowth. One appealing idea is that the ligand-bound Breathless FGFR at the surface of the terminal cell generates a spatial cue that directs polarized growth of cytoplasmic extensions toward hypoxic, FGF-secreting cells. Such a spatial cue could be used to direct vesicular traffic to the growing ends of terminal cell extensions, both for polarized growth of the new branch and construction of a lumen within it. The spatial cue might be a modified membrane PI because PI signaling functions downstream in many RTK signaling pathways Although Branchless-Btl signaling likely controls and coordinates many of the events in terminal branch morphogenesis, it is unlikely to be the sole control and coordination mechanism because not all of the morphogenesis events occur at the same time and place. Lumen formation (tubulogenesis) occurs after cytoplasmic outgrowth, and lumen maturation including clearance and gas filling occur even later, in some cases days after the lumen has formed. Likewise, branch and lumen maintenance are late steps in the process. Although there may be delays built into some of the effector pathways downstream of Breathless to stagger its effects, other factors likely also contribute to the timing and spatial organization of the events. For example, ecdysone signaling may gate the timing of lumen clearance and gas filling, and there are presumably cell intrinsic cues that direct transport vesicles carrying integrins and other basolateral markers to the plasma membrane of growing buds, and vesicles carrying apical markers and lumenal components to internal positions.trachealess gave a pruned phenotype. This implies that trachealess is required not only for its well established role in the initiation of tracheal development pha-4One surprising finding of our clonal analysis of known tracheal genes was that terminal cell clones of the tracheal master regulator small potatoes, bulgy, balloon) and others that cause local constriction of multicellular and autocellular tubes . An especially intriguing set of genes  are those required to form autocellular junctions and lumens. These may encode specialized components or regulators of autocellular junctions and tubes, such as proteins required for a cell to wrap on or seal to itself.We identified a number of genes required for proper formation of the larger branches of the tracheal system that form earlier than terminal branches and from which they arise. For example, we identified mutants required for proper shape of multicellular tubes, including mutations that cause tracheal dilatations , suggests that there are 110–240 genes dependent on the master regulator for expression, at least at certain stages of development. Although it is not known how many of these downstream genes are required for organ morphogenesis, or what fraction of organ morphogenesis genes are both selectively expressed and downstream targets of organ master regulators, these genomic results are in line with the estimate from our genetic studies that organ morphogenesis programs, even ones for relatively simple organs like the Drosophila tracheal system, are likely to involve several hundred genes. The approach used here, involving a clonal analysis in the tracheal system of all mutations that do not survive late enough in development as homozygotes to assess their tracheal function, has begun to be extended to the other major chromosomes to identify the rest of the tracheal morphogenesis program Genomic profiling of developing and mature organs indicates that there are hundreds of differentially expressed genes among different organs, and genetic profiling to identify downstream genes of organ master regulators such as the D. melanogaster strains used in the screen and meiotic mapping experiments were: (1) btl-GAL4, UAS-GFP; Pr, Hs-hid/TM3Sb, Tub-GAL80, (2) a newly isogenized btl-GAL4, UAS-GFP; FRT2A,FRT82B, (3) y w FLP122; btl-GAL4, UAS-DsRED; FRT82B cu UASi-GFPhp/TM6B, (4) y w FLP122; btl-GAL4, UAS-DsRED; UASi-GFPhp th st FRT2A/MKRS (5) y w ey-FLP; cell-lethal, GMR-hid FRT2A/MKRS, (6) y w ey-FLP; FRT82B, cell-lethal, GMR-hid/MKRS, (7) y w FLP122; breathless-GAL4, UAS-lumGFP, UAS-DsRED; FRT82B TubGal80, (8) y w FLP122; breathless-GAL4, UAS-lumGFP, UAS-DsRED; TubGal80 FRT2A; (9) a newly isogenized ru h th st cu sr e ca; (10) ru h th st cu sr e Pr ca/TM6B. Other strains were: FRT2A, FRT82B (from Trudi Schüpbach); Hs-hid , onto which Pr was recombined; TM3Sb, Tub-GAL80 (from Stefan Luschnig); and strains used in complementation tests and deficiency mapping experiments (see http://flybase.bio.indiana.edu) and are available from http://flystocks.bio.indiana.edu.ents see . All oth1118w embryos. The second chromosome insertion (line 5A) was recombined with breathless-GAL4 and used here.UAS-DsRed. This Gal4-dependent DsRed transgene was constructed by inserting a 0.7 kb Kpn I-Xba I restriction fragment containing the DsRed coding sequences from pDsRed (Clontech) between the corresponding sites of the vector pUAST . The intron fragment was then excised with Xho I and Kpn I and inserted at those sites in pUAST. Note that the trachealess intron contains an Eco RI site, leaving Bgl II, Not I and Xho I as the only unique restriction sites 5′ of the trachealess intron, and Kpn I and Xba I as the only unique sites 3′ of the trachealess intron. To generate RNAi constructs, a ∼500 bp fragment from the gene of interest is inserted in the forward orientation just upstream of the intron, and in the reverse orientation downstream of the intron, as described below for UAS-GFP(RNAi). Gal4-driven expression of this transgene results in tissue specific transcription of the self-complementary RNA, which is predicted to form a double-stranded “hairpin” conformation that initiates the RNAi response.pUASTi. This P element vector for generating RNAi transgenes was constructed by PCR amplification ru h th st FRT2A (from Stefan Luschnig) to generate the UASi-GFPhp th st FRT2A chromosome, and an insertion on 3R (insertion 4A) was recombined onto FRT82B cu sr e ca (from S. Luschnig) to generate the FRT82B cu UASi-GFPhp chromosome.UAS-GFP(RNAi). This Gal4-dependent GFP(RNAi) transgene was created by PCR amplification  and insertion of an ∼500 bp fragment of GFP  between the Not I and Xho I sites upstream of the intron in pUASTi, and amplification of the same fragment  and insertion (in the reverse orientation) between the Kpn I and Xba I sites downstream of the intron, to create plasmid pUASTi-GFP(RNAi). Transgenic flies were generated as above, and insertions were identified on all major chromosomes. For this study, an insertion on 3L (insertion B) was recombined onto breathless-GAL4 bearing second chromosome for use here.UAS-lumGFP. This Gal4-dependent transgene expressing secreted  GFP was constructed by inserting an Nhe I-Kpn I restriction fragment with the GFP coding sequence from plum-GFP breathless-GAL4 breathless-GAL4, UAS-GFP; Pr, Hs-hid/TM3, Sb, Tub-GAL80 virgin females. F1 males were each mated to two virgins of the genotype used in the P cross. After five days, parents were removed from the F1 cross and on days five and six F2 larvae were heated to 38°C for 1.5 hours to induce Hs-hid and eliminate animals carrying that chromosome. In the few cases (∼2–3%) where animals carrying Pr, Hs-hid survived, virgins of the appropriate genotype were selected to generate a stock of FRT2A, FRT82B*/TM3, Sb, Tub-GAL80 . If animals homozygous for the treated third chromosome (non-Sb) were not detected in the F3 or subsequent generations, a lethal mutation was assumed to be present. Two hundred mutagenized chromosomes were assayed in three small-scale pilot screens, and 4100 mutagenized chromosomes were assayed in a final large-scale screen.A standard F3 EMS mutagenesis screen was performed . StrainsSibling F2 flies (described above) were allowed to mate and were brooded to produce two clutches of F3 individuals, one-quarter of which should be homozygous for the mutagenized third chromosome. The first F3 brood (after five days) was used to maintain the stock and assess for presence of a lethal mutation; the second F3 brood (at 12 days) was screened for tracheal phenotypes (see below). If less than four pairs of flies were obtained in the F2 generation, screening was postponed for a generation. For tracheal phenotype screening, F3 larvae were washed out of their food vials with distilled water and examined under an M2 Zeiss or a Leica fluorescence stereomicroscope. Homozygous third instar larvae were identified by tracheal expression of GFP, and tracheal morphology was analyzed; at least three homozygous larvae were examined for each mutant line. Animals that appeared to have tracheal defects were heat-killed (70–75°C for 3–5 s), mounted in 50% glycerol and examined under a Zeiss Axioplan 2 compound fluorescence microscope. Lines in which a tracheal defect was detected were retested to confirm the phenotype. If no reproducible phenotype was found, the line was discarded. Mutant lines that did not give rise to viable homozygous third instar animals were analyzed in genetic mosaics as follows.y, w, FLP122; breathless-GAL4, UAS-DsRED; UASi-GFPhp, th, st, FRT2A/MKRS virgin females, and to y, w, FLP122; breathless-GAL4, UAS-DsRED;FRT82B, cu, UASi-GFPhp/ TM6B virgin females, to test mutants on 3L or 3R, respectively. For each arm (3L and 3R) of every mutant stock, a cross with 20–40 pair matings was done. Embryos (0–4 hr old) were heat-treated as above for 0.75–1 hr to induce FLP-mediated recombination; animals 2 hrs old or less at the time of heat treatment typically do not survive. Crosses were maintained at 25°C for five days and then mosaic animals were examined under a fluorescence stereomicroscope. All tracheal cells are marked by expression of UAS-DsRED; mutant tracheal cells also express GFP. This GFP marking of mutant cells was achieved by inducing recombination between a chromosome arm carrying the mutation of interest and the homologous chromosome arm carrying UASi-GFPhp (see above). Daughter cells homozygous for the mutagenized chromosome arm lack the GFP(RNAi) transgene and thus express GFP. Animals of the correct genotype were selected, heat killed, and analyzed for tracheal defects as above. Under these clone induction conditions, ∼60±8 (mean +/- SEM) tracheal clones were generated per animal . Among dorsal branch clones (n = 127 clones), 50% appeared to be composed of a single cell, 37% of two cells, 10% of three cells, and 3% of four or five cells.Males from the mutant stock established in the F3 screen were crossed to y, w, ey-FLP; *, GMR-hid FRT2A/MKRS, or y, w, ey-FLP; FRT82B, *, GMR-hid/MKRS, where * is the undergrowth mutation. Adult progeny lacking Sb  were scored for eye size. Mutations unable to support normal eye development were presumed to affect general cell growth and viability genes (“housekeeping” genes) and were discarded.Mutations that caused undergrowth defects in tracheal clones were tested for growth defects in eye development using the EGUF/HID technique that generates eye imaginal discs composed exclusively of mitotic clones of a single genotype y w FLP122; breathless-GAL4, UAS-lumGFP; TubGal80 FRT2A/MKRS or y w FLP122; breathless-GAL4, UAS-lumGFP; FRT82B TubGal80/MKRS virgin females, and mosaic analysis was carried out as described above in the Genetic Mosaic Screen.Mutants with no detectable lumen under brightfield optics were tested for presence of liquid-filled or discontinuous lumens using the lumGFP transgene described above, which expresses GFP with a signal peptide that we found accumulates in liquid-filled tracheal lumens but is not detectable in normal, gas-filled lumens. Males from the mutant stocks were crossed to 338miracle-gro on cell size was determined using ImageJ software to measure the maximal cross-sectional area in a stack of 2D optical sections through the soma of mutant and wild type control terminal cell clones  Initial mapping was carried out by complementation tests against a panel of chromosomal deficiencies spanning the third chromosome, and by meiotic recombination mapping using visible recessive markers miracle gro/warts terminal cell phenotype to that of activated Breathless FGFR, the MARCM system +) clones of tracheal cells expressing λ-Breathless miracle-gro/warts and blistered/pruned/SRF, a downstream transcription factor in the Breathless pathway, virgins of the genotype y, w, hsFLP122; l(2)3267bs, btl-Gal4, UAS-GFP/CyO; FRT82B cu, UAS-GFP(RNAi) were crossed to males of the genotype l(2)3267bs, btl-Gal4, UAS-GFP/CyO; FRT82B 338miracle gro/warts/MKRS. Mutant animals homozygous for l(2)3267bs were identified by the strong pruned phenotype of unmarked (GFP-) terminal cells, and marked (GFP+) 338wts mutant terminal cell clones in these animals were examined and scored as above.To compare the Figure S1trachealess resemble those for the house keeping gene Aats-gln. Fluorescence (A–D) and brightfield  images of larval dorsal branch terminal cell  or dorsal trunk  clones  homozygous mutant for the house keeping gene glutamine aminoacyl tRNA synthetase  or the tracheal master regulator 10512trachealess . Mutant DB terminal cells are small and lack terminal branches  and air-filled lumens . Control DB terminal cells  are shown at left in the images. Homozygous 05461Aats-gln mutant dorsal trunk tracheal cells (yellow cells in B) are smaller than control wild-type dorsal trunk cells (yellow cells in C). Bars in A  and C , 50 µm.Tracheal terminal cell clones mutant for (TIF)Click here for additional data file.Table S1Molecularly identified tracheal genes. Previously identified Drosophila genes with defined tracheal phenotypes. Each of the genes has been assigned as either a presumptive tracheal patterning (P) or morphogenesis (M) gene.(DOC)Click here for additional data file.Table S2Polymerase chain reaction (PCR) primers. Primers were used to generate the modified pUAST vector, pUASTi, and the GFP(RNAi) construct, pUASTi-GFPhp.(DOC)Click here for additional data file.Table S3Additional tracheal morphogenesis mutants. Genes that were identified in the screen but represented by a single allele, and for which the molecular identity of the gene remains unknown. Abbreviations and gene mapping methods are given in the footnotes of (DOC)Click here for additional data file.


Alternative splicingof the IgIII loop of fibroblast growth factorreceptors (FGFRs) 1–3 produces b- andc-variants of the receptors with distinctlydifferent biological impact based on theirdistinct ligand-binding spectrum. Tissue-specific expression of these splice variantsregulates interactions in embryonic development,tissue maintenance and repair, and cancer.Alterations in FGFR2 splicing are involved inepithelial mesenchymal transition that producesinvasive, metastatic features during tumorprogression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1–3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application.  They mThe FGFR-genes consist of up to 20 exons that together code for a highly conserved protein domain structure , 10. Eacα-loop) has been described for FGFR1. This event creates a high-affinity oncogenic FGFR-variant that activated distinct signaling cascades [Deletion of the Ig loop I . FGFFGFRs serve an essential role in embryology from gastrulation  to organDeeper insight in the specific roles of the IIIb and IIIc receptor variants of the FGFRs 1–3 is provided by analysis of their distinct spatial expression patterns. Extensive studies of mouse embryonic development demonstrate characteristic localizations of FGFR isoforms during organogenesis. This is observed throughout the body , 21, butThe distinction is best observed for FGFR2 whose IIIb/IIIc choice is strictly tissue specific . During β-catenin target gene and expressed in the stem cell compartment at the bottom of colonic crypts [ For FGFR3 the distinction is less clear as isoform expression is not strictly tissue specific. While the IIIb form is restricted to epithelia, the IIIc variant can be found in both the mesenchyme and the epithelium . An examc crypts , 37. Simc crypts . Howeverc crypts . This spIn the past several years evidence has accumulated that demonstrates a central role for deregulated FGF/FGFR signaling in a large number of malignant tumors A FGFR2-IIIb to IIIc switch is related to increased invasiveness in bladder and prostate cancers . In the β-induced EMT FGFR1-IIIc is upregulated as an independent mesenchymal marker and the reciprocally downregulated IIIb receptor is FGFR2 [For other receptors the contrast is less clear. However, FGFR1-IIIc has been upregulated in several carcinomas where it is regarded as a strong oncogene , 48. In is FGFR2 , 51.in vivo and in vitro and also blocks colorectal tumor cell migration [In colorectal cancers the reciprocally downregulated receptor is FGFR3-IIIb. Restoration of its expression inhibits growth under some conditions . The mecigration . For several other tumor types investigation of FGFR IIIb/IIIc expression patterns are still ongoing. An interesting example was found in ovarian cancer cells most of which express both the IIIb and the IIIc forms of FGFR 2 and 3. By contrast immortalized ovarian surface epithelial cells express mainly the IIIc forms while FGFR2-IIIb is the main receptor type in nonimmortalized ovarian epithelial cells (unpublished observation). Even though not completely understood, this does indicate a high degree of plasticity in this cell type and seems to support the hypothesis that ovarian cancers may arise from mesenchymal fimbrial cells . In contrast to carcinomas, very little is known of the role of FGF/FGFR signaling in the disease progression in soft-tissue sarcomas (STS). To date, there are fewer than 10 publications covering any aspect of FGFR signaling in STS. Research is hampered by the low incidence of these tumors in humans, so we have initiated comparative studies of STS in humans, dogs, and cats. The dog, with an STS incidence rate estimated to be 12-fold to 33-fold higher than the incidence in humans –56, is aPreliminary data have been collected by our research group from human and animal cell lines and patient tissues. We could identify all FGFRs and their splice variants in human and canine STS cell lines as well as in canine spontaneous tumor samples. Specifically, in human and canine STS cells FGFR1-IIIc is the FGFR with the highest transcript levels .  Feline sarcomas differentially express FGFRs depending upon the underlying cause of tumor formation—that is, injection site sarcomas versus spontaneous sarcomas (unpublished data). As these studies progress we will be able to address the question whether STS require alterations in FGFR expression patterns to achieve a metastatic phenotype and whether a “switch” between the FGFR IIIc or IIIb splice variants has an impact in this process.β (TGFβ), WNT signaling, and activation of tyrosine kinase receptors, for example, by hepatocellular growth factor (HGF), or FGF family members [Expression profiling in both cell lines and tissues indicates that the splicing choice for FGFR-IIIb and IIIc variants is strictly regulated. For FGFR2, the choice appears mutually exclusive and maintained even in the absence of a functional alternative exon . This st members , 63. It Two pieces of information exist that link FGF signaling itself with alternative splicing. (1) In the prostate there are indications that loss of FGFR2-IIIb stimulation by its ligand FGF7 contributes to the switch—indicating that the activated receptor reinforces its own expression ; (2) in Isoform expression patterns indicate that the choice is made differently, or at least not as stringently, for FGFRs 1 and 3—with FGFR1-IIIc being upregulated in carcinomas without a concomitant loss of the IIIb variant , 48 and In this context it is interesting that downregulation of FGFR3-IIIb expression in colorectal carcinoma cells is mediated by aberrant splicing that produces out-of-frame nonsense transcripts . There iAt the transcript level, regulation of splicing is achieved by auxiliary cis-elements that bind regulatory proteins to either enhance or silence splicing of adjacent exons . The cisMost known splicing regulators are RNA-binding proteins (RBP), such as the serine/arginine-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. These RBPs are fairly ubiquitously expressed, albeit with some differences in expression between tissues . DetaileFor FGFR2 exon IIIb and exon IIIc splicing, previous studies identified a number of auxiliary cis-elements and RPBs that regulate the splice event . Suiewed in . More reiewed in –88. Prec[in vivo . Similarin vitro , 91. Thiin vitro . Newer s in vivo . Applyin in vivo . We have in vivo , 49. BasDuring the past few years most FGFRs have been identified as targets for cancer therapy, whose blockade by genetic or chemical means inhibits tumor growth . InAntibody-based therapies are being developed for FGFR3 for use in bladder cancer and multiple myeloma cell models and have been entered into clinical trials . Currenthttp://clinicaltrials.gov/ identifier NCT00687505).Ligand traps have been developed from the extracellular domains of FGFRs or from soluble splice variants. They contain the ligand-binding site of the respective receptor and trap all IIIb- or IIIc-specific FGFs that signal through it see . IIIc-spAccumulating evidence suggests that, beyond triggering cell migration, EMT is capable of conferring stem-cell-like features such as enhanced tumor cell survival and therapy resistance. Inducing factors and pathways other than FGF include among others TGFß, EGF, IGF, and HGF (reviewed in ). All ofCommon down-stream mediators that have been implicated in alternative splicing regulation are the kinases in the raf-Map-kinase and the ras-PI3K-Akt pathways . TTargeting EMT-inducing signaling pathways and cellular kinases will not be specific for splicing events due to their pleiotropic roles and interactions in cell regulation. A thorough knowledge not only of the kinases' role in splicing regulation, but also of all their additional downstream substrates will be an essential prerequisite for employing such strategies .Based on the emerging detailed information on the splicing apparatus described above, targeting on the splicing process that underlies EMT may be an innovative strategy in cancer therapy. Most importantly, it should impact not only FGFR-dependent growth and survival signals but also all the additional pro-metastatic alterations in EMT . For this approach three different strategies seem feasible: (1) splice site modulation, (2) targeting of hnRNPs or SR proteins, and (3) targeting the spliceosome in general . Splice-Targeting the hnRNPs or SR proteins known as splice modulators constitutes an indirect approach . SeveralThe third approach, targeting the spliceosome in general, is based on the fact that malignant cells have higher metabolic rates than normal cells and thus require increased splicing activity. Therefore, they are more sensitive to splicing modulation or inhibition . A serieAlternative splicing of FGFRs producing IIIb/IIIc variants has long been known to have strong physiological and pathophysiological impact—specifically in tumor development and progression. Observations on prostate, bladder, and, lately, in colon cancer indicate that altered splice choices are related to altered cell behavior. Specifically the IIIc isoform was associated with more aggressive tumors, probably be due the broader ligand specificity of this splice forms. With FGFR2, the switch to a IIIc variant is a marker of EMT which produces invasive, metastasizing tumors.Comparative analysis of FGFR splice patterns in a larger panel of malignancies should provide more insight in both general and tumor type specific consequences of FGFR splice choices. From this, new diagnostic/prognostic markers as well as therapeutic targets should arise.Over the past years exciting new tools have been developed that enable high-throughput analysis of splice variants as well as a thorough investigation of the cellular mechanisms underlying FGFR splice choices. The cellular splice machinery has been characterized in sufficient detail to permit targeting not only of tumor-specific splice variants but also of the splicing process itself for cancer therapy.in vivo and in vitro. Application and further development of these new tools and technologies should help to enlighten the role of FGFR splice variants in cancer with emphasis on malignant progression.Splice-reporter constructs expressing fluorescent/luminescent gene products even permit real-time observation of splice choices in single cells


Bladder lymphomas are rarely primary tumours and more commonly associated with systemic lymphoma, either as nonlocalised bladder lymphoma or as secondary bladder lymphoma. Primary bladder lymphomas (PBL) tend to be low-grade mucosa-associated lymphoid tissue (MALT) type, contrasting with diffuse large cell or follicular centre cell types more commonly seen in secondary bladder lymphoma. Bladder involvement by systemic lymphoma infers poor prognosis and patients often have no localising symptoms . Other treatments are preferred over surgery for all bladder lymphomas, except where diagnosis is uncertain or for relief of irritative bladder symptoms. We describe a unique case of systemic high-grade B-cell lymphoma with simultaneous cutaneous renal and bladder lesions at presentation.  A 70-year-old man presented to the dermatologists with a 2-year history of painless enlarging scalp nodules, which had rapidly worsened over the previous 6 months . He had The patient described neither lower urinary tract symptoms nor episodes of macroscopic haematuria and was a nonsmoker. A urine cytology specimen revealed atypical cells more consistent with lymphoid than epithelial/transitional cell origin . At cystTo date there are few case reports of nonprimary bladder lymphoma, reflecting its rarity as a presenting pathological entity. To our knowledge we describe the first reported case of disseminated lymphoma with simultaneous cutaneous, renal, and bladder lesions demonstrable at presentation.The incidence of non-Hodgkin's lymphoma (NHL) is increasing and maintains a geographical variability: in 2007 there were 10,900 people diagnosed with NHL in the UK [Lymphomas of the bladder are rare and account for 0.2% of all bladder neoplasms , while HMore commonly, although relatively few case studies exist, lymphoma of the bladder is seen in association with systemic lymphoma; this can either be as bladder involvement with active systemic lymphoma , as reported in this case study, or as lymphoma recurrence in the bladder after a period of remission from previous lymphoma (secondary lymphoma) . It is rNonprimary bladder lymphomas tend to be a different histological type than those found in the PBL group and are commonly of the diffuse large cell or follicular centre cell types . In the Non-Hodgkin's lymphoma involving the bladder can be successfully treated using chemotherapy, radiotherapy, immunotherapy, surgery, and combinations of these modalities . The treThe indolent nature of the MALT lymphomas means that local radiotherapy can be used with curative intent, such as in PBL. Surgical resection has a limited role in primary bladder lymphoma, beyond obtaining biopsy specimens, and rarely adds anything to management or prognosis. There are reports, however, of patients having undergone cystectomy due to unclear diagnosis, or to relieve irritative symptoms . There iIn this case, the patient's disease constituted stage IV B. He underwent eight cycles of CHOP/R chemotherapy followed by radical radiotherapy to scalp and brain. Follow-up MRI showed dramatic improvement in the scalp and dural mass with minimal residual dural enhancement. A repeat CT showed complete remission with no residual disease in either kidneys or bladder. The patient remained in complete remission for 14 months but then developed a gradual deterioration in his balance and thereafter a marked peripheral neuropathy. Repeat imaging confirmed recurrent widespread cerebral metastasis. He died following a cardiac arrest before palliative radiotherapy could be given.


Postirradiation lumbosacral syndrome is a radiculopathy induced by radiation injury to the spinal cord. Its usual presentation is motor deficit and or sensory loss involving the lower limbs. Visceral involvement has not been reported previously.We describe a case of severe hypotonic bladder caused by radiation-induced spinal cord injury following treatment of stage Ι testicular seminoma in a 38-year-old Caucasian man who had undergone radical orchidectomy and prophylactic paraaortic lymph node irradiation for stage Ι seminoma. Three years later he had clinical and urodynamic findings of hypotonic bladder. The magnetic resonance imaging results suggested a radiation-induced injury.Such an unusual presentation of the syndrome of postirradiation lumbosacral radiculopathy can impose a clinical challenge to practicing clinicians. Future studies are required to further delineate the mechanism of injury and further management plans. Postirradiation lumbosacral syndrome is a radiculopathy secondary to radiation injury of the spinal cord manifesting as motor and/or sensory loss involving the lower limbs. This case report describes a 38-year-old Caucasian patient with severe hypotonic bladder caused by radiation-induced spinal cord injury following treatment of stage Ι testicular seminoma. Our 38-year-old Caucasian patient had undergone radical orchidectomy and prophylactic paraaortic (PA) lymph node irradiation for stage Ι seminoma. Three years following radical orchidectomy and prophylactic PA lymph node irradiation, the patient presented with clinical and urodynamic findings of hypotonic bladder. The magnetic resonance imaging (MRI) findings suggested a radiation-induced injury. There is a paucity of systematic studies detailing the mechanism of injury of postirradiation radiculopathy, in particular bladder detrusor muscle involvement.A 38-year-old Caucasian man presented four years ago to our urology service with right-sided groin pain, and, on clinical examination, bilateral undescended testes were discovered. Clinically, the right testicle raised suspicions of malignancy, and a scrotal ultrasound confirmed a solid testicular mass. Tumor markers, including serum β-human chorionic gonadotropins, α-fetoprotein and lactate dehydrogenase, were normal. A right-sided radical orchidectomy was performed, and the left testicle appeared clinically healthy and was brought down into the left hemiscrotum and underwent successful three-point orchiopexy. Both the chest X-ray and abdominal computed tomography (CT) scan were negative for metastasis. The tumor histology results revealed an 8 cm seminoma confined to the testicle and stained positive for placental alkaline phosphatase. There were no syncytiotrophoblastic elements and no vascular or lymphatic invasion. The tumor was diagnosed as stage Ι testicular seminoma.The patient received precautional PA field radiotherapy. Treatment was given five days per week for four weeks with daily fractions of 2 Gy up to a target dose of 25 Gy. The patient's follow-up protocol consisted of clinical evaluation, chest X-ray, tumor markers and an abdominal CT scan. The results of these investigations have remained normal to date.2O at maximum capacity with an inability to micturate for voiding cystometry. A trial of pharmacotherapy with bethanechol chloride for four weeks at a dose of 10 mg thrice daily failed to improve both clinical and urodynamic findings. Consequently, the patient was commenced on clean intermittent self-catheterization, which he is managing well to date. A gadolinium-enhanced brain and dorsolumbosacral MRI scan showed in the lower two thoracic vertebrae (T11-T12) and all lumbar vertebrae (L1-L5) an increased signal in the marrow fat consistent with previous irradiation and with no obvious mass lesion in the spinal cord or vertebrae  are the most common malignant tumors 17%) affecting men under the age of 45 years, with seminoma comprising 40% of GCTs. Prophylactic radiotherapy to the PA lymph nodes following orchidectomy for stage Ι seminoma provides a treatment modality that is the most cost-effective and is associated with the lowest risk of tumor recurrence (1% to 3%) [7% affectCommon side effects of radiotherapy include gastrointestinal toxicity, decreased sperm count and leukemia . This caThe urinary bladder derives its innervations via the lumbar (sympathetic) and sacral (parasympathetic) regions of the spinal cord. A spinal cord lesion involving the lumbosacral roots typically presents as a lower motor neuron lesion with clinical and urodynamic findings of a flaccid neuropathic bladder .Radiation-induced spinal cord injury principally involves the white matter. Several factors, such as radiation dose, fractionation or linear energy transfer, modify its occurrence and severity. The basic process for white matter injury as a result of irradiation involves radiation-induced vascular damage resulting in vascular hyperpermeability and venous exudation .Postirradiation spinal cord injury is associated with long latency periods of up to 25 years following radiation treatment. The natural history of this disorder is one of relentless deterioration, occasionally punctuated by one to two year periods of stability .In our patient, only the involvement of the urinary bladder and the absence of spinal cord or vertebral column masses on MRI scans clearly argue against metastatic disease. The urodynamic findings were those of a flaccid bladder, and the MRI findings suggest that the origin of the patient's spinal injury was radiation-induced. Our patient received a radiation dose of 25 Gy in accordance with the departmental treatment protocol. Although this radiation dose is generally considered to be a high dose according to current standards, it was effective and was associated with reduced side effects . CurrentA hypotonic bladder presenting suddenly in a patient who has undergone prophylactic radiotherapy for testicular seminoma may be the initial presentation of postirradiation lumbosacral syndrome.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.Both authors read and approved the final manuscript.


Mitf) [ortho-decanoate from Thymelaea hirsuta [Mitf gene and the melanogenic enzymes’ genes [Mitf gene expression, however, has not yet been determined. In melanogenesis, the Mitf gene expression can be regulated through the cAMP pathway or the Wnt signaling pathway. This study aimed to determine the mechanism underlying the inhibitory effect of HB on Mitf gene in B16 murine melanoma cells.Skin pigmentation serves as protection against ultraviolet-induced skin damage through melanin’s optical and chemical filtering properties . AlthougMitf) . Previou hirsuta  has antis’ genes . The exaTotal RNA was isolated from B16 murine melanoma cells  and used for DNA microarray analysis, using chips of 528 spots loaded with 265 genes prepared by Genopal™ , to determine the expressions of genes for melanogenesis, membrane-bound receptors, tyrosine kinase regulation, melanosome transport, and other cell signal regulation-related genes (including the housekeeping and negative control genes). To validate the results, real-time PCR, using TaqMan FAST 7500  and specific TaqMan primers  for the differentially-expressed genes, was performed.Mitf gene and the melanogenic enzymes’ genes were downregulated, verifying our previous report [Mc1r) of the cAMP pathway was downregulated while most of the genes that were upregulated are those involved in the Wnt signaling pathway (Table Results showed that the expressions of the s report . In addiMC1R and stimulate melanin production through the cAMP/PKA signalling pathway [In mouse, peptide hormones from the pituitary gland bind to the  pathway , by induMc1r gene of the cAMP pathway. HB may therefore be used as a treatment for hyperpigmentation due to its significant melanogenesis downregulation effects in B16 cells or as a pretreatment for melanotic melanomas.The results obtained suggest that the significant antimelanogenesis effect of hirsein B is through the inhibition of the expression of the


Objective: Screening for full face transplantation candidates includescomputed tomographic vascular mapping of the external carotid distribution for potentialarterial and venous anastomoses. The purpose of this study is to illustrate the benefitsand drawbacks of cine computed tomographic imaging for preoperative vascular mappingcompared with best arterial and venous phase static images. Methods: Twoimage data sets were retrospectively created and compared for diagnostic findings. Thefirst set of images was the clinical cine computed tomographic acquisition including allphases. The second set of images was composed of the best arterial and best venous phasesextracted from the cine loop and determined by the quality of contrast enhancement. Foreach patient, the benefits and drawbacks of the cine loop were documented in consensus bya plastic surgeon and a radiologist. Results: Cine loop analysis identifiedretrograde arterial filling not illustrated on the static images alone. Cine assessmentidentified most of the major vessels necessary for surgery, whereas the static imagesdepicted small vessels more clearly, particularly in the crowded vessel takeoffs.Conclusions: Cine computed tomographic images provide data on direction ofblood flow, which is important for preoperative planning. Combination of cine computedtomographic and the best static images will allow comprehensive vascular assessmentnecessary for future successful full face transplantation. Full face transplantation for eligible patients3-7-10,Standard-of-care vascular screening for face transplant candidates uses wide-areadetector4Three previously reported full face transplant candidatesIn brief, subject 1 was a 25-year-old man who suffered burns secondary to high-voltageinjury to the face and scalp and subsequently underwent conventional reconstruction withmultiple free flaps and split-thickness skin grafts. Subject 2 was a 30-year-old man aftera motor vehicle accident and high-voltage injury to the face. Subject 3 was a 57-year-oldwoman who was left disfigured by a chimpanzee attack, suffering substantial damage to hercentral face and requiring multiple surgical reconstruction attempts.The CT protocol has been previously described.,For each subject, 1- to 2-mm maximum intensity projection cine angiography12The cine and best static image sets were retrospectively compared for diagnostic findingsby the consensus reading from 2 experienced readers (a plastic surgeon and a radiologist).For each patient, the clinical benefits and drawbacks of the cine, when compared with thebest static imaging data sets, were documented. Since display in standard  anatomic planes does not adequately depict the complex relationship betweenstructures, arbitrary cut planes were used for the assessment.The total imaging time was less than 45 minutes for all 3 subjects. All images wereacquired with less than 100 mL of contrast material, and the estimated, effective totalradiation dose was less than 10 mSv for all subjects.The cine loop  that are relevant to major reconstructive surgery for anastomosis. Ininjured tissues that demonstrate substantial collateralization, however, webs ofcollateralized veins appeared as a thin sheet of contrast, making it difficult to identifysmall veins and their detailed anatomy. In this regard, static images allow more directviewing of the trajectories of individual vein segments and their precise paths  and 4.,14-Candidates for face transplantation require presurgical planning that includes mapping ofthe vessels being considered for anastomoses to the donor allograft vasculature. Astransplantation expands from partial3This study demonstrates the value of cine loop CT for this clinical indication. Earlier,less complex reformations of these data have been used to measure vascular transittimes.,Noninvasive cine angiography was initially developed using sophisticated magnetic resonancemethods.17As the technique and surgical expertise for face transplantation developed and began todemonstrate unrivaled functional, aesthetic, and psychosocial outcomes, wide-area detectorCT methods were introduced and enabled cine imaging where each frame in the cine loop wasacquired instantaneously and over a volume up to 16 cm in the craniocaudal dimension.4,22,24Risks of CT imaging can be separated into those related to contrast administration19There are several limitations to our study. First, we acknowledge the small patient cohort.However, as the procedure becomes more available, future studies will include more subjects.Second, we did not correlate the imaging findings with the findings at surgery, as it isbeyond the scope of this work. Future work will investigate the overall vascular correlationbetween surgery and computed tomography.In summary, the cine CT assessment gives information on blood flow that is difficult toobtain from traditional static CT images only. The cine CT, combined with the static images,would provide comprehensive preoperative vascular mapping necessary for full facetransplantation. Future studies will evaluate the preoperative findings from these CT imagesin relation to the posttransplant vascular flow.


Poecilia reticulata) from different environments, who do not show complete reproductive isolation despite adaptive divergence. We investigate patterns of mating isolation between populations that do and do not exchange migrants and show evidence for both by-product and reinforcement mechanisms depending on female ecology. Specifically, low-predation females discriminate against all high-predation males thus implying a by-product mechanism, whereas high-predation females only discriminate against low-predation males from further upstream in the same river, implying selection to avoid maladaptive mating. Our study thus confirms that mechanisms of adaptive speciation are not necessarily mutually exclusive and uncovers the complex ecology-geography interactions that underlie the evolution of mating isolation in nature.Local adaptation to different environments can promote mating isolation – either as an incidental by-product of trait divergence, or as a result of selection to avoid maladaptive mating. Numerous recent empirical examples point to the common influence of divergent natural selection on speciation based largely on evidence of strong pre-mating isolation between populations from different habitat types. Accumulating evidence for natural selection's influence on speciation is therefore no longer a challenge. The difficulty, rather, is in determining the mechanisms involved in the progress of adaptive divergence to speciation once barriers to gene flow are already present. Here, we present results of both laboratory and field experiments with Trinidadian guppies ( Renewed interest in speciation mechanisms has revealed that divergent natural selection can be a powerful promoter of mating isolation, such that stronger pre-zygotic barriers to gene flow evolve between populations from more divergent environments, i.e. “ecological speciation” within a species – Trinidadian guppies (Poecilia reticulata) - where any reproductive barriers would be those that evolve early in the process of diversification.We used laboratory and field experiments to resolve how interactions between ecology (adaptation to different environments) and geography  influence the evolution of mating isolation. When reproductive isolation is complete or nearly complete, it becomes more difficult to determine how barriers to gene flow initially arose - although there is no guarantee that those same barriers will complete it. For this reason, we focus on differentiated populations Crenicichla alta) – a large piscivore, whereas upstream guppies coexist with only the killifish (Rivulus haarti) – a predator of mild to minimal effect. In contrast, the downstream reaches of rivers on the northern slopes of the mountains, which meet the Carribean sea, were colonized by piscivorous fish of marine origins, such as the mountain mullet (Agonostomus monticola) and the goby (Gobiomorus maculatum), whereas guppies in upstream sites again coexist with the R. haarti and another predator of minimal effect; freshwater prawns (Macrobrachium sp.)The rivers of Trinidad's Northern mountain range offer an excellent system to integrate evolutionary processes  and environmental contexts (ecology and geography) in the study of speciation. In general, predation intensity varies along the upstream-downstream axis, with sharp changes occurring across waterfalls that prevent upstream colonization by predatory fishes Regardless of the particular predator assemblage, high- and low-predation populations within a river show adaptive divergence in many traits, including male colour, behaviour, and life history Our laboratory experiment employed no-choice mating trials to test whether females are more likely to mate with males from similar predation environments, and whether any such assortment is influenced by the geographic context of the source populations. This experiment used laboratory-reared guppies from paired high- and low-predation populations in three rivers  Two different source populations of low-predation males were chosen to test for differences in geography and phenotype independent of predation environment. One source population was located less than 1 km upstream from the focal high-predation site thus reflecting an interaction that occurs in nature . The other was located approximately 3.5 km from the high-predation site: 2 km downstream in the Marianne main stem and then 1.5 km upstream in a separate tributary. Given that guppy dispersal is primarily downstream We employed both field and laboratory experimental designs using multiple paired high- and low- predation populations in order to isolate the roles of ecology, geography, and both inter and intra sexual selection on the progress or limitations to the evolution of reproductive isolation. Rather than exposing one mechanism in particular, we found that the above factors all interact. Specifically, our experiments revealed three main interactions. First, we found differences in patterns of mating isolation between females from the different predation environments. Second, both experiments showed that dispersal is necessary to elicit mating isolation only when migrants are maladapted, and third, that mating isolation appears to be strongest in competitive and social contexts.3,123 = 7.34, p = 0.01) between female predation type (high or low) and cross type. We therefore subdivided the data set to determine the nature of this divergence. We first consider low-predation females in the laboratory experiment and then high-predation females in the laboratory and field experiments. Overall, ecology largely determines mating patterns in low-predation females, whereas ecology and geography interact to determine mating patterns in high-predation females . On the one hand, high-predation males outcompeted parapatric low-predation males for successful fertilizations with resident females, siring 90.54% of the offspring . On the other hand, high-predation males sired only 65% of the offspring when in competition with allopatric low-predation males, which did not deviate from random mating .High-predation females in the laboratory experiment discriminated against low-predation males from the same river (IMI  = 0.395), but not against low-predation males from different rivers . This effect was not consistent between enclosures however, as indicated by a significant enclosure by orange interaction . The nature of this interaction is illustrated in 1, 25 = 30.51, p<0.001) and sired the majority of the offspring, male mating success was positively related to orange colour . In contrast, although males from the ‘allopatric’ low-predation population are substantially more orange than high-predation males . Finally, in the laboratory trials, there as no indication that variation in orange among males generally influenced the observed patterns of mate choice .To further investigate the basis of the observed patterns of mating isolation, we examined the influence of individual male phenotype on his mating success. If mating isolation were evolving as a consequence of divergence in sexually selected traits, then males more phenotypically divergent from the female's native population should be discriminated against most strongly, regardless of predation type Our results reinforce the importance of examining interactions between ecological and geographical factors in the early stages of divergence and speciation On the one hand, low-predation females routinely discriminated against high-predation males from the same or different rivers (possible by-product evolution), and this was strongest when they were from a different river. Given that low-predation populations were likely originally colonized from high-predation populations in the same river High-predation females, on the other hand, only discriminated against low-predation males when they were from the same river in both field and laboratory experiments. For these females in a high-risk environment, mating isolation does not appear to be a by-product of general adaptation to an environment of increased mortality, but rather due to selection against the regular influx of potentially maladapted migrant males or their resulting ‘hybrid’ offspring (i.e. reinforcement).Although high-predation females consistently show a reduced response to upstream low-predation males and indices of mating isolation for these pairings are the highest observed, the magnitude of this effect is relatively low compared with similar studies Furthermore, mating preferences for particular trait values do not appear to predict patterns of mating isolation in high-predation females, which provides further support to rejection the by-product hypothesis in favour of reinforcement in high-predation. Male orange colour is potentially a main target of both mate choice All experiments and housing conditions of animals were conducted with the approval of the Canadian Council for Animal Care (CCAC) and McGill University's Animal Care Committee (Protocol # 4570). Import and export permits of guppies were graciously provided by the Department of Fisheries and Agriculture, Trinidad, W.I.All experiments used the offspring of females collected from six populations along Trinidad's Northern Range mountains. These populations were paired upstream (low-predation) and downstream (high-predation) localities in three separate rivers same predation and same river, (2) same predation and different river, (3) different predation and same river, and (4) different predation and different river .Although 36 possible pair-wise combinations were possible between the six populations, this was not logistically feasible. We therefore followed previous related studies of ecological speciation nt river . Each maVariation in female preference (FIR) was analyzed with generalized linear mixed models (GLMM) that included male identity as a random factor. Fixed factors included female predation type (high or low), female river of origin , male river cross type , male predation cross type , and all possible interactions. By-product mating isolation would be indicated by a significant effect of male predation cross type that was consistent across all rivers; i.e., with a non-significant interaction with female river. Significance was assessed using restricted maximum likelihood as implemented in SAS.http://rsbweb.nih.gov/ij/). The influence of male orange colouration on relative mating success was examined in two ways. First, male trait values were included as a factor in the GLMM above. Second, linear regressions were used within each female population to assess the relationship between female FIR for a given male and the difference between his phenotype from the average trait value in the female's population. A negative correlation would support the hypothesis that mating isolation is driven by divergent selection on male colour since foreign males with phenotypes closest to a female's local resident males would have the highest mating success.Male body and colour spot size were measured from digital photographs using standard protocols An index of mating isolation (IMI) was calculated as the difference between the average preference (FIR) of females from a given population for their local males relative to foreign males of a given cross type (FIR own – FIR foreign/FIR own + FIR foreign) Juveniles were collected from a high-predation section of the Marianne River (MH) and raised to maturity in the laboratory with the sexes kept separate. Forty-six of the resulting virgin females were split randomly into two experimental enclosures in their home environment . Enclosures were constructed by isolating side-channel habitats with chicken wire and mesh fabric, therefore excluding both local guppies and aquatic predators but allowing for otherwise natural habitat conditions and water flow. Local high-predation males (MH) and foreign low-predation males Click here for additional data file.Table S2Geographic information of site locations and sample sizes for populations used in the field enclosure experiment. Grid references are from the Trinidad National Grid System 1∶25,000 map series. Also shown are least-square means (+/- standard errors) and results of analysis of variance in male relative orange area. Superscripts indicate homogeneous subsets from post-hoc Tukey tests examining variation among populations.(DOC)Click here for additional data file.Table S3Indices of ecological and geographical mating isolation by female population. Because three types of foreign crosses were performed (see text), multiple indices can be calculated. The three shown here inform different hypotheses about the roles of ecology, geography and their interaction in mating isolation. “Parapatric ecological mating isolation” compares female preferences for a local male to a male from the same river but different predation type; “allopatric ecological mating isolation” compares local preferences to preferences for males from a different river and predation type; “geographic mating isolation” compares local preferences to preferences for males from a different river but the same predation type.(DOC)Click here for additional data file.


The diagnostic efficacy of biochemical and imaging modalities for investigating the causes of Cushing's syndrome are limited. We report a case demonstrating the limitations of these modalities, especially the inability of functional imaging to help localize the site of ectopic adrenocorticotropic hormone secretion.A 37-year-old Arabian woman presented with 12 months of progressive Cushing's syndrome-like symptoms. Biochemical evaluation confirmed adrenocorticotropic hormone -dependent Cushing's syndrome. However, the anatomical site of her excess adrenocorticotropic hormone secretion was not clearly delineated by further investigations. Magnetic resonance imaging of our patient's pituitary gland failed to demonstrate the presence of an adenoma. Spiral computed tomography of her chest only revealed the presence of a non-specific 7 mm lesion in her left inferobasal lung segment. Functional imaging, including a positron emission tomography scan using 18-fluorodeoxyglucose and gallium-68-DOTA-D-Phe1-Tyr3-octreotide, also failed to show increased metabolic activity in the lung lesion or in her pituitary gland. Our patient was commenced on medical treatment with ketoconazole and metyrapone to control the clinical features associated with her excess cortisol secretion. Despite initial normalization of her urinary free cortisol excretion rate, levels began to rise eight months after commencement of medical treatment. Repeated imaging of her pituitary gland, chest and pelvis again failed to clearly localize a source of her excess adrenocorticotropic hormone secretion. The bronchial nodule was stable in size on serial imaging and repeatedly reported as having a nonspecific appearance of a small granuloma or lymph node. We re-explored the treatment options and endorsed our patient's favored choice of resection of the bronchial nodule, especially given that her symptoms of cortisol excess were difficult to control and refractory. Subsequently, our patient had the bronchial nodule resected. The histological appearance of the lesion was consistent with that of a carcinoid tumor and immunohistochemical analysis revealed that the tumor stained strongly positive for adrenocorticotropic hormone. Furthermore, removal of the lung lesion resulted in a normalization of our patient's 24-hour urinary free cortisol excretion rate and resolution of her symptoms and signs of hypercortisolemia.This case report demonstrates the complexities and challenges in diagnosing the causes of adrenocorticotropic hormone -dependent Cushing's syndrome. Functional imaging may not always localize the site of ectopic adrenocorticotropic hormone secretion. The diagnostic efficacy of biochemical and imaging modalities for localizing the anatomical site of ectopic adrenocorticotropic hormone (ACTH) secretion are limited. Somatostatin receptor scintigraphy (SRS), using the ligand 111-indium-pentetreotide, has traditionally been the functional imaging technique used, but it's usefulness has been questioned . HoweverA 37-year-old Arabian woman was referred to our endocrinology clinic with 12 months of progressive weight gain of 30 kg, hirsutism, acne, alopecia, lethargy, amenorrhea and marked anxiety. An examination revealed features of Cushing's syndrome with rounded face, buffalo hump, abdominal striae and proximal muscle weakness. Investigations confirmed excess cortisol production. Her 24-hour urinary free cortisol excretion was 1870 nmol/day , her midnight salivary cortisol level was 121 nmol/L  and after a 1 mg overnight dexamethasone suppression test her serum cortisol level was 597 nmol/L . Her serum ACTH level was elevated at 55.8 and 55.1 ng/L on two separate occasions , consistent with a diagnosis of ACTH-dependent Cushing's syndrome. Magnetic resonance imaging (MRI) of her pituitary gland did not reveal the presence of an adenoma.Localizing the source of excess ACTH secretion was challenging. Inferior petrosal sinus (IPS) sampling was difficult due to a left petrosal sinus anatomical variation. However, it demonstrated a central to peripheral ACTH gradient of less than three, consistent with ectopic ACTH secretion. This diagnosis was supported by failure of cortisol suppression (472 nmol/L) after an 8 mg overnight dexamethasone suppression test. Computed tomography (CT) of her chest, abdomen and pelvis only revealed a well circumscribed 7 mm left inferior basal lung segment lesion. This was reported to most likely represent a benign granuloma or a small lymph node Figure .Due to the wide range in sensitivity and specificity of the high dose dexamethasone suppression test  and the inability to successfully catheterize her left IPS, further dynamic biochemical tests were performed . A five-Serial CT scanning of her chest, abdomen and pelvis over 18 months failed to definitively localize a source of ectopic ACTH production. The well circumscribed 7 mm left inferior basal lung segment lesion was reported as stable in size over this time. A repeat MRI of her pituitary gland once again did not reveal the presence of an adenoma.Given the failure of biochemical or imaging techniques to localize the site of excess ACTH secretion, medical therapy was initiated with ketoconazole. However, combination treatment with metyrapone was required after eight months due to rising 24-hour urinary free cortisol levels and progressive symptoms of weight gain, lethargy, depression and anxiety. Despite combination medical therapy there was still a progressive rise in 24-hour urinary free cortisol levels (Figure Given the failure of medical therapy to control her symptoms, other potential treatment options were discussed with our patient. These included progressing to bilateral adrenalectomy or resection of the lung lesion, which was the only possible anatomical site of ectopic ACTH secretion located so far. Unfortunately, the lung lesion was reported to be lying adjacent to the pericardium which negated a minimally invasive surgical approach to remove it. Despite this our patient still strongly favored proceeding to resection of the lung lesion even though this would require an open procedure. Hence, an open thoracotomy to remove the lung lesion was performed 18 months after her initial presentation.Histological examination of the resected nodule showed a 9 mm well circumscribed tumor surrounding a bronchus, with features consistent with a carcinoid tumor. Immunohistochemical analysis revealed that the tumor stained strongly positive for chromogranin, synaptophysin and ACTH. Postoperative recovery was uneventful and perioperative corticosteroid replacement was progressively weaned. Clinically, her symptoms of Cushing's syndrome slowly abated. She achieved a normal 24-hour urinary free cortisol excretion off all treatment four months after surgery. Our patient remains well with no clinical or biochemical evidence of cortisol excess seven months after her surgery.This case illustrates the difficulty in diagnosing a pituitary or an ectopic source of ACTH-dependent Cushing's syndrome. A wide variability in the sensitivity and specificity of current biochemical dynamic tests has been reported Table . None ofFurthermore, functional imaging may not always assist in localizing an anatomical site of excess ACTH secretion Table . In someWhen all modalities fail to localize a source of ectopic ACTH, the role of clinical judgment plays a significant role. Ongoing monitoring of the patient, combined with a relevant discussion of risks and benefits of different therapeutic options led to a decision to proceed to removal of the small bronchial nodule. This nodule was subsequently confirmed to be an ACTH-secreting carcinoid tumor. Embarking on this decision despite there being no definitive preoperative confirmation that the nodule was the source of her ectopic ACTH production resulted in a cure of our patient's Cushing's syndrome.We have shown the limitations of the currently available diagnostic tools in differentiating pituitary or ectopic sources of ACTH-dependent Cushing's syndrome. Furthermore, despite significant advances in radiological and nuclear medicine imaging modalities, the localization of the site of ectopic ACTH may still not be possible.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EG, AC and RM analyzed and interpreted the patient's data and were involved in the patient's care. LG was a major contributor to writing the manuscript. All authors read and approved the final manuscript.


Although a rare condition, rosette formation in malignant melanoma has been previously documented. The present study describes the second documented case of malignant melanoma with perivascular pseudorosettes. A 38-year-old male presented with a black nodule on his back. Histopathological study revealed diffuse proliferation of neoplastic cells in the dermis and subcutis. A section of the tumor (~30%) was composed of a conventional malignant melanoma component. The remaining area was comprised of medium-sized polygonal cells with slightly eosinophilic cytoplasm and small-to-medium, round nuclei. Melanin pigment was rarely observed. A noteworthy observation was the presence of perivascular pseudorosette formations, which were characterized by their radial arrangement around the blood vessels, with a perivascular, anuclear zone. Immunohistochemically, the neoplastic cells were diffusely positive for S-100 protein and Melan-A and focally positive for HMB-45. Clinicopathological analyses of cases of malignant melanoma with rosette formations revealed that the types of rosette included the Homer-Wright type (two cases), perivascular pseudorosettes (two cases) and an unclassifiable type (one case). Immunohistochemical analysis is a useful method for forming a diagnosis as Melan-A or HMB-45 are generally expressed in all cases. Rosette formation in malignant melanoma is a distinct histopathological variant and may be an under-recognized phenomenon. Therefore, its recognition is significant for obtaining an accurate diagnosis of malignant melanoma. Malignant melanoma occasionally exhibits a variety of cytomorphological and architectural features and includes the balloon cell, rhabdoid, small cell, signet-ring cell, myxoid and adenoid (pseudoglandular) types . AlthougA 38-year-old male with no past history of malignant melanoma presented with a gradually enlarging nodule on his back. A physical examination revealed that the nodule was black and measured 35×30 mm in diameter. The left axillary lymph nodes were enlarged. Computed tomography demonstrated multiple nodules in the liver, which were clinically suspected to be metastatic lesions. A total resection of the back nodule and the left axillary lymph nodes was performed.The formalin-fixed, paraffin-embedded tissue blocks of the resected skin specimen and lymph nodes were cut into 3-μm thick sections, deparaffinized and rehydrated. Each section was stained with hematoxylin and eosin and used for immunostaining. The immunohistochemical analyses were performed using an autostainer  according to the manufacturer’s instructions. The following primary antibodies were used: A mouse monoclonal antibody against CD56 , a mouse monoclonal antibody against chromogranin A , a mouse monoclonal antibody against glial fibrillary acid protein , a mouse monoclonal antibody against HMB-45 , a mouse monoclonal antibody against Melan-A , a rabbit polyclonal antibody against S-100 protein  and a mouse monoclonal antibody against synaptophysin .The histopathological study of the resected back nodule revealed a diffuse proliferation of neoplastic cells accompanied by scattered geographical necrotic foci from the entire dermis to the subcutis. The upper section of the lesion (~30% of the tumor) was composed of a conventional malignant melanoma component. The section was comprised of large polygonal neoplastic cells that contained large nuclei with or without conspicuous nucleoli. The majority of these cells contained the melanin pigment within the cytoplasm . The remImmunohistochemical studies revealed that S-100 protein and Melan-A were diffusely expressed in the two components . HMB-45 The left axillary lymph nodes contained metastatic malignant melanoma with the two components and their immunohistochemical characteristics were the same as those of the back lesion.According to these findings, a diagnosis of malignant melanoma with perivascular pseudorosettes accompanied by lymph node metastases was made.In diagnostic pathology, rosettes are classically referred to as discrete cell clusters showing peripheral nuclear palisading and a central space. Although rosettes are commonly considered to be of neuroepithelial differentiation, they are occasionally observed in other types of tumors, including neuroendocrine carcinomas, granulosa cell tumors and thymomas. Certain histopathological subtypes of rosettes are well known, including the Homer-Wright type and perivascular pseudorosettes. Homer-Wright rosettes are characterized by a radial arrangement of cells with centrally situated fibrillary material and are often observed in neuroblastomas, medulloblastomas and primitive neuroectodermal tumors. Perivascular pseudorosettes are composed of columnar cells arranged radially around the blood vessels, with a perivascular anuclear zone. This structure is a well-known characteristic feature of an ependymoma –4.et al described melanin pigment within the cytoplasm of the neoplastic melanocytes forming the Homer-Wright rosette, melanin pigment was barely observed within the cytoplasm of the rosette-forming tumor cells in the present case and in the case reported by Falconieri et al(Although rare, rosette formation has been previously documented in malignant melanomas –4. Tableeri et al. TherefoAlthough the mechanism of rosette formation in malignant melanoma remains unclear, it has been speculated that the neoplastic melanoma cells share the same capabilities for differentiation as neural crest precursors .In conclusion, the present study describes the second documented case of a malignant melanoma with perivascular pseudorosettes. Rosette formation in malignant melanoma is a distinct histopathological variant and may be an under-recognized phenomenon. Additional studies are required to clarify the clinicopathological features of this variant of melanoma, whose recognition is significant for obtaining an accurate diagnosis of a malignant melanoma.