Atoh1-cre; Atoh1f/f); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons , and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1−/−). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed  for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei.We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised ( Experimental tracing of developing afferent innervation from individual end organs to the brain show initially segregated projections of all major sensory organs  ears in which no hair cells ever differentiate  mice lose Atoh1 expression in the periphery, and subsequently hair cells, resulting in loss of a peripheral target  (ACURF #1103057).Atoh1−/− mice were bred from heterozygotes as previously described ; Atoh1f/f CKO mice ; Atoh1f/f CKO mice , which generated a 280 bp product. Two internal control primers were included in the PCR reaction that produced a 330 bp product (forward: 5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′ and reverse: 5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′). The Atoh1 allele-specific primers (forward: 5′-AGC GAT GAT GGC ACA GAA G-3′ and reverse: 5′-GAA GTC AGG TCG TTG CTA AC-3′) generated a 300 bp product from the wild-type Atoh1 coding region and a 500 bp product from the floxed allele.Mice were genotyped using PCR analysis of DNA obtained from tails. The All postnatal mice and pregnant females for collecting embryos were anesthetized by injection of a lethal dose of Avertin (1.25% of 2.2.2-tribromoethanol at a dose of 0.025 ml/g of body weight) and then perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) using a peristaltic pump. Heads were isolated and fixed for at least 24 h in 4% PFA before further processing.For all conditions, both ears of at least three mutants and littermate controls from at least two litters were examined for the stages indicated in results for each phenotype. Between mutant variation was so limited relative to control animals allowing the generation of compelling qualitative evidence displayed in our data.Beta-galactosidase detection to express LacZ was run as previously described . In fact, cochlear projections remain confined within the cochlear nucleus at P7 without aberrant targeting (Figure In contrast to Atoh1-cre; g Figure . Since cg Figure , analysig Figure . Likewisg Figure  as well g Figure . The affg Figure . Howeverg Figure .These data suggest that the initial auditory and vestibular afferent segregation and cochleotopic afferent segregation in mouse embryos is independent of hair cell development and may not depend on formation/maintenance of a normally developed central target either, consistent with previous reports on rhombomere-specific cochlear nucleus deletions (Maricich et al., Atoh1-LacZ positive cells still form in the rhombic lip (Figure Atoh1 mutants, our data show segregation of vestibular afferents from cochlear afferents is complete in Atoh1−/− and this is despite the fact that cochlear afferents have neither a peripheral nor a central differentiated target whereas the vestibular afferents only miss the peripheral target (Figures Atoh1−/− mice shows a segregation of base versus apex (Figures While p Figure  at the tp Figure . As in t Figures . In addi Figures  as in co Figures  or anima Figures . In summAtoh1 mutants demonstrate that neither peripheral nor central target cell differentiation is necessary for cochlear afferent segregation from vestibular afferents or for some degree of a cochleotopic segregation. Cochleotopic connections from specific areas of the cochlea to the cochlear nuclei are the basis for tonotopic representation of the spiral ganglion projection and thus for perception of different sound frequencies (Rubel and Fritzsch, Atoh1 mutations with some retention of cochlear nuclei (Figures Atoh1−/− mice of afferents that have neither a central nor a peripheral cellular target, this suggests that the molecular basis of afferent targeting restricts cochlear afferents to the area of the cochlear nuclei, even if this area lacks fully differentiated target cells.The results presented here from various  Figures , 3 and t Figures  indicatiCombined, these data support the idea that cochlear afferent projections are specified by means that require either the temporal pattern of development of spiral ganglion neurons (basal turn neurons exit the cell cycle prior to apical turn; Ruben, Fibers not only need to find their target, neurons need to interact with target cells to ensure their survival through the neurotrophic support molecules released from target cells (Huang and Reichardt, These data have implications on the evolution of the vertebrate auditory system, which has mostly focused on the terrestrial middle ear (Reichert, BF and KE wrote the paper; IJ, NP, and JK contributed images and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9's utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications. We offer perspective on how to utilize these tools for currently untested research endeavors and analyze strengths and weaknesses of novel CRISPR systems in plants. Advanced CRISPR functionalities and delivery options demonstrated in plants are primarily reviewed but new technologies just coming to the forefront of CRISPR development, or those on the horizon, are briefly discussed. Topics covered are focused on the expansion of expression and delivery capabilities for CRISPR-Cas9 components and broadening targeting range through orthogonal Cas9 and Cpf1 proteins. Sequence specific nucleases (SSNs) are quickly being incorporated into new and broadening research and development programs. Functional genomics studies, genetic engineering pipelines, molecular breeding activities and many other areas of plant research are all being impacted Voytas, . Of the in vivo  at genomic loci Once DSBs have been catalyzed by Cas9-gRNA complexes two possible fates proceed depending on cell type, target site, and DNA repair machinery. The vast majority of DSBs are hastily repaired by non-homologous end joining (NHEJ) mechanisms  based promoter  35S promoter or Arabidopsis Ubiquitin 10 (AtUbi) promoter to drive Cas9 expression in dicots. Maize or rice Ubiquitin  promoters are most commonly used for constitutive Cas9 expression in monocots. Constitutive promoters such as 35S and Ubi are useful for testing proof of concept studies or preliminary investigations but result in high levels of somatic cell mutation and mosaic T0 or T1 plants. When producing heritable transgenic lines, it is desirable to use various egg or germline specific promoters. This has been shown to be important for plants such as Arabidopsis thaliana, which is generally transformed using Agrobacterium mediated floral dip that targets ovule cells for transformation  be present in the host nucleus at sufficient concentration. If both elements are to be transcribed r Figure . The majgRNAs are typically expressed under small nuclear RNA promoters such as the U6 and U3 promoters. These small nuclear RNA promoters are constitutively transcribed by RNA polymerase III (Pol III) and require a specific 5′ nucleotide to initiate transcription (5′-Guanine for U6 or 5′-Adenine for U3)  is efficient enough for many CRISPR based applications. However, for applications that require more control over mutation heritability, involve potentially toxic mutations or warrant spatial or temporal control over genome editing, then tissue-specific or inducible gRNA expression is preferable. Multiple groups have tested germline-specific promoters for driving Cas9 expression in order to obtain high frequency germinal editing in Since the first reports of CRISPR function in plants, mixed dual promoter systems have become the overwhelming standard for CRISPR-Cas9 experiments. However, possible limitations of this strategy for multiplex applications has led to the very recent developments of different expression systems that modify either the gRNA promoter or condense multiple gene stacks to polycistronic genes.Arabidopsis , Neisseria meningitidis (Nm), and Staphylococcus aureus (Sa), which have been demonstrated previously to function in mammalian cells  was developed based on reports of the type V CRISPR-Cas system found in various bacteria  and was shown to exhibit heterologous RGEN activity in mammalian cells . Cpf1 recognizes a thymine rich (TTTN) protospacer adjacent motif (PAM) sequence while Cas9 recognizes Guanine rich (NGG) PAMs. Cpf1 cleaves DNA distal to its PAM as opposed to Cas9 which cleaves DNA close to its PAM (Figure A major development in mammalian cell genome editing has been the report of a novel class II CRISPR RNA guided nuclease that cleaves DNA leaving 4-5-nt overhanging “sticky” ends as opposed to the blunt end digestion of Cas9 nucleases Figure . Cpf1 (CM Figure . BecauseM Figure  and Cpf1M Figure , it is lM Figure . FurtherM Figure . Cpf1 alM Figure , which pDrosophila (Port and Bullock, Recently, genome-wide targeting specificity of the Cpf1 system was comprehensively analyzed in human cells by two independent groups and both groups concluded Cpf1 is a highly specific nuclease system suitable for precise genome editing (Kim, D. et al., LL and YQ conceived and wrote the draft. AM drafted Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Pediatric residents report a lack of confidence and competence with procedural skills at graduation. Training programs could benefit from improved approaches to target these needs. Using the Institute for Healthcare Improvement (IHI) Model for Improvement and three Plan-Do-Study-Act (PDSA) cycles, we examined the impact of a procedure simulation boot camp on self-reported procedural confidence and competence as well as the longitudinal impacts of these sequential interventions on Accreditation Council for Graduate Medical Education (ACGME) Graduating Resident Survey (GRS) results.Methods: Three rapid cycle interventions were performed in successive academic years. The interventions included 1) increased awareness of available procedural experiences, 2) institution of procedural educational conferences, and 3) implementation of a senior resident procedure boot camp. Senior resident self-reported procedural confidence was measured before and after the boot camp. Procedural competence was measured using the ACGME GRS. Results: Thirty-two of 34 senior residents (94%) completed the 2016 ACGME GRS, similar to the response rates of 2014 (92%) and 2015 (94%), and 30 of 34 third-year residents participated in the procedure boot camp (88%). Resident confidence and competence with procedural skills improved after the institution of the quality improvement intervention. ACGME GRS-reported competency increased in bag and mask ventilation (77% to 94%), neonatal endotracheal intubation (39% to 69%), peripheral IV placement (10% to 50%), and umbilical catheter placement (35% to 53%).Conclusion: A quality improvement intervention with three rapid PDSA cycles was successful in improving senior pediatric resident confidence and competence with ACGME required procedural skills. The Accreditation Council for Graduate Medical Education (ACGME) Residency Review Committee for Pediatrics mandates that pediatric residents demonstrate procedural competence in 13 procedures . The pediatric residency program consists of approximately 112 total residents, 36 per year. Typically half of the graduates from this program pursue careers in general pediatrics or hospital medicine in the Pacific Northwest, and many graduates work in practices in small to mid-size cities without a nearby Children’s Hospital or ready access to pediatric specialists. This quality improvement (QI) effort was undertaken to address ACGME GRS scores for procedural competence that were below the national mean for two consecutive years. Program leadership also recognized that many of these procedural skills were necessary for optimally training the pediatric workforce for our region. The project was approved by Seattle Children’s Institutional Review Board.Before this intervention, residents received informal procedural training by supervisors often immediately before or during a procedure. Formal education was provided every two years in the Pediatric Advanced Life Support (PALS) course and once during the intern year in the Neonatal Resuscitation Program (NRP) course. During the July 2014 – June 2015 academic year, we focused on improving resident awareness of their procedural training. In coordination with the faculty-resident education committee, we worked to improve resident identification of existing procedural training during residency. Based on qualitative graduating resident feedback, we identified procedures that residents felt least competent with to improve our procedural education curriculum targeting these procedures.Our second PDSA cycle utilized resident feedback, as well as ACGME GRS results, to design a series of teaching conferences during the July 2015 – June 2016 academic year focusing on procedural skills that were easily taught in a large group setting. We held eight procedural lunchtime conferences covering splinting, laceration repair, and immunizations. Resident feedback on the content was sought after each conference and improvements were made on a monthly basis.Our third PDSA cycle was the implementation of a senior resident simulation boot camp in January 2016. Based on the ‘Learn-See-Practice-Prove-Do-Maintain’ pedagogy for procedural skills training, residents were asked to review preparatory materials, including written materials and instructional videos for each procedure, prior to boot camp . During Residents completed two anonymous surveys, one immediately prior to boot camp and one immediately following the boot camp. In the pre-boot camp survey, residents were asked to indicate the number of times they had performed specific procedures during their residency, their confidence in performing each procedure, and to rate their self-percieved competence. In the post-boot camp survey, residents rated their confidence and their self-percieved competence in performing each procedure. Estimations of confidence and competence utilized a 5-point Likert-type scale (1 = "not confident" / "not competent" to 5 = "very confident" / "very competent").Graduating senior resident procedural competence was determined based on results of the ACGME GRS performed in the spring of each academic year. Competency was considered a response of “Agree” or “Strongly Agree” to the question “I feel well prepared to perform the following procedures without supervision.”Survey data were analyzed using matched variables and compared using paired t-tests. Differences were considered statistically significant if P < 0.05. ACGME survey data were evaluated using descriptive statistics, including means and standard deviations. Data analysis was performed using Microsoft Excel (XP)  and Stata, version 12 .Thirty of 34 third-year pediatric residents in our residency program participated in the procedure boot camp (88% participation rate). All residents who attended the boot camp completed pre-boot camp and post-boot camp surveys (100% completion rate). The estimated total numbers of procedures performed during residency are shown in Table Resident self-assessed procedural confidence and competence are shown in Table Thirty-two of 34 senior residents (94%) completed the 2016 ACGME GRS, which was similar to the response rates from our program to the GRS in 2014 (92%) and 2015 (94%). Procedures, which were taught in a large group setting in the Procedural Educational Curriculum (PDSA Cycle 2), demonstrated a variable change in resident competency. Competency was increased for immunization administration (26% to 69%) and laceration repairs (87% to 94%) but unchanged for splinting (74% to 75%).Four ACGME required procedures were taught during the half-day boot camp (PDSA cycle 3). For all, the percentage of graduating residents reporting competence increased from 2014 to 2016 for bag and mask ventilation (reported competence increased from 77% to 94%), neonatal endotracheal intubation (39% to 69%), peripheral intravenous catheter placement (10% to 50%), and umbilical catheter placement (35% to 53%) , decreases in self-reported competency for reduction of dislocations (81% to 69%), and foreign body removals (87% to 81%), and increases in resident competency for delivery room resuscitation (87% to 100%), bladder catheterization (16% to 31%), and incision and drainage of an abscess (71% to 87%).Our study found that a quality improvement intervention enhanced procedural confidence and competence among graduating pediatric residents. We found a positive impact of our procedural simulation training on ACGME procedural competency scores, with the most significant changes in procedural competence seen in skills taught in a simulation-based procedural boot camp (PDSA cycle 3). We believe these results reflect the impact of the boot camp training and not the result of general trends in resident training or observation biases. This lends validity to the impact of the simulation-based boot camp training as an effective methodology.As residents’ experiential training faces growing time restrictions, the implementation of adjuvant educational methods is critical. The lack of procedural competence seems to be a growing concern and may be a reflection of limited opportunities . As we hWe showed that participation in a simulation boot camp improved resident self-reported procedural confidence and competence. Our results support the results of other studies in this area. Cohen, et al. reported that a simulation-based mastery learning boot camp for internal medicine interns improved clinical skills scores on five parts of a clinical skills examination as compared to historical controls . KrajewsThe next steps of this QI project include expanding the procedural education curriculum to include more procedures and more sessions. We are planning for monthly large group procedure teaching sessions in the July 2016 - June 2017 academic year, as well as increasing participation in the simulation-based procedural boot camp by including second-year residents. We plan to continue frequent PDSA cycles to optimize resident procedure training, with the goal of graduating procedurally competent residents.Our educational intervention had several limitations. First, as residents were aware of our ongoing efforts to improve procedural competence and that procedure competency was an important issue to the residency program, there may have been an element of reporting bias. Secondly, we chose to assess both ‘confidence’, defined as feeling well prepared to perform the procedure, as well as ‘competence’, defined as the ability to perform a procedure correctly without direct supervision. Previous research into procedural training has shown that residents may be competent without reporting confidence in their procedural ability . ConversThe lack of pediatric resident procedural competence is a growing concern, both at our institution and nationwide, as residents perform fewer procedures in the clinical setting. A quality improvement educational intervention with several PDSA cycles was successful at improving resident reported procedural competence. Procedural simulation training, which included large group education sessions and targeted boot camps, was successful in increasing graduating resident reported procedural competence.


We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis ( The mammalian foregut endoderm, for example, gives rise to several organs with important physiological functions including liver, pancreas, stomach, lungs and thyroid. Identification of signalling pathways and intrinsic factors involved in the specification, morphogenesis and functional maturation of foregut-derived organs is crucial for understanding of the molecular mechanisms leading to developmental disorders in organogenesis and adult organ function2. This problem can be well exemplified with the case of developmental disorders in thyroid morphogenesis, commonly referred to as thyroid dysgenesis (TD), which can present as either thyroid ectopy, thyroid hypoplasia, athyreosis or hemiagenesis3. Although TD is the most prevalent cause of congenital hypothyroidism in human newborns, the pathogenic mechanisms underlying TD remained obscure in most cases4. One reason for this is our still limited understanding of the molecular signalling cascades orchestrating normal thyroid morphogenesis6.During vertebrate embryonic development, the definitive endoderm transforms into a primitive gut tube from which various organ primordia will bud at predefined locations along the rostro-caudal axis11. In addition, the application of NGS in clinical research and diagnostics provided a plethora of genome-wide data on genetic variants and spurred the identification of new candidates for disease-causing genes13. However, up to date, the actual biological function during organ and tissue formation is often far from being clear for many of the newly identified genes.At the molecular level, one important resource for the understanding of organogenesis and developmental disorders is knowledge about organ- and cell type-specific gene expression programs during critical developmental periods. In this respect, the introduction of next generation sequencing (NGS) techniques has allowed for unprecedented high-throughput transcriptome analyses of developing embryos, organs and tissues in diverse species14. In combination with a suitable animal model, the CRISPR/Cas technique provides currently the most promising platform to facilitate rapid and scalable approaches towards a functional characterization of novel genes in a developmental context.Thus, one has to appreciate that information-rich analyses based on NGS techniques created new bottlenecks for developmental biologists including the need to identify rapid and scalable approaches to characterize the biological function for larger panels of candidate genes with hitherto unknown function during organ development. The most direct approach to study gene function is still the generation of loss-of-function models for a given gene followed by an analysis of developmental and functional phenotypes of the organ or tissue of interest. The recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system as a highly efficient genomic editing technology revolutionized the way new genetic models can be realized including the rapid production of mutant or knock-out animal modelsDrosophila with physiological and anatomical characteristics of higher vertebrates17. Several salient features make this model system particularly attractive for studies on gene function during early development. Foremost, zebrafish embryos and larvae develop externally and thus permit the monitoring of developmental processes and their pathological deviations in real time19. Further advantages include high fecundity, a rapid embryonic and larval development, and optical transparency of embryos and larvae. Importantly, a powerful molecular and genetic toolbox permits efficient transgenesis, real-time imaging of tissue and cell behaviours and forward and reverse genetic approaches to generate mutant models22.The zebrafish is one such powerful animal model to decipher cellular and molecular mechanisms of vertebrate organogenesis as it combines the genetic tractability of 23, little progress has been made in assigning specific functions to most of the newly identified genes during thyroid morphogenesis. This holds true for processes including thyroid precursor cell specification, thyroid bud formation, detachment and relocalization, early thyroid growth and functional maturation6. Most of our current knowledge about the development of functional thyroid tissue from endodermal precursors stems from studies in murine models but recent studies in zebrafish revealed that many aspects of thyroid morphogenesis are well conserved from fish to men24.In this study, we harnessed the advantages of the zebrafish model to develop a reverse genetics strategy to functionally characterize genes involved in thyroid development and function. Compared to other endoderm-derived organs such as liver and pancreas, our understanding of the molecular mechanisms underlying thyroid morphogenesis is much more limited. Although recent transcriptomic studies have disclosed gene expression patterns in foregut-derived organ buds including the thyroid27, we combined optimized mutagenesis conditions with a novel phenotyping strategy and an information-rich genotyping approach into a reverse genetic screening protocol to identify genes involved in thyroid development and function. For this purpose, we generated a new zebrafish reporter line that permitted a rapid and sensitive detection of thyroid phenotypes related to thyroid dysgenesis as well as dyshormonogenesis in live fish. When applying this 6-day screening protocol to target loci encoding for genes with specific functions in thyroid development and function, we achieved a robust recovery of loss-of-function phenotypes in somatic mutants including cases of athyreosis, thyroid hypoplasia and goitrous thyroid enlargement.Stimulated by the high efficiency of the CRISPR/Cas9 system to generate biallelic gene disruption in F0 zebrafish embryos29. This is particularly true for small organs, such as the zebrafish thyroid, which are hardly detectable by brightfield microscopy of live embryos. To exploit the advantageous properties of transgenic zebrafish, we generated a novel reporter line expressing a nuclear-located EGFP (nlsEGFP) variant under the control of the zebrafish thyroglobulin (tg) promoter30. The Tg(tg:nlsEGFP)ulb4 line showed very promising properties for sensitive analyses of thyroid morphogenesis in live embryos by using various microscopic modalities ulb1 line, embryos of the Tg(tg:nlsEGFP) line showed an early onset of reporter expression around 36 hpf and EGFP expression was restricted to thyroid follicular cells (TFC) at all developmental stages examined  and Tg(tg:mCherry) fish confirmed identical spatiotemporal expression patterns of both reporters yet distinct subcellular expression domains  embryos and larvae for confocal live imaging to characterize changes in thyroid morphology during thyroid development from 55 hpf to 11 dpf and established a three-dimensional atlas of normal thyroid development  embryos and larvae from 52 hpf with either 30 mg/L phenylthiourea (PTU) or 5.0 µg/L thyroxine (T4) and monitored thyroid morphogenesis and thyroid growth in treated versus untreated fish (controls) by confocal live imaging.Thyroid-stimulating hormone (TSH), secreted by pituitary thyrotrophic cells, is the primary physiological regulator of vertebrate thyroid function and growthtshb expression and results in enhanced TSH stimulation of thyroid tissue33. Here, we demonstrate that PTU treatment induces a goitrous thyroid enlargement in zebrafish larvae that was readily detectable by live imaging of Tg(tg:nlsEGFP) larvae at 6 dpf  synthesis, causes increased pituitary dpf Fig. . Notably33, thyroids of PTU-treated larvae did not differ from controls in terms of overall size and shape, TFC number and follicular morphology  to assess T4 treatment effects on feedback signalling along the pituitary-thyroid axis in this experimental model  embryos and larvae raised in the presence or absence of T4 closely recapitulated observations made for endogenous tg mRNA expression  larvae displayed first marked differences in thyroid reporter expression at 100 hpf. At this stage, the reporter signal was very dim in T4-treated larvae  embryos provides a sensitive and rapid means of thyroid phenotyping.Live imaging of ion Fig. . Similarvae Fig.  and confvae Fig. . Althougvae Fig. . The atrvae Fig. . Quantitvae Fig. , these aTg(tg:nlsEGFP) fish over a period of 6 days in combination with functional assays performed in fixed 6 dpf larvae. For the monitoring of live Tg(tg:nlsEGFP) embryos and larvae, we considered 55 hpf, 80 hpf and 6 dpf as relevant developmental stages to detect possible effects on early thyroid morphogenesis (55 hpf), growth expansion (80 hpf) and folliculogenesis (6 dpf). For the present study, we further selected whole-mount immunofluorescence (WIF) of colloidal T4 as an integrative endpoint to characterize thyroidal function in fish sampled at 6 dpf36. In addition, all thyroid phenotyping was done for fish that are not affected in their gross development . Notably, meaningful thyroid phenotyping according to the criteria outlined above relies on a fairly normal development and growth of the experimental fish and particularly on the absence of gross dysmorphogenic alterations of the pharyngeal region  were available for phenotypic assessment. All sgRNAs used in this study fulfilled these criteria. Moreover, the far majority of fish injected with different sgRNAs displayed normal morphological development and growth up to 6 dpf  and nkx2.4b (ZDB-GENE-000830-1) genes encode for transcription factors which act as key regulators of early thyroid morphogenesis38. Similar to their mammalian counterparts Pax8 and Nkx2.15, zebrafish models with impaired function of pax2a  and nkx2.4b (transient gene knock-down by morpholino injection) display defects in early thyroid morphogenesis resulting in athyreosis38.The zebrafish pax2a and the homeodomain region in exon 2 of nkx2.4b with specific sgRNAs and assessed whether we can recover thyroid phenotypes in somatic mutants. When analysed at 55 hpf, many Tg(tg:nlsEGFP) embryos injected with sgRNAs targeting either pax2a or nkx2.4b displayed complete absence of the thyroid (athyreosis) or severe thyroid hypoplasia , thus phenocopying the thyroid phenotype described in noi mutants38. To further compare thyroid phenotypes between noi mutants and pax2a crispants, we analysed thyroid marker expression in pax2a crispants at 28 hpf (nkx2.4b) and 55 hpf (tg) by WISH. Consistent with previous studies of noi mutants38, we found that the early thyroid anlage was formed in all pax2a crispants  but expression of thyroid markers becomes severely impaired or is absent in about half of the pax2a crispants analysed at 55 hpf  was slightly lower  up to 98.5% . When comparing indel allele frequencies between individual larvae displaying athyreosis and hypoplasia, we observed a reduced frequency of frameshift alleles in pax2a crispants displaying less severe thyroid phenotypes (hypoplasia) whereas no such tendency was apparent from the sequencing of nkx2.4b crispants  alleles. The latter observations strengthen the notion that our phenotyping strategy provides high sensitivity to recover phenotypes linked to thyroid dysgenesis. In the case of pax2a crispants we additionally confirmed effective frameshift mutagenesis at the protein level by demonstrating strongly reduced pax2a immunoreactivity using an antibody directed against an C-terminal epitope , we extracted genomic DNA from whole larvae and assessed the mutagenesis efficiency for at least 4 individual crispant larvae from each phenotypic category  using Illumina HiSeq technology. Total numbers of HiSeq-derived sequences, mapped reads and allele frequencies for individual larvae are provided as Supplementary Information DUOX1/2 and DUOXA1/2 genes. In the mammalian thyroid, heterodimeric DUOX/DUOXA complexes generate the H2O2 required for TH synthesis and genetic alterations of the DUOX/DUOXA system can lead to congenital hypothyroidism40. In contrast to mammals, the zebrafish genome contains only a single duox gene (ZDB-GENE-091117-14) and a single duoxa gene . To date, functional zebrafish models were limited to morpholino-mediated knockdown of duox gene function and the assessment of developmental processes unrelated to thyroid function41.We next addressed the question whether our mutagenesis assay can sensitively recover phenotypes arising from disturbed TH synthesis. For this purpose, we targeted zebrafish orthologues of mammalian duox gene. For zebrafish duoxa, we targeted a region close to the N-terminus of the protein to maximize the likelihood that frameshifts create a non-functional allele. When assessed at 55 and 80 hpf, neither duox nor duoxa crispants presented gross thyroid phenotypes  than in duoxa crispants . Thus, the T4 immunofluorescence assay contributed to the overall sensitivity of the mutagenesis assay by recovering additional milder phenotypic variants. In conjunction with the thyroid phenotypes observed following chemical blockade of TH synthesis, the results from duox and duoxa crispant analyses corroborate the capacity of our mutagenesis-phenotyping strategy to sensitively detect genetic alterations that impair thyroidal TH synthesis.In this study, we targeted a sequence in exon 23 of the pes Fig. . This fiduox and duoxa crispants displaying dyshormonogenetic goiter confirmed a high mutagenesis efficiency of the sgRNAs used in this study .Illumina sequencing of individual udy Fig. . Total n42. Murine Tshr knock-out models display hypothyroidism and develop thyroid hypoplasia in the postnatal period43. Morpholino knock-down of zebrafish tshr does not affect early thyroid morphogenesis but tshr morphants show a hypofunctional thyroid phenotype33. Against this background information, we targeted regions in exon 4 and exon 10 of the zebrafish tshr gene (ZDB-GENE-110524-4), respectively, and assessed the capacity of our mutagenesis-phenotyping strategy to recover mutant phenotypes.Regulation of thyroid function by TSH is mediated via binding of TSH to its cognate membrane receptor (TSHR) and subsequent activation of several intracellular second messenger systemstshr crispants at 55 and 80 hpf did not reveal gross deviations from normal thyroid development  and 98.5% . All of the observed frameshift indels detected in the vicinity of the gRNA target site in exon 4 introduce premature stop codons and are expected to result in truncated, non-functional tshr variants lacking all 7 transmembranes domains. Collectively, the hypoplastic/hypofunctional phenotypes recovered in tshr crispants are very consistent with observations previously reported for murine and zebrafish models with impaired TSHR signalling43.For tshr gene and the high penetrance of the associated thyroid hypoplasia phenotype, we next addressed the question of whether somatic mutants provide a valuable model to perform additional functional experiments. For this purpose, we treated tshr crispants and non-injected siblings with PTU (30 mg/L) from 52 hpf throughout 6 dpf and assessed all larvae at 6 dpf for the presence of a goitrous thyroid enlargement  analysed in this experiment. The proportion of tshr crispants not responding to PTU-induced TSH overstimulation compares well with the proportion of tshr crispants displaying thyroid hypoplasia (54.5%) in the initial mutagenesis assay . Despite the presence of such mosaicism at the tissue level, the robust tissue responses observed in somatic mutants in our experiments support the notion that somatic mutants provide a valuable experimental system to address first hypotheses regarding the putative role of target genes in classical thyroidal signalling pathways.Given the high mutagenic efficiency of the gRNA targeting exon 4 of the ent Fig. . In contispants 5.3% analyion Fig. . Overallsee Fig.  as evideduoxa sgRNA injection experiments to adulthood, inbred F0 founders (without prior testing of germline transmission of mutations) and analysed the F1 progeny for thyroid phenotypes. F0 inbreeding experiments consistently confirmed the presence of goitrous thyroid phenotypes in F1 larvae of many crosses, yet with a variable and non-Mendelian distribution. To generate duoxa germline mutants with defined genetic lesions, we next outcrossed F0 founders to WT fish, raised their F1 progeny to adulthood and screened adult F1 fish for genome modifications at the duoxa locus by Sanger sequencing. We identified several duoxa mutant alleles in F1 fish, including deletions and insertions at the target site, and selected a mutant allele harboring a 11 bp deletion (duoxa Δ11) in exon 2 site for further analyses  including 18 incorrect amino acids at the C-terminus. The deletion allowed for rapid genotyping by PCR  or heterozygous carriers of the duoxa Δ11 allele (N = 49/85). Together, these data corroborate Mendelian inheritance of the dyshormonogenic goiter phenotype and confirm that the thyroid phenotype is genetically linked to the duoxa Δ11 allele. Homozygous duoxa Δ11 larvae uniformly presented goitrous thyroids with undetectable colloidal T4 by WIF  as a gene with high expression in the developing zebrafish thyroid  larvae but observed no significant differences between mean TFC numbers of control larvae  and adamtsl2 crispants .Live imaging of adamtsl2 crispants compared to the efficient cleavage of the BclI site observed in non-injected controls  compared to sgRNA-ex13 , this elevated toxicity did not cause detectable alterations in thyroid morphogenesis/function. Given that In this study, we developed a reverse genetic screening strategy to identify genes involved in thyroid morphogenesis and thyroid function. Our approach capitalizes on the high mutagenic efficiency and sequence-specificity of the CRISPR/Cas system, the possibility to monitor the evolution of organ abnormalities in transgenic zebrafish and the sensitive analysis of mutagenesis efficiency by Illumina HiSeq sequencing.27. By targeting genes with known function in early thyroid morphogenesis 38, functional thyroid maturation (tshr)43 and TH synthesis 40, we demonstrate in this study that this capacity of the CRISPR/Cas mutagenesis system can be exploited to robustly recover distinct thyroid phenotypes in somatic mutants. Moreover, the rapid development of zebrafish allowed us to limit our phenotypic analyses to the first few days of development permitting a cost- and time-efficient screening strategy.Injection of active Cas9/sgRNA ribonucleoprotein complex into zebrafish one-cell stage embryos reportedly permits bi-allelic invalidation of target genes directly in injected F0 fish and allows to recapitulate loss-of-function phenotypesTg(tg:nlsEGFP) line include an overall strong signal intensity facilitating the use of stereomicroscopes for phenotypic analyses and a nuclear-located reporter signal permitting rapid analyses of thyroid cell numbers and changes in cell morphology by confocal live imaging.In its current version, the mutagenesis assay is designed to comprehensively cover phenotypic alterations that might arise from defects in early thyroid morphogenesis , early TSH-independent growth of the thyroid primordium, TSH-dependent functional maturation and TH synthesis. To sensitively detect thyroid phenotypes in a rapid and efficient manner, we developed a new transgenic thyroid reporter line permitting a non-invasive monitoring of thyroid development from 55 hpf to 6 dpf using simple fluorescence microscopy equipment. Particularly advantageous properties of the versus functional maturation).Consistent with the functional roles of the genes targeted in this study, monitoring of thyroid reporter fish allowed us to recover severe dysgenesis phenotypes as early as 55 hpf while dyshormonogenesis phenotypes evolved only after the onset of endogenous TH synthesis (around 80 hpf) and became manifest until 6 dpf. Thus, repeated phenotypic analyses over the course of the first six days of development provided first mechanistic information as to the possible functional roles of the genes under study. Accordingly, this knowledge, once fostered with data for additional genes will then allow to customize the observation periods depending on the primary purpose of the screening , we noted that thyroid tissue of zebrafish larvae rapidly responds to hypothyroid conditions by developing large hyperplastic goiters (due a sustained TSH overstimulation). From these data, we conclude that zebrafish provides an excellent model system to recapitulate the development of dyshormonogenic goiters. On the other hand, the atrophic hypofunctional thyroids observed in tshr crispants corroborate the capacity of our phenotyping strategy to detect conditions of diminished TSH signalling. Interestingly, the late onset of a hypoplastic thyroid phenotype in zebrafish tshr crispants closely replicates the sequence of thyroid anomalies reported for murine Tshr knock-out models43. In summary, the spectrum of very specific thyroid phenotypes recovered in somatic crispant models underscores the efficiency of the selected mutagenesis-phenotyping approach to comprehensively cover different aspects of thyroid morphogenesis and functional maturation. Results from our studies also corroborate the conservation of molecular mechanisms regulating thyroid organogenesis in zebrafish and mammals. Thus, this reverse genetics screening strategy holds great promise as an efficient tool for the identification of new genes involved in thyroid development and thyroid function.In the case of genetic defects resulting in impaired TH synthesis , we recommend to use a positive injection control that would generate a predictable percentage of a specific thyroid phenotype. Based on our results, any of the sgRNAs used in this study would qualify for such a positive control.in vivo mutagenesis studies revealed little if any evidence for genome editing at off-target sites as a relevant confounding factor in experiments applying CRISPR/Cas-technology25. However, as we did not attempt to specifically analyse loci bearing some level of sequence similarity to our target sequences, we cannot rule out the existence of off-target activities for any of the Cas9/sgRNA ribonucleoprotein complexes that were injected in the course of the current studies. Give the intended use of our reverse genetics strategy as a first screening tool to identify candidate genes, we rather favour another option to rule out off-target effects as the cause for the phenotypic alterations detectable in F0 fish, that is by performing classical segregation analysis of phenotypic traits in the offspring of stable mutants as we have shown for the dyshormonogenic goiter phenotype in the duoxa Δ11 germline mutant line. Particularly when targeting genes with hitherto unknown function in thyroid development/function, this will be critically important before definitive conclusions are drawn on the role of any given gene.False positive findings, on the other hand, might occur due to various reasons including possible genome editing activity at off-target sites, unspecific toxicities of the editing reagents or artefacts occurring during experimental manipulation . So far, results from an increasing number of adamtsl2, we identified a gene that is expressed in zebrafish thyroid cells but that causes no detectable alterations in thyroid development or functional capacity if targeted by various sgRNAs. Implementing the proposed positive and negative control groups in routine injection protocols will strengthen the validity of the powerful approach of F0 mutagenesis.Nevertheless, it is desirable to implement some quality measures in protocols for each individual injection experiment such as negative injection controls. To date, there is no consensus how such a “mock” control might look like. While injecting Cas9 protein together with a non-targeting sgRNA might be one option to establish a negative injection control, we favoured the use of a sgRNA that would cause editing of a real sequence without causing a thyroid phenotype. With 26, T7 endonuclease assays26, hetero-duplex mobility assays44, high-resolution melt analysis45, PCR based methods46, classical cloning followed by Sanger-Sequencing25 as well as deep-sequencing techniques27. For our studies, we identified Illumina HiSeq sequencing of whole body genomic DNA extracted from individual crispants as the method of choice. In combination with CrispRVariants software, we found that this approach provided critically relevant information to confirm that mutagenesis efficiencies of individual sgRNAs are sufficiently high to achieve bi-allelic invalidation of the target gene and to obtain additional information about the frequency and distribution of in-frame and frameshift indels.One of the most critical elements of any mutagenesis strategy relying on phenotypic analyses of somatic mutants is to carefully control the mutagenesis efficiency of the injected sgRNAs. This is particularly important to rule out false negative results as a result of insufficient bi-allelic genome editing. Several methods are available for the detection, quantification, and characterization of indels in the target region including restriction enzyme assaysThyroid cells represent a very small cell population in zebrafish larvae and unless costly and laborious techniques based on cell sorting and single cell sequencing are used, it will remain impossible to reconstruct the allelic composition of thyroid cells in F0 crispants. We therefore stress that data generated by our sequencing of genomic DNA extracted from whole larvae do by no means reflect the allelic composition of thyroid cells. When confronted with the difficulties inherent to the interpretation of possible phenotype-genotype relationships in genetically mosaic samples, we found it worthy to perform our sequencing analyses for individual larvae rather than for pools of larvae. Given the limited accessibility to target cells populations, such as thyroid cells, for direct genotyping analyses, we consider it critical to derive genetic information at the next higher level of organisation that is accessible for analysis  in order to generate more knowledge and advance our understanding how patterns of allelic composition relate to phenotypic differences. At this early phase of F0 mutagenesis assay development, we also think that pooling of several crispants prior to sequencing might bear the danger of diluting or masking information that could be relevant for the interpretation of the assay outcome.In our study, the information-rich data of Illumina HiSeq sequencing allowed for some interesting observations. First, across the various genes targeted in our study, we found that fish presenting the most severe thyroid phenotypes have very high frequencies of mutant alleles  suggesting that bi-allelic invalidation of the target genes was likely achieved in thyroid cells. Fish presenting weaker phenotypes or no detectable phenotype had a much more variable frequency of edited alleles. Although we so far analysed only a limited number of larvae, it seemed that the distribution and frequency of edited alleles detected in whole larvae alone is not necessarily a strong predictor of phenotypic severity of the actual thyroid phenotypes. Whether this reflects the small number of samples analysed or indicate that the predictive value will depend on the gene in question remains elusive so far.vice versa. We currently explain these examples by genetic mosaicism within the thyroid cell population. Although genome editing occurs very early during zebrafish development, cells within a given lineage can have distinct allelic compositions47 and our confocal imaging confirmed that mosaicism can even be observed within the small zebrafish thyroids. Assuming that the thyroid is composed of progeny of several progenitor cells with diverse patterns of genetic mosaicism, then the presentation of a phenotype might depend on various factors such as relative size of the mosaic patches, potential growth rate advantages of normal, hypomorphic or knock-out allele carriers, and whether the deleterious mutations occurs in a gene acting in a cell-autonomous or non-cell-autonomous fashion. More studies targeting a wider range of genes will be necessary to address these critical questions. The interpretation of phenotypic patterns in mosaic F0 zebrafish mutants is a new and challenging research topic and might benefit a great deal from incorporating knowledge generated in other well studied models of cellular mosaicism. Of particular interest might be a comparison with models of X-linked diseases and the various factors that determine how female mosaicism of X inactivation in mammals can affect phenotypic presentation48.Of particular interest to us was the identification of some outlier fish, that is individual crispant fish lacking a detectable phenotype despite relatively high frequency of frameshift mutations at the target site and In summary, we describe a new approach to identify genes involved in thyroid development and function based on CRISPR/Cas mutagenesis in zebrafish embryos. Our phenotyping strategy based on live monitoring of organ development in mutagenized reporter fish proved a robust and sensitive means to recapitulate classical thyroid phenotypes. Our results strengthen the concept that somatic mutants provide a valuable model system for phenotypic screens. Pending the availability of suitable reporter models, the general screening strategy can be adapted for studies on other endoderm-derived organs.49 and staged according to hours (hpf) or days post fertilization (dpf) as described50. For screening and live imaging, embryos and larvae were anesthetized in 0.02% tricaine. The following zebrafish lines were used in this study: wild-type AB strain (WT), pigmentless Casper strain51, Tg(tg:mCherry)ulb130 and Tg(tg:nlsEGFP)ulb4 (this study). All zebrafish husbandry and all experiments were performed under standard conditions in accordance with institutional (Université Libre de Bruxelles-ULB) and national ethical and animal welfare guidelines and regulation. All experimental procedures were approved by the ethical committee for animal welfare (CEBEA) from the Université Libre de Bruxelles (protocol 578 N).Zebrafish embryos were collected by natural spawning and embryos and larvae were raised at 28.5 °C under standard conditions52 was used to generate a reporter construct in which expression of a nuclear-localized EGFP variant is driven by a fragment of the zebrafish tg gene promotor previously shown to permit thyroid-specific transgene expression30. The tg promotor sequence30 was sub-cloned into the multi-cloning site (XhoI and BamHI) of p5E-MCS (Tol2kit vector 228) to generate a p5E-tg vector. Next, a Tol2tg:nlsEGFP vector was assembled by recombining p5E-tg, pME-nlsEGFP (Tol2kit vector 385), and the destination vector pTol2DestR4R2pA in a LR reaction53. The destination vector contained flanking Tol2 ends so that the final Tol2tg:nlsEGFP vector could serve as the transposon donor vector in subsequent Tol2 transgenesis. Capped mRNA encoding for transposase was generated by in vitro transcription of NotI-linearized pCS-zT2TP plasmid54 using mMessage mMachine SP6 kit (Ambion).The Gateway cloning technology-based Tol2kittg:nlsEGFP vector (25 ng/µL) and Tol2 transposase mRNA (35 ng/µL) in one-cell stage WT embryos. Injected embryos were screened at 55 hpf for EGFP expression in thyroid cells and F0 fish with thyroid-specific EGFP expression were raised to adulthood. F0 founders with germline transmission of the transgene were crossed with WT fish to generate stable transgenic Tg(Tol2tg:nlsEGFP) lines. We initially maintained six founder lines over two generations and selected one line with robust reporter expression in all thyroid cells for the experiments reported in this study. This Tg(tg:nlsEGFP)ulb4 line is maintained in both WT and Casper backgrounds.Transgenic embryos were produced by co-injection of Tol2Tg(tg:nlsEGFP) embryos and larvae were treated with phenylthiourea  or thyroxine  from 52 hpf onwards in order to induce hypo- and hyperthyroid conditions, respectively. Stock solutions of 100 mg/L PTU and 5 mg/L T4 were diluted in embryo medium to make experimental treatment solutions of 30 mg/L PTU and 5.0 µg/L T4. A control group was maintained in embryo medium. Embryos and larvae were treated in 90 mm petri dishes containing 25 mL of treatment solution and solutions were renewed every other day.Zebrafish http://crispr.dfci.harvard.edu/SSC/) was used to identify single guide RNA (sgRNA) sequences with high on-target activity55. The CRISPR design web tool available at http://crispr.mit.edu was used to predict potential off-target sites56. sgRNAs with highest on-target activity and lowest predicted off-target score were selected for the experiments.The Sequence Scan for CRISPR software  with gene-specific oligonucleotides containing the SP6 promoter sequence (GCGATTTAGGTGACACTATA) followed by a 20 base target sequence without the PAM (see Supplementary Table DNA templates for sgRNA synthesis were produced using the PCR-based short-oligo method as describedin vitro from purified PCR products by using SP6 RNA-polymerase (NEB). 20 µL reactions contained 1 µg DNA template, 1x reaction buffer, 2.5 mM of each rNTP (Roche), 40 U RNase Inhibitor (Thermo Fisher Scientific), and 6 U Large Fragment of DNA Polymerase I (Thermo Fisher Scientific). After pre-incubation for 15 min at room temperature, 20 U SP6 RNA-polymerase were added and reactions were run over night at 40 °C. After treatment with 3 U DNase I , sgRNAs were purified using High Pure PCR Cleanup Micro Kit (Roche). Concentration of sgRNAs was measured by Nanodrop (Thermo Fisher Scientific) and integrity was checked by gel electrophoresis. Aliquots of sgRNA solutions were stored at − 80 °C until use.sgRNAs were synthesized Tg(tg:nlsEGFP) embryo. For each sgRNA, at least three independent injection experiments were performed with spawns from different founder fish.Cas9 protein  and sgRNA  were mixed in 200 mM KCl solution containing 0.15% phenol red (Sigma) and incubated for 5 min at room temperature. Approximately 3 nL of active sgRNA-Cas9 ribonucleoprotein complex were injected per one-cell stage Injected embryos (crispants) and non-injected siblings (controls) were raised in 90 mm petri dishes (approximately 50 embryos per dish) containing 25 mL of embryo medium. Between 24–26 hpf, all crispant and control embryos were dechorionated by a gentle 20 min treatment with pronase (0.6 mg/mL). Thyroid development was monitored in live zebrafish at 55 hpf, 80 hpf and 6 dpf by visual inspection using a M165 FC fluorescence stereomicroscope (Leica). For the duration of the phenotypic analysis, embryos and larvae were anesthetized in 0.02% tricaine. All control and crispant fish were inspected for gross developmental defects, deviations in size, shape or location of the thyroid, and for the overall intensity of the fluorescent reporter signal.For documentation of thyroid phenotypes, embryos and larvae were immobilized in 1% low-melting point agarose (Lonza) containing 0.016% tricaine and imaged on Fluoro-Dish glass bottom dishes (WorldPrecisition Instruments). Images of the ventral pharyngeal region were acquired using a DMI600B epifluorescence microscope equipped with a DFC365FX camera and LAS AF Lite software (Leica). If indicated, confocal live imaging was performed with a LSM 510 confocal microscope (Zeiss) using Zen 2010 D software (Zeiss).36, images of the thyroid region were acquired for live Tg(tg:nlsEGFP) fish at 6 dpf using a DMI600B epifluorescence microscope. Using LAS AF imaging software, pixel sum values were obtained for fluorescent thyroid tissue  and adjacent non-fluorescent regions . For each fish analysed, the intensity of ROI-1 was corrected by subtracting the intensity of ROI-2. Values of background-corrected fluorescence signal intensities from individual fish were used in comparisons between treatment groups and controls.For quantification of PTU and T4 treatment effects on fluorescence signal intensitiesadamtsl2, nkx2.4b, slc5a5, tg and tshb riboprobes were generated by PCR , anti-DIG antibody conjugated to alkaline phosphatase  and BM Purple (Roche) as substrate for alkaline phosphatase stainings. Stained embryos were postfixed in 4% PFA (Sigma) and embedded in 90% glycerol for whole-mount imaging using an Axiocam digital camera mounted on an Axioplan 2 microscope (Zeiss).Embryos and larvae were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde  over night at 4 °C, washed in PBS containing 0.1% Tween 20, gradually transferred to 100% methanol, and stored at −20 °C until use. DNA templates for synthesis of 33 using the following antibodies: rabbit anti-T4 polyclonal antibody , chicken anti-GFP polyclonal antibody , rabbit anti-pax2a polyclonal antibody , cy3-conjugated donkey anti-rabbit IgG antibody , and Alexa Fluor 488-conjugated goat anti-chicken IgG antibody . Stained specimens were postfixed in 4% PFA, gradually transferred to 100% glycerol and phenotypically analysed using a M165 FC fluorescence stereomicroscope. Whole mount imaging of stained specimens was performed with a DMI600B epifluorescence microscope equipped with a DFC365FX camera.Embryos and larvae were fixed in 4% PFA (pH 7.3) over night at 4 °C and stored in PBS containing 0.1% Tween 20 at 4 °C until use. WIF experiments were performed essentially as describedFour to six individual larvae per phenotypic category were selected for genotypic analysis by Illumina HiSeq sequencing. Individual larvae were lysed in 50 mM NaOH at 95 °C for 10 min and the lysate was neutralized with 0.5 M Tris (pH 8.0). Two rounds of PCR were performed to produce barcoded libraries for Illumina sequencing. First, gene specific primers 60 to classify variants as either no-variant, single-nucleotide variant (SNV), frameshift indel, in-frame indel, splice-site variant, or chimeric reads. Total numbers of HiSeq-derived sequences as well as absolute and relative allele frequencies detected in individual larvae are provided as accompanying Supplementary Information  using the protocol described above. DNA from individual fish was used as a template for subsequent PCRs using primers spanning the target sites of adamtsl2 sgRNAs  encompassing the target region in exon 1 were digested with Bcl1 (NEB #RO160S) for one hour at 50 °C. Reaction products (expected size of restriction fragments: 111 bp and 95 bp) were loaded on 1.0% agarose and on 12% polyacrylamide gels and analysed for the presence of undigested bands (loss of restriction site).The sgRNA target site in exon 1 of the Cell number and relative fluorescence data were assessed for normal distribution  and analysed either by unpaired student’s t-test or by one-way ANOVA followed by post hoc Tukey–Kramer’s multiple comparison test. Differences were considered significant at p ≤ 0.05. Statistical analyses were performed using the software package Graph-Pad Prism 5.01.All data generated or analysed during this study are included in this manuscript and its Supplementary Information files.Supplementary Information


Understanding central mechanisms underlying drug-induced toxicity plays a crucial role in drug development and drug safety. However, a translation of cellular in vitro findings to an actual in vivo context remains challenging. Here, physiologically based pharmacokinetic (PBPK) modeling was used for in vivo contextualization of in vitro toxicity data (PICD) to quantitatively predict in vivo drug response over time by integrating multiple levels of biological organization. Explicitly, in vitro toxicity data at the cellular level were integrated into whole-body PBPK models at the organism level by coupling in vitro drug exposure with in vivo drug concentration–time profiles simulated in the extracellular environment within the organ. PICD was exemplarily applied on the hepatotoxicant azathioprine to quantitatively predict in vivo drug response of perturbed biological pathways and cellular processes in rats and humans. The predictive accuracy of PICD was assessed by comparing in vivo drug response predicted for rats with observed in vivo measurements. To demonstrate clinical applicability of PICD, in vivo drug responses of a critical toxicity-related pathway were predicted for eight patients following acute azathioprine overdoses. Moreover, acute liver failure after multiple dosing of azathioprine was investigated in a patient case study by use of own clinical data. Simulated pharmacokinetic profiles were therefore related to in vivo drug response predicted for genes associated with observed clinical symptoms and to clinical biomarkers measured in vivo. PICD provides a generic platform to investigate drug-induced toxicity at a patient level and thus may facilitate individualized risk assessment during drug development.The online version of this article (doi:10.1007/s00204-016-1723-x) contains supplementary material, which is available to authorized users. Drug-induced toxicity is a major clinical problem  is presented. PICD integrates in vitro toxicity data into drug-specific whole-body PBPK models to translate drug-induced in vitro findings to an actual in vivo situation, thereby predicting drug-specific response profiles induced by different dose levels administered in vivo. At the cellular level, in vitro toxicity data are coupled with equivalent PBPK-simulated concentration–time profiles at the organism level to allow a quantitative description of time-resolved in vivo drug response of key cellular processes and biological pathways. Applying PICD in clinical research allows the quantitative prediction of patient-specific drug response by specifically incorporating patient physiology in individualized PBPK models. In brief, PICD aims for a translation of preclinical in vitro toxicity data into an in vivo context and hence allows risk assessment for individual patients during drug development.PICD is exemplarily applied on the hepatotoxicant azathioprine in humans and rats. As input, human and rat PBPK models of azathioprine are developed and in vitro toxicity data are analyzed Fig. . ExpliciHere, the development of PICD—an integrative multiscale approach—is shown. The application of PICD allows predicting in vivo drug response by integrating multiple levels of biological organization, thereby using whole-body PBPK models at the organism level to couple interstitial PK profiles at the organ level with in vitro toxicity data at the cellular level Fig. . The useIn an initial step, a drug-specific PBPK model is developed to identify in vivo doses that are directly related to in vitro drug exposure Fig. . The in To couple interstitial concentration–time profiles with in vitro toxicity data, in vivo doses are identified by PBPK simulations for intravenous drug administration such that the in vitro drug exposure in the assay equals the interstitial area under the curve in the liver at each experimental time point Fig. . Note thdlow, dmiddle and dhigh), which thus represent the in vivo equivalents to in vitro concentrations . Drug response values are next calculated and assigned to doses dlow, dmiddle and dhigh by linearly interpolating dose–response curves . Moreover, since validated PBPK models allow extrapolating PK simulations to different dosage regimens, PICD is applicable to predict drug response not only for the in vivo equivalent doses administered intravenously Fig.  but alsoFU) were used to parametrize the initial reference PBPK model for humans . Functional enrichment analysis was then applied to find significantly overrepresented terms (GO) and pathways  . Gene response values defined as absolute log2 fold change were calculated to quantify changes in significantly affected terms and pathways. Since the drug response values reflect the extent of activation or inhibition of functionally related genes in an in vivo situation, they were used to predict drug-induced cellular changes over time in both rats and humans.At the cellular level, in vitro toxicity data is required for PICD to predict in vivo drug response over time. Human and rat gene expression and enrichment analysis was performed in the same way. Time course gene expression profiles of primary human and rat hepatocytes from Open TG-GATEs were analyzed to obtain quantitative toxicity data of azathioprine  were used. The developed rat PBPK model  together with the in vitro toxicity data obtained in rat hepatocytes  served as input for the application of PICD to predict in vivo drug response in rats. When applying PICD on rats, a corresponding in vivo dose was determined for each of the nine in vitro treatments . In the in vivo rat study, the minimum toxic dose identified in a 4-week toxicity study was used as highest dose while the low and middle dose were selected by diluting the high dose with a factor of three and ten, respectively . In contrast, applying PICD obviously increased the concordance with in vivo measurements for all biological pathways and cellular processes . The correlation results for the individual pathways and cellular processes can be found in Data S2.In vivo drug response of cellular processes and biological pathways significantly regulated in rats (Data S1) were then predicted for all three doses orally administered in the in vivo rat study and were subsequently correlated with corresponding in vivo observations  to concentrations exposed in vitro (Table S2). Since the highest in vitro concentration was defined at the onset of toxicity  was expected to cause the experimentally observed toxic effects at the cellular level. The remaining two concentrations were further selected by diluting the toxic concentration by a factor of five and twenty-five, respectively  were coupled with interstitial PK profiles simulated in the liver for the same drug Fig. . To thisp < 0.05, one-sample t test). High responsive pathways induced by all equivalent in vivo doses after 1 day showed significant increases in drug response between 8 and 24 h  following administration of the in vivo equivalent doses Fig. , Fig. S6Here, PICD was applied to predict in vivo drug response in humans induced by in vivo doses derived from in vitro concentrations . To assess the increase in toxicity, drug response values calculated at different time points were correlated with global cytotoxicity values . For this correlation analysis, patient 17, who remained asymptomatic after receiving a heavy overdose, was not considered (Table S3).Analyzing patient-specific drug response profiles indicated an early increase in gene response for every patient , was regarded in a patient case study to investigate acute toxicity after single dosing of azathioprine.When regarding the various cases of acute azathioprine overdoses  by azathioprine in at least one time point Fig. e. In botConsidering the first process following acute azathioprine overdose, CHEK1 responsible for cell cycle arrest and repairing damaged DNA  was treated orally with 50 mg of azathioprine once daily over a period of 7 years Fig. a. DuringTo compare changes at the cellular level with observed clinical symptoms, temporal expression patterns following oral administration of azathioprine were predicted for fifteen genes that are associated with jaundice Fig. b. This wBiochemical markers measured shortly before, during and after the occurrence of jaundice indicated significant elevations Fig. d. LevelsIn this second patient case study, PICD provided the contextualization of simulated pharmacokinetics, predicted gene expression changes induced by the therapeutic dose and in vivo measurements of biochemical markers.In this study, the integrative multiscale approach PICD is presented, which allows a time-resolved description of drug-specific response profiles at the cellular level induced by in vivo drug administration at the organism level. Conceptually, PBPK models validated with blood plasma concentration–time data were used to simulate unbound drug concentrations in the interstitial space of the liver that in turn corresponds directly to the extracellular medium of in vitro experiments. Applied consistently, the systematic approach of using PBPK modeling for contextualization of in vitro toxicity data, which was exemplarily applied here for azathioprine, thus enables a generic workflow for the analysis of toxic effects of arbitrary drugs at patient level.Predicted in vivo drug response induced by the identified doses Fig. , Fig. S6To initially validate PICD, a rat PBPK model was built  by performing a cross-species extrapolation from humans to rats using the validated human PBPK model Fig. . This mePrediction of drug-induced cellular changes in response to interstitial PK profiles is not limited to hepatotoxicants. The compartmentalization of PBPK models enables the prediction of interstitial drug concentrations in multiple tissues or organs, for example, the heart. In this case, cellular changes obtained from compound-treated cardiomyocytes could likewise be used to get insights into adverse effects of cardiotoxic compounds in an in vivo context. Time series gene expression profiles from a toxicogenomics database  and cell viability values both measured in vitro  and the maximum velocity (vmax) were used to characterize the kinetic behavior of such active processes. Abundances of relevant enzymes and transporters in multiple compartments were quantified by using tissue-specific gene expression data . The latter parameters describing the physiology and anatomy of the organism are provided by the PBPK modeling software . Once sufficient model accuracy is reached, quantified in general by visual inspection, model validity can be confirmed by extrapolating the initial reference PBPK model to different dosage regimens or different patient populations. Note that all model parameters of the reference PBPK model were left unchanged for the validation step, except anthropometric parameters characterizing the specific patient subgroup. In this study, the quality of the developed PBPK model of azathioprine was assessed by comparing simulated PK data with different experimental concentration–time profiles from the literature .Raw data were downloaded from TG-GATEs . Furthermore, patient 19 was considered in a patient case study, thereby investigating acute toxicity after single dosing of azathioprine.PICD was used for different clinical cases of acute azathioprine overdose reported between 1995 and 2013  and pathways  identified for human and rat hepatocytes as well as correspondent p-values. The list of GO terms presents all terms except those removed by the filtering method (PDF 2465 kb)Data S1 Correlation results for significantly regulated pathways and cellular processes in rats. Correlation analysis was applied between predicted drug response profiles and in vivo measurements in rats for significantly regulated pathways and cellular processes. Correlation analyses were performed by calculating Pearson’s correlation coefficient r and the corresponding p-value p (PDF 283 kb)Data S2 Deleted GO terms. Go terms for the three sub-ontologies  that have been deleted after applying the presented filtering method of GO terms  (PDF 2417 kb)Data S3 Supplementary Materials (DOCX 37 kb)Schematic representation of a multiscale whole-body PBPK model. Schematic representation of a multiscale whole-body PBPK model including 15 different tissues and organs that are connected by blood flow. Sub-compartmentalization is exemplarily presented for a default compartment (PNG 1415 kb)Figure S1 PBPK model assessment. Simulated concentration-time profiles were compared to experimental data. Observed vs. predicted plots including RMSD value, coefficient of determination (R2), and the equation of the linear regression were generated for the reference and validated PBPK model. Simulated concentration-time profiles were compared to experimental data. Observed vs. predicted plots including RMSD value, coefficient of determination (R2), and the equation of the linear regression were generated for the reference and validated PBPK model (PNG 1248 kb)Figure S2 Rat PBPK model. Blood plasma concentration-time profiles of azathioprine and 6-mercaptopurine were simulated for rats  and for humans  after oral administration of 100 mg of azathioprine. The rat PBPK model of azathioprine was developed by considering rat-specific anatomy and physiology in the human PBPK model according to  In vitro gene expression data obtained in primary human hepatocytes. (B) In vitro gene expression data obtained in primary rat hepatocytes. (C) In vivo gene expression data obtained in rats (PNG 446 kb)Figure S4 Correlation between observed in vivo drug response and both predicted in vivo drug response and observed in vitro drug response. Predicted in vivo drug response (blue) induced by the identified toxic dose  Correlation of significantly affected KEGG pathways. (B) Correlation of significantly affected toxicity-related pathways. (C) Correlation of significantly affected biological processes. (D) Correlation of significantly affected cellular components. (E) Correlation of significantly affected molecular functions. (F) Correlation of genes considered in both case studies (PNG 1750 kb)Figure S5 Predicted in vivo drug response. In vivo drug responses of significantly affected GO terms and toxicity-related pathways (Benjamini-Hochberg corrected p < 0.01) following in vivo drug administration of dlow, dmiddle, and dhigh. The color scale depicts predicted in vivo drug responses. (A) Go terms of biological processes. (B) Go terms of cellular components. (C) Go terms of molecular functions. (D) Toxicity-related pathways (PNG 725 kb)Figure S6 Predicted in vivo drug response of DNA replication. (A) Drug response map exemplarily shown for DNA replication reflecting time- and dose-dependent effects following administration of azathioprine at dose levels dlow, dmiddle and dhigh (black lines). The color scale depicts predicted in vivo drug responses. (B) Predicted in vivo drug response over time induced by doses dlow, dmiddle and dhigh (PNG 147 kb)Figure S7 Predicted in vivo cytotoxicity over time. In vivo cytotoxicity values over time induced by the therapeutic dose were predicted for both replicates (gray area). The mean cytotoxicity values are shown as solid line (PNG 18 kb)Figure S8 Workflow for PBPK model development and validation. After parametrizing compound-specific properties and organism-specific parameters in the reference PBPK model, the model quality is evaluated by comparing simulated drug concentrations with experimental data from literature. If a sufficient model accuracy has been reached, a subsequent validation step enables reliable model extrapolations. Amongst others, this validation step ensures accurate predictions of concentration-time profiles in various compartments. Otherwise, the PBPK model is revised in a refinement step thereby adjusting key model parameters or adding more active transport processes or metabolizing reactions to improve the description of physiological processes governing the fate of the considered compound within the body (PNG 184 kb)Figure S9 Filtering gene ontology terms. The presented graph illustrates an exemplary subgraph of the complete GO graph. In total, four biological processes were identified as significantly enriched (blue and green nodes). After applying the filtering procedure, three terms were filtered out (blue nodes) while one remained for further analysis (green). Note that the green node represents the highest specialization (PNG 754 kb)Figure S10 Toxicity-related biological pathways. Symbols as well as human and rat Entrez IDs for 370 genes showing high response to toxic compounds were grouped in thirteen different biological pathways. The genes and the functional gene grouping terms were taken from the Human Molecular Toxicology PathwayFinder RT2 ProfilerTM PCR Array . Rat Entrez IDs were identified through the use of QIAGEN’s Ingenuity Pathway Analysis  (DOCX 41 kb)Table S1 Dose identification. In vitro concentration, exposure duration, resulting in vitro exposure, and identified in vivo dose for the specific treatments for rats and humans (DOCX 26 kb)Table S2 Clinical cases of acute azathioprine overdose. Anthropometric parameters , administered dose, and observed symptoms including assigned Poisoning Severity Scores (PSS)  (DOCX 27 kb)Table S4 Interaction network. Interactions between genes involved in DNA damage and repair processes. The interactions were identified through the use of QIAGEN’s Ingenuity Pathway Analysis  (DOCX 26 kb)Table S5 Genes related to jaundice. Symbol, Entrez gene name, type, human and rat Entrez identifier and assigned relation for all genes associated with jaundice. Functional classifications and assigned relations on jaundice were taken from QIAGEN’s Ingenuity Pathway Analysis  (DOCX 26 kb)Table S6 Below is the link to the electronic supplementary material.


This study investigated sensory and motor nerve excitability properties to elucidate the development of diabetic neuropathy. A total of 109 type 2 diabetes patients were recruited, and 106 were analyzed. According to neuropathy severity, patients were categorized into G0, G1, and G2+3 groups using the total neuropathy score-reduced (TNSr). Patients in the G0 group were asymptomatic and had a TNSr score of 0. Sensory and motor nerve excitability data from diabetic patients were compared with data from 33 healthy controls. Clinical assessment, nerve conduction studies, and sensory and motor nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. In the G0 group, sensory excitability testing revealed increased stimulus for the 50% sensory nerve action potential (P<0.05), shortened strength-duration time constant (P<0.01), increased superexcitability (P<0.01), decreased subexcitability (P<0.05), decreased accommodation to depolarizing current (P<0.01), and a trend of decreased accommodation to hyperpolarizing current in threshold electrotonus. All the changes progressed into G1 (TNSr 1–8) and G2+3 (TNSr 9–24) groups. In contrast, motor excitability only had significantly increased stimulus for the 50% compound motor nerve action potential (P<0.01) in the G0 group. This study revealed that the development of axonal dysfunction in sensory axons occurred prior to and in a different fashion from motor axons. Additionally, sensory nerve excitability tests can detect axonal dysfunction even in asymptomatic patients. These insights further our understanding of diabetic neuropathy and enable the early detection of sensory axonal abnormalities, which may provide a basis for neuroprotective therapeutic approaches. Type 2 diabetes mellitus (DM) is an alarming health concern worldwide . Among iDespite recent evidence suggesting that intensive therapy might reduce the risk of developing diabetic neuropathy, once it has developed, even strict glycemic control cannot reverse neuropathic symptoms and pathological changes . This faMany aspects of the pathogenesis of diabetic neuropathy remain to be explored, but recently, a number of well conducted studies have broadened our understanding on the subject –9. AlthoNerve excitability testing is a useful tool to provide further understanding regarding the pathogenesis of diabetic neuropathy. Previously, we demonstrated that the test can provide valuable electrophysiological data that added to our understanding of how diabetes causes dysfunction in motor nerves. It was also able to detect motor axonal dysfunction in diabetic patients even before the onset of diabetic neuropathy . NeverthClinical assessments, conventional nerve conduction studies (NCS), and nerve excitability testing were performed in patients with type 2 DM. All the patients met the American Diabetic Association criteria for diabetes diagnosis . PatientStandard neurological examinations were performed for all patients. To quantify the neuropathic symptoms, the TNSr was obtained in each patient . The redConventional NCSs were performed for all subjects. To determine the diagnostic utility of sensory nerve excitability test in early diabetes, we compared nerve excitability test parameters of HCs with patients who had yet to develop clinically relevant NCS changes for diabetic neuropathy, as defined in criterion 3 of the American Academy of Neurology diagnostic criteria . RoutineNerve excitability studies were performed by stimulating the nerve median at the wrist according to previously described protocols, with skin temperature over the wrist maintained at least 32.0 degrees Celsius , 17. PaiThe nerve excitability protocol incorporated the following recordings: 1) a stimulus response (SR) curve; 2) strength-duration (SD) relationship which also determined rheobase and strength-duration time constant (SDTC); 3) threshold electrotonus (TE) utilizing subthreshold 100-ms polarizing currents in both depolarizing  and hyperpolarizing  directions to change the potential difference across the internodal membrane; and 4) recovery cycle (RC) using a paired pulse paradigm with a supramaximal conditioning stimulus followed by a test stimulus at interstimulus intervals from 2 to 200 ms. Superexcitability was measured as the maximal threshold reduction, and subexcitability as the maximal threshold increase after an interstimulus interval of 10 ms.Nerve excitability data of diabetic patient groups and HCs were compared with one-way ANOVA with post hoc analysis. Correlation studies were performed with Pearson R. Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows version 21 .We obtained adequate sensory nerve excitability testing results from 109 patients. Of these, 2 were excluded for having carpal tunnel syndrome, and 1 was excluded for having comorbid myopathy . The demA summary of conventional NCS results is shown in Sensory and motor axonal excitability indices in patients are listed in Comparison of sensory and motor SR curve is shown in The sensory and motor SD relationship can be seen in the threshold charge vs stimulus width plot in The results of RC are shown in In agreement with the previous study by Sung et al., 2012, the motor RC curve showed no significant difference in superexcitability. The motor subexcitability was significantly decreased in the G1 group  and worsened as the disease progressed , a change confirmed by one-way ANOVA between groups . The motor mean RRP also showed a trend of prolongation in the late stage (G2+3 group: 3.47±0.12).The plotting of sensory and motor TE is shown in TE in motor axons showed a gradual “fanning-in” in later stages of diabetes both toward depolarizing and hyperpolarizing current. “Fanning-in” toward depolarizing current was seen in TEd(peak) showing more significant decrement in the G2+3 group . In addition, “fanning-in” toward hyperpolarizing current could be seen as TEh (90–100 ms) showing changes in the G2+3 group . One-way ANOVA results were consistent with “fanning-in” in TEd(peak)  and TEh(90–100 ms)  across groups.To assess the early changes in sensory nerve excitability indices of patients prior to developing conventional NCS changes, we compared the sensory excitability indices of normal controls (n = 33) with patients who had not yet developed clinically relevant NCS changes for diabetic neuropathy (n = 78) using an unpaired t-test. There are several significant differences between HCs and the patients in their sensory nerve excitability indices , includiThe correlations between HbA1c and nerve excitability parameters were investigated in patients who had not yet developed clinical neuropathy. The results of the analysis show correlations between HbA1c level and motor subexcitability  as previously reported ; sensoryFurthermore, in all diabetic patients, longer duration since diabetic diagnosis was correlated with increased latency in sensory  and motor axons , and increased motor TEh(90–100 ms) To further dissect the relationship between sensory and motor axons in the same DM patient, the correlation between paired sensory and motor excitability parameters was also evaluated: sensory superexcitability was statistically correlated to motor superexcitability   and motoThis study is the first to explore the progression of diabetic neuropathy in sensory axons in asymptomatic patients with severe diabetic neuropathy, using an array of examinations including standard neurological examinations to quantify the neuropathic symptoms, TNSr, and nerve excitability testing. Substantial insights into the underlying pathophysiological mechanisms were obtained utilizing these tests. The findings suggest that sensory axons developed nerve dysfunction prior to and in a different fashion than that of motor axons. These striking results enable us to detect abnormalities in sensory axons at asymptomatic stages. In the following section, the mechanisms involved and underlying voltage-gated ion channel functions that may contribute to the differences between sensory and motor axons and the phenotype of diabetic neuropathies will be discussed.+/K+ pump, culminating in distal axonal degeneration [Abnormal excitability parameters developed over the course of the disease, reflecting gradual progression from metabolic alteration, to impairment of ion conductances, to further structural defects including dysfunction of the Naneration . (Fig 5A+/K+ pump or nodal Na+ channel impairment in the beginning of diabetic neuropathy. In contrast to motor axons, only increased stimulation threshold and decreased subexcitability were noted in G0. Sensory axons are liable to damage caused by hyperglycemia and mitochondrial dysfunction [This study indicates that sensory axons develop axonal dysfunction earlier than motor axons: more sensory nerve excitability parameters were changed in the early stage in the G0 cohort, reflecting Nafunction . In G2+3+/K+ pump [Hofmeijer and colleagues found that sensory axons were more vulnerable to ischemia and hyperglycemic hypoxia than motor axons, presumably due to the greater sensory nerve dependency on the Na/K+ pump . Pump ac/K+ pump . It has /K+ pump , 21. By In sensory axons of DM patients, right shifting of the SR-curve, shortening of SDTC, down-shifting of RC curve, together with a trend of RRP shortening and TE “fanning-out,” suggested that the sensory axon is possibly in a state of hyperpolarization . These c+ conductances in sensory axons, which could produce hyperpolarization. Reduction of nodal Na+ permeability has been observed in later stages of diabetic neuropathy in animal models [+ permeability might be due to progressive redistribution of nodal Na+ channels across the paranodal barrier into the paranodal and internodal domains [+ channel expression might also be affected in DM by post-translational modification [Sensory axons might be hyperpolarized at the early stage of DM due to the reduction of Nal models –26. This domains . Na+ chafication .+ conductance in diabetes. Transient Na+ channels were probably also affected, as a trend of shortening in RRP was observed as the disease progressed, which could be responsible for the parathesia that patients suffer.This study has shown that SDTC was progressively shortened, reflecting a decrement of persistent Na+/K+ pump activity [The motor nerve excitability dysfunction seen in this study are in agreement to those previously reported , 28, 29.activity , 29. Theactivity . Nerve sactivity , 31.Previous studies found evidence that glycemic control is associated with changes in axonal properties , 32–34. Correlation between numerous sensory and motor excitability parameters in the paired sensory and motor study confirmed concurrent pathological sensori-motor axonal changes in the same patients. While correlation between sensory superexcitability and motor superexcitability suggested that nodal and internodal potassium channels are affected in the same patients, the correlation between sensory subexcitability and motor subexcitability suggested slow potassium channels in the paranodal region might also be similarly affected . PreviouThis study revealed that sensory nerve excitability indices showed axonal dysfunction earlier than motor. Sensory nerve excitability testing could be a potential screening tool for the early detection of peripheral nerve involvement. Among all parameters, sensory superexcitability appeared to be the most sensitive in the detection of early sensory axonal dysfunction. Clearly, more studies are needed to further establish the diagnostic utility of sensory excitability indices in diabetic neuropathy. Earlier detection of sensory axonal dysfunction would prompt earlier neuroprotection for diabetic neuropathy. Taken together, insights from this study provide a basis for new therapeutic approaches aimed at delaying or reversing diabetic neuropathy with the potential to further change clinical practice such that early neuroprotection can be initiated.