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RIPPLY2 is an essential part of the formation of somite patterning during embryogenesis and in establishment of rostro-caudal polarity. Here, we describe three individuals from two families with compound-heterozygous variants in RIPPLY2 (NM_001009994.2): c.238A > T, p.(Arg80*) and c.240-4 T > G, p.(?), in two 15 and 20-year-old sisters, and a homozygous nonsense variant, c.238A > T, p.(Arg80*), in an 8 year old boy. All patients had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of SCDO6 including scoliosis, mild facial asymmetry, spinal spasticity and hemivertebrae. The nonsense variant can be classified as likely pathogenic based on the ACMG criteria while the splice variants must be classified as a variant of unknown significance. With this report on two further families, we confirm RIPPLY2 as the gene for SCDO6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum., (© 2021 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

During vertebrate embryonic development, early skin, muscle, and bone progenitor populations organize into segments known as somites. Defects in this conserved process of segmentation lead to skeletal and muscular deformities, such as congenital scoliosis, a curvature of the spine caused by vertebral defects. Environmental stresses such as hypoxia or heat shock produce segmentation defects, and significantly increase the penetrance and severity of vertebral defects in genetically susceptible individuals. Here we show that a brief exposure to a high osmolarity solution causes reproducible segmentation defects in developing zebrafish (Danio rerio) embryos. Both osmotic shock and heat shock produce border defects in a dose-dependent manner, with an increase in both frequency and severity of defects. We also show that osmotic treatment has a delayed effect on somite development, similar to that observed in heat shocked embryos. Our results establish osmotic shock as an alternate experimental model for stress, affecting segmentation in a manner comparable to other known environmental stressors. The similar effects of these two distinct environmental stressors support a model in which a variety of cellular stresses act through a related response pathway that leads to disturbances in the segmentation process., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Several epidemiologic studies in humans have shown a relationship between pregestational obesity and congenital malformations in offsprings. However, there are no experimental evidence in animal models of obesity and pregnancy that reproduce the teratogenesis induced by this pathological condition., Objective: To evaluate the effect of monosodium glutamate-induced obesity on embryonic development., Methods: Female rats received subcutaneously (4 mg/g body weight) monosodium glutamate (MSG) solution or saline solution 0.9% (vehicle control) at days 2, 4, 6, 8, and 10 of life. At 90 days of age, all animals were mated, and on day 11 of pregnancy, the animals were killed. Biochemical variables (glucose, triglycerides, total cholesterol, and insulin) were determined in plasma of dams and embryo homogenates (DNA and protein content, advanced oxidation protein products). Embryos were evaluated for malformations, crown-rump length, and somite number., Results: Obese rats presented higher triglyceride levels as compared to nonobese rats. Increased proportion of malformed embryos, decreased crown-rump length, somite number, DNA, and protein content were observed in offspring of obese rats., Conclusion: The model of obesity induced with MSG reproduces the maternal obesity-induced teratogenesis. The hypertriglyceridemia observed in MSG obese pregnant rats could be related to increased birth defect., (© The Author(s) 2016.)

Background: The reduced cost and improved efficiency of whole genome sequencing (WGS) is drastically improving the development of cats as biomedical models. Persian cats are models for Leber's congenital amaurosis (LCA), the most severe and earliest onset form of visual impairment in humans. Cats with innocuous breed-defining traits, such as a bobbed tail, can also be models for somite segmentation and vertebral column development., Methods: The first WGS in cats was conducted on a trio segregating for LCA and the bobbed tail abnormality. Variants were identified using FreeBayes and effects predicted using SnpEff. Variants within a known haplotype block for cat LCA and specific candidate genes for both phenotypes were prioritized by the predicted variant effect on the proteins and concordant segregation within the trio. The efficiency of WGS of a single trio of domestic cats was evaluated., Results: A stop gain was identified at position c.577C > T in cat AIPL1, a predicted p.Arg193*. A c.5A > G variant causing a p.V2A was identified in HES7. The variants segregated concordantly in a Persian - Japanese bobtail pedigree. Over 1700 cats from 40 different breeds and populations were genotyped for the AIPL1 variant, defining an allelic frequency in only Persian -related breeds of 1.15%. A sub-set of cats was genotyped for the HES7 variant, supporting the variant as private to the Japanese bobtail breed. Approximately 18 million SNPs were identified for application in cat research. The cat AIPL1 variant would have been considered a high priority variant for evaluation, regardless of a priori knowledge from previous genetic studies., Conclusions: This study represents the first effort of the 99 Lives Cat Genome Sequencing Initiative to identify disease--causing variants in the domestic cat using WGS. The current cat reference assembly is efficient for gene and variant identification. However, as the feline variant database improves, development of cats as biomedical models for human disease will be more efficient, providing an alternative, large animal model for drug and gene therapy trials. Undiagnosed human patients with early-onset blindness should be screened for this AIPL1 variant. The HES7 variant should further calibrate the somite segmentation clock.

mib(nn2002), found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mib(nn2002) is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mib(nn2002) homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mib(nn2002) homozygotes partially rescued the anterior somite specification. In the process of characterizing mib(nn2002) mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mib(nn2002) has a chromosomal deletion with the size of about 9.6 Mbp.

Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis., (Copyright © 2015. Published by Elsevier Ltd.)

Although Mayer-Rokitansky-Küster-Hauser syndrome is a rare condition with a reported incidence of 1/4500 female live births, it represents the second most common cause of primary amenorrhea and has psychologically devastating consequences. The radiologist plays a pivotal role in both making the accurate initial diagnosis of this condition and assessing findings that may contribute to treatment planning. The purpose of this article is to provide an overview of the capabilities of ultrasound and magnetic resonance imaging (MRI) for the diagnosis and management of this syndrome with emphasis on the relevant clinical and surgical findings and to describe potential associated abnormalities and differential diagnosis., (Copyright © 2013 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Objective: To analyze the histologic and immunohistochemical structure of uterine rudiments focusing on the endometrium in a representative group of patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome compared with a control group., Design: Prospective comparative study., Setting: University hospital., Patient(s): Forty-two patients with MRKH syndrome and 13 control subjects., Intervention(s): Representative biopsies or whole uterine rudiments were removed during surgery and processed by a standardized procedure including immunohistochemical staining and analysis., Main Outcome Measure(s): Histologic structure, tissue types, hormone receptor expression, endometrial proliferative capacity, and type in correlation with cycle phase., Result(s): Twenty-two of the uterine rudiments showed a duct-like structure or small cavity, 17 of which contained endometrial epithelium and CD10-positive stroma. All rudiments contained an intact myometrial layer. Tubal epithelium and stroma were found in three rudiment samples. No significant differences were observed with regard to estrogen receptor (ER) or progesterone receptor (PR) expression in endometrium or myometrium. Interestingly, patients showed predominantly basalis-like endometrium with specific lack of CD90 expression and significantly lower proliferation compared with controls., Conclusion(s): All typical uterine tissues can be found in uterine rudiments of patients with MRKH syndrome. Expression of hormonal receptors in the latter and controls did not differ significantly. Endometrium shows predominantly basalis-like features in uterine rudiments., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To analyze magnetic resonance imaging (MRI) findings of Müllerian remnants in young females clinically suspected of Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome in a primary amenorrhea workup., Materials and Methods: Fifteen young females underwent multiplanar T2- and transverse T1-weighted MRI at either a 1.5T or 3.0T MR imager. Two gynecologic radiologists reached consensus decisions for the evaluation of Müllerian remnants, vagina, ovaries, and associated findings., Results: All cases had bilateral uterine buds in the pelvic cavity, with unilateral cavitation in two cases. The buds had an average long-axis diameter of 2.64 ± 0.65 cm. In all cases, bilateral buds were connected with fibrous band-like structures. In 13 cases, the band-like structures converged at the midline or a paramedian triangular soft tissue lying above the bladder dome. The lower one-third of the vagina was identified in 14 cases. Fourteen cases showed bilateral normal ovaries near the uterine buds. One unilateral pelvic kidney, one unilateral renal agenesis, one mild scoliosis, and three lumbar sacralization cases were found as associated findings., Conclusion: Typical Müllerian remnants in MRKH syndrome consist of bilateral uterine buds connected by the fibrous band-like structures, which converge at the midline triangular soft tissue lying above the bladder dome.

Objective: To describe the spectrum and frequency of renal and urological abnormalities in a cohort of patients with uterine anomalies., Material and Methods: We conducted an ethics committee approved review of 164 women referred to the imaging department from a specialist gynaecological anomaly clinic. Imaging acquired over 8 years was assessed and this was sufficient for assessment of the morphology of the gynaecological and renal tracts in 113 patients., Results: Absent unilateral kidney was the most common abnormality (31.8%), most frequent in Type 3 uterine anomalies. Absent kidneys occurred in other anomaly types at lower frequency. Pelvic ureteric remnants were found in 9 of 36 patients with absent kidneys; these inserted ectopically, most commonly into the vagina. Urological abnormalities were found in 11 patients with two kidneys, including ectopic ureters, scarred kidneys and dysplastic kidney. Obstructed hemivaginas were associated commonly but not invariably with an absent kidney., Conclusion: Previously unreported renal and urological abnormalities have been described in patients with congenital uterine anomalies. These have significant clinical impact. Ectopic ureters can cause incontinence, and potentially cause pain and become infected. Knowledge of the ureteric course, including ureteric remnants, is essential before complex laparoscopic and vaginal surgery., (Copyright © 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

The Mayer-Rokitansky-Kuster-Hauser is a rare congenital anomaly characterized by lack of vaginal and uterine development variable and normal ovaries. It results from agenesis or hypoplasia Müller duct system. Cervicovaginal agenesis as part of the complex syndrome, is even rarer. We report two cases: adolescent patient with primary amenorrhea, cervicovaginal agenesis and chronic pelvic pain, and a 28-year-old patient with primary amenorrhea, congenital absence of uterus and vagina.

This study has documented the major types of lineage progenitor cells at the second level of cell differentiation after the establishment of the primary germ layers in ectopic human embryos in vivo. These correspond to stages 8 and 9 of embryogenesis (weeks 3-4) in the Carnegie collection. The aim of this study was to provide images of fine structure of tissue progenitor cells to compare them with current imaging of their equivalent stem cells identified using fluorescent stem cell markers. These include neural, mesenchymal, endodermal, ectodermal (epidermal) and haematopoietic progenitor cells, including those for amniotic, yolk sac and chorionic tissues that are used in current stem cell research. Neural induction by the notochord has been imaged. This study should give valuable clues to understand the pattern of cell differentiation of embryonic stem cells (ESC) in vitro, which are more or less mimicked in ESC colonies, embryoid bodies and neurospheres as documented in the literature. The fine structure of week-3 and week-4 human ectopic embryos is presented to demonstrate progenitor tissue cells that will eventually form the brain, spinal cord, skin, gut, heart, blood, muscle, bone and other tissues of the human body later on in development. These images should help stem cell researchers using fluorescent markers and other techniques to identify embryonic and adult stem cells in culture., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Cell growth, proliferation, differentiation and survival are influenced by the availability of oxygen. The effect of hypoxia on embryonic cells and the underlying molecular mechanisms to maintain cellular viability are still poorly understood. In this study, we show that hypoxia during Xenopus embryogenesis rapidly leads to a significant developmental delay and to cell apoptosis after prolonged exposure. We provide strong evidence that hypoxia does not affect somitogenesis but affects the number of mitotic cells and muscle-specific protein accumulation in somites, without interfering with the expression of MyoD and MRF4 transcription factors. We also demonstrate that hypoxia reversibly decreases Akt phosphorylation and increases the total amount of the translational repressor 4E-BP, in combination with an increase of the 4E-BP associated with eIF4E. Interestingly, the inhibition of PI3-kinase or mTOR, with LY29002 or rapamycin, respectively, triggers the 4E-BP accumulation in Xenopus embryos. Finally, the overexpression of the non-phosphorylatable 4E-BP protein induces, similar to hypoxia, a decrease in mitotic cells and a decrease in muscle-specific protein accumulation in somites. Taken together, our studies suggest that 4E-BP plays a central role under hypoxia in promoting the cap-independent translation at the expense of cap-dependent translation and triggers specific defects in muscle development.

Background: The aim of this retrospective study was to describe the spectrum of genital and associated malformations in women with Mayer-Rokitansky-Küster-Hauser syndrome using evaluated diagnostic procedures and the Vagina Cervix Uterus Adnex - associated Malformation classification system (VCUAM)., Methods: 290 women with MRKH syndrome were clinically evaluated with using clinical examinations, abdominal and perineal/rectal ultrasound, MRI, and laparoscopy., Results: Classification of female genital malformation according to the Vagina Cervix Uterus Adnex - associated Malformation classification system was possible in 284 women (97.9%). Complete atresia of Vagina (V5b) and bilateral atresia of Cervix (C2b) were found in 284 patients (100%). Uterus: bilateral rudimentary or a plastic uterine horns were found in 239 women (84.2%). Adnexa: normal Adnexa were found in 248 women (87.3%). Malformations: associated malformations were found in 126 of 282 evaluable women (44.7%), 84 women (29.6%) had malformations of the renal system. Of 284 women with Mayer-Rokitansky-Küster-Hauser syndrome 212 women (74.7%) could be classified as V5bC2bU4bA0. The most frequent classification was V5bC2bU4bA0M0 (46.8%) diagnosed in 133 of 284 women., Conclusions: Complete atresia of vagina and cervix were found in all patients, variable malformations were found with uterus and adnexa. A variety of associated malformations were present, predominantly of the renal system. It is therefore recommended that all patients with genital malformations should be evaluated for renal abnormalities.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

The objective of this study was to investigate the embryotoxicity of diclofenac. Zebrafish (Danio rerio) embryos at 12 hpf were treated with different dosages of diclofenac (0-2,000 ppm) for different time courses (12-72 hr). Results showed no evident differences in survival rates or morphological changes between the mock-treated control (0 ppm) zebrafish embryos and those with 1-ppm diclofenac-exposure (12-24, 12-36 hpf). In contrast, after higher doses (5 and 10 ppm) of exposure, embryos displayed some defective phenotypes, including malformed somite boundary, a twisted body axis, and shorter body length. In addition, diclofenac-treated embryos exhibited significantly reduced frequencies of spontaneous in-chorion contractions in comparison with mock-control littermates (mock-control: 13.20 ± 2.24 vs. 5-10 ppm diclofenac: 6.66 ± 1.35-3.03 ± 1.84). Subtle changes were easily observed by staining with specific monoclonal antibodies F59 and phalloidin to detect morphological changes in muscle fibers and formation of F-actin, respectively. Our data show that diclofenac treatment disturbs actin organization and muscle fiber alignment, thus causing malformed somite phenotypes., (© 2011 Wiley-Liss, Inc.)

Basement membranes (BMs) are a complex, sheet-like network of specialized extracellular matrix that underlies epithelial cells and surrounds muscle cells. They provide adherence between neighboring tissues, permit some flexibility of these adherent structures, and can act as a store for growth factors and as a guide for cell migration. The BM is not just a static structure; its deposition and remodeling are important for many processes including embryonic development, immune response, and wound healing. To date, dysfunction in BM deposition or remodeling has been linked to many human congenital disorders and diseases, affecting many different tissues in the body, including malformations, dystrophies, and cancer. However, many questions remain to be answered on the role BM proteins, and their mutations, play in the pathogenesis of human disease. In recent years, the zebrafish (Danio rerio) has emerged as a powerful animal model for human development and disease. In the first part of this chapter, we provide an overview of described defects caused by BM dysfunction in zebrafish, including development and function of notochord, muscle, central nervous system, skin, cardiovascular system, and kidney. In the second part, we will describe details of methods used to visualize and assess the structure of the BM in zebrafish, and to functionally analyze its different components., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Inositol phosphate (IP) kinases constitute an emerging class of cellular kinases linked to multiple cellular activities. Here, we report a previously uncharacterized cellular function in Hedgehog (Hh) signaling for the IP kinase designated inositol hexakisphosphate kinase-2 (IP6K2) that produces diphosphoryl inositol phosphates (PP-IPs). In zebrafish embryos, IP6K2 activity was required for normal development of craniofacial structures, somites, and neural crest cells. ip6k2 depletion in both zebrafish and mammalian cells also inhibited Hh target gene expression. Inhibiting IP(6) kinase activity using N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine (TNP) resulted in altered Hh signal transduction. In zebrafish, restoring IP6K2 levels with exogenous ip6k2 mRNA reversed the effects of IP6K2 depletion. Furthermore, overexpression of ip6k2 in mammalian cells enhanced the Hh pathway response, suggesting IP6K2 is a positive regulator of Hh signaling. Perturbations from IP6K2 depletion or TNP were reversed by overexpressing smoM2, gli1, or ip6k2. Moreover, the inhibitory effect of cyclopamine was reversed by overexpressing ip6k2. This identified roles for the inositol kinase pathway in early vertebrate development and tissue morphogenesis, and in Hh signaling. We propose that IP6K2 activity is required at the level or downstream of Smoothened but upstream of the transcription activator Gli1.

Three new minor components, the pyridoacridine alkaloids 1-hydroxy-deoxyamphimedine (1), 3-hydroxy-deoxyamphimedine (2), debromopetrosamine (3), and three known compounds, amphimedine (4), neoamphimedine (5) and deoxyamphimedine (6), have been isolated from the sponge Xestospongia cf. carbonaria, collected in Palau. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-6 were evaluated in a zebrafish phenotype-based assay. Amphimedine (4) was the only compound that caused a phenotype in zebrafish embryos at 30 muM. No phenotype other than death was observed for compounds 1-3, 5, 6.

Somite is sequentially generated in a head-to-tail order by segmentation of the mesenchymal tissue called presomitic mesoderm (PSM). The segmentation occurs periodically at the anterior end of the PSM, and this periodic segmentation has been suggested to be regulated by a molecular clock. In mouse PSM, the segmentation-related genes change their expression every 120 minutes, and this cyclic expression is essential for regular somite segmentation. In this study, a molecular mechanism of segmentation clock involving Wnt and Delta-Notch signaling pathways is modeled, and reality of the model structure is investigated through simulating biological findings. One dimensional array of the cellular clock models is constructed to simulate spatio-temporal dynamics of the gene-expressions in the PSM. The simulation result suggests that the Wnt gradient across the PSM is involved in the dynamics under concern.

Spina bifida, or failure of the vertebrae to close at the midline, is a common congenital malformation in humans that is often synonymous with neural tube defects (NTDs). However, it is likely that other etiologies exist. Genetic disruption of platelet-derived growth factor receptor (PDGFR) alpha results in spina bifida, but the underlying mechanism has not been identified. To elucidate the cause of this birth defect in PDGFRalpha mutant embryos, we examined the developmental processes involved in vertebrae formation. Exposure of chick embryos to the PDGFR inhibitor imatinib mesylate resulted in spina bifida in the absence of NTDs. We next examined embryos with a tissue-specific deletion of the receptor. We found that loss of the receptor from chondrocytes did not recapitulate the spina bifida phenotype. By contrast, loss of the receptor from all sclerotome and dermatome derivatives or disruption of PDGFRalpha-driven phosphatidyl-inositol 3' kinase (PI3K) activity resulted in spina bifida. Furthermore, we identified a migration defect in the sclerotome as the cause of the abnormal vertebral development. We found that primary cells from these mice exhibited defects in PAK1 activation and paxillin localization. Taken together, these results indicate that PDGFRalpha downstream effectors, especially PI3K, are essential for cell migration of a somite-derived dorsal mesenchyme and disruption of receptor signaling in these cells leads to spina bifida.

Background: Nitric oxide (NO), produced by the nitric oxide synthase family of enzymes, mediates multiple signaling functions, and when unchecked, NO causes pathological damage. Exposure of embryos to a variety of teratogens, including carbon monoxide (CO), has been shown to increase reactive intermediates, such as NO, and recent work showed that either the excess or absence of NO caused morphological defects. While endogenous NO is known to regulate many adult tissues, its role during embryonic organogenesis and/or in mediating responses to teratogen exposure has not been explored., Methods: We have examined here the presence of NO during normal chick embryonic organogenesis, and investigated the teratogenicity of NO through the application of sodium nitroprusside (SNP), which mimics NO overproduction, and NG-monomethyl-L-arginine (L-NMMA), which inhibits endogenous NOS activity., Results: Topical treatment with SNP or L-NMMA for 18 h resulted in morphological defects, specifically in the neural tube and somites, which corresponded to sites of altered apoptosis. The location of NO was histochemically correlated with the observed morphological defects. Coadministration of SNP or L-NMMA with CO showed functional coregulation and interaction between NO and CO in chick embryonic development., Conclusions: Our results showed that regulation of NO is essential for normal axial development, that sites of altered NO expression correlate to those of altered apoptosis and dysmorphogenesis, and that CO coadministration resulted in a rectification of normal NO expression. Collectively, these results suggest that alteration in endogenous NO/CO signaling is responsible, at least in part, for the observed NO-induced teratogenesis., (2007 Wiley-Liss, Inc.)

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of alveolar RMS by influencing the commitment and differentiation of the myogenic cell lineage.

Over the past decade, the Notch signaling pathway has been shown to be crucially important for normal metazoan development and to be associated with several human inherited and late onset diseases. The realization that altered Notch signaling contributes at various levels to human disease lead in May to the first meeting dedicated solely to Notch signaling in vertebrate development and disease in Madrid, Spain. Hosted by the Cantoblanco Workshops on Biology and organized by Tom Gridley, José Luis de la Pompa and Juan Carlos Izpisúa Belmonte, the meeting covered diverse aspects of this important signaling pathway.

On the basis of two recently studied human fetuses and the historical records and remnant 19th century skeletons in the Museum Vrolik in Amsterdam, we have begun an analysis of an unusual form of somite dysgenesis. This disorder includes vertebral and costal segmentation defects with or without (distal) limb malformation and deformities, anogenital anomalies, unusual colonic atresia, abdominal wall and diaphragmatic defect, Central nervous system abnormality with large head, and severe neurohypotrophic lower limb deformities. This study suggests the existence of an axial vertebral/costal dysgenesis complex with apparently or nearly normal number of cervical vertebrae. There also is some overlap with lumbosacral agenesis but different from the autosomal recessive entities Jarcho-Levin syndrome or spondylocostal dysostosis. To date, no associated heart defects have been noted.

Background/purpose: Recent biological studies have elucidated the molecular mechanism of muscle development, in which various regulatory factors (myogenic regulatory factors [MRFs]) play key roles during embryogenesis. To investigate the development of anorectal malformations (ARMs), we studied MRF expressions in myogenic cells in the pelvic floor using murine embryos affected with ARM., Methods: Anorectal malformation embryos were obtained from the 10.5th embryonal day (E10.5) to the 7.0th postnatal day (D7.0) in a natural mutant strain (Sd/+, RSV/Le). Serial frozen sections were prepared for immunohistochemistry using specific antibodies to M-cadherin, myoD, Myogenin, myosin heavy chain, and alfa-actin molecule., Results: In normal mice, embryonal caudal somites differentiated into myogenic stem cells and migrated to the pelvic floor between E11.0 and E14.0. In the ARM mice, however, caudal somites were irregularly arranged and MRF expressions in myogenic cells were markedly decreased in the dorsocaudal region at E11.5 to E13.0, leading to hypoplastic pelvic floor muscles., Conclusions: The maldevelopment of pelvic floor muscles in ARM is derived from a deficient supply of myogenic stem cells, with impaired MRF expression. These results suggest that myogenic stem cells, available from bone marrow contents, may be used for postnatal muscle regeneration to reinforce the pelvic floor muscle function in children with ARM.

Background: Notch signaling influences binary cell fate decisions in a variety of tissues. The Notch1 receptor is widely expressed during embryogenesis and is essential for embryonic development. Loss of global Notch1 function results in early embryonic lethality, but the cell type responsible for this defect is not known. Here, we identify the endothelium as the primary target tissue affected by Notch1 signaling., Methods and Results: We generated an endothelium-specific deletion of Notch1 using Tie2Cre and conditional Notch1(flox/flox) mice. Mutant embryos lacking endothelial Notch1 died at approximately embryonic day 10.5 with profound vascular defects in placenta, yolk sac, and embryo proper, whereas heterozygous deletion had no effect. In yolk sacs of mutant embryos, endothelial cells formed a primary vascular plexus indicative of intact vasculogenesis but failed to induce the secondary vascular remodeling required to form a mature network of well-organized large and small blood vessels, which demonstrates a defect in angiogenesis. These vascular defects were also evident in the placenta, where blood vessels failed to invade the placental labyrinth, and in the embryo proper, where defective blood vessel maturation led to pericardial and intersomitic hemorrhage. Enhanced activation of caspase-3 was detected in endothelial and neural cells of mutant mice, which resulted in enhanced apoptotic degeneration of somites and the neural tube., Conclusions: These findings recapitulate the vascular phenotype of global Notch1-/- mutants and indicate an essential cell-autonomous role of Notch1 signaling in the endothelium during vascular development. These results may have important clinical implications with regard to Notch1 signaling in adult angiogenesis.

The developmental and genetic etiology of most congenital vertebral malformation disorders remains unknown. The objective of this study was to evaluate and classify congenital vertebral defect cases into groupings based on developmental etiology for clinical genetic studies. This classification is intended to be distinct from but complementary to traditional groupings based on spinal curvature or progression. In the first step of this analysis, the authors identified 84 cases of vertebral segmentation disorders by radiologic screening and prospectively recruited 39 of these patients into a clinical genetic study. Next, the authors quantified the extent of contiguous defects and organized cases by craniocaudal localization. Finally, the authors used available clinical association data to identify syndromic and nonsyndromic subcategories, and identified a high rate of orthopaedic and neurologic associations in nonsyndromic patients. This type of analysis has identified subgroups of patients with multiple, contiguous segmental defects and orthopaedic associations that are particularly suitable for further genetic analysis.

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD., (Copyright 2004 Wiley-Liss, Inc.)

Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2(-/-) mice as a model for the developmental pathology of RS. Our results demonstrate that vertebral malformations in Ror2(-/-) mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2(-/-) mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2(-/-) mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2(-/-) mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome.

Background: Congenital axial skeletal defects affect two to three individuals per 1,000 live births. Without strong evidence for heritability, the cause is assumed to be multi-factorial. Carbon monoxide (CO), an increasingly prevalent environmental toxicant, is a potential environmental component in the etiology of these defects. The chick embryo is a useful model for the characterization and assessment of the mechanism(s) of action of basic developmental mechanisms., Methods: We have determined a critical period and dose for CO teratogenicity and established a model of CO-induced axial skeletal dysmorphogenesis in the chick embryo. The resulting phenotypes reveal a spectrum of axial skeletal defects ranging from minor defects of the vertebral canal and inter-vertebral discs, to thoraco-lumbar scoliosis, to a tailless phenotype reminiscent of caudal dysgenesis syndrome. These axial skeletal defects have been related to earlier developmental defects in somitogenesis, including errors in segmentation and epithehalization and the expression of the somitic epithelialization factor, Paraxis. We have examined patterns of cell death and apoptosis in CO exposed chick embryos to assess the target tissue(s) involved in the teratogenicity of CO., Results: With respect to the embryonic axis, the neural tube was found to be the most sensitive to CO-induced apoptosis, followed by the somitic mesoderm and Hensen's node., Conclusions: We hypothesize that the somitic defects and the resulting axial skeletal dysmorphogenesis are caused by disrupted neural tube or ectoderm functions related to somite formation and maintenance. We also hypothesize that CO-induced dysmorphogenesis at this critical period of somitogenesis is caused by the overabundance of CO acting endogenously as a cellular signal, while coincidentally exerting its influence as a toxicant of oxygen delivery or utilization.

The observation of elevated maternal plasma homocysteine concentration in pregnancies affected by neural tube defects (NTD) suggests that folate metabolism may be disturbed in NTD cases. In addition, studies on the chick embryo suggest that hyperhomocysteinaemia may contribute directly to the development of NTD. In order to test the hypothesis that homocysteine may cause NTD, we cultured mouse embryos in the presence of homocysteine thiolactone during the period of cranial neural tube closure. At doses of 0.5 mM or above, exposure to homocysteine thiolactone caused growth retardation, blisters and abnormalities of somite development. Despite the teratogenic effects of homocysteine we did not detect any increase in the incidence of neural tube defects. Neither was there an effect on the incorporation of thymidine into DNA, a potential marker of alterations in the folate or homocysteine/methionine cycles. These observations suggest that homocysteine is unlikely to be a direct cause of NTD in humans. Rather, the elevated levels of homocysteine in human NTD pregnancies may reflect a disturbance in folate-related metabolism.

Serial sections of 99 human embryos from Carnegie stages 8-23 were investigated and 38 graphic reconstructions were evaluated. At stage 9 somite 1 is of appreciable size and is separated from the otic disc, as also in the next several stages by rhombomeres and pharyngeal arches 3 and 4, thereby differing from the chick. At stage 10 somite 1 begins to differentiate into sclerotome and dermatomyotome. At stage 11 spinal neural crest begins to develop. At stage 12 parts of somites 1-4 are being transformed into the hypoglossal cell cord. It is stressed that the numbers of somites present at stages 9-12 are part of the definition of those stages. At stage 13 dense and loose zones begin to be detectable rostrally in the sclerotomes and also, although out of phase, in the perinotochord. Spinal ganglia begin to develop in phase with the somites. At stages 14-16 the maximum number of somites observed was 38-39 rather than 42-44, as usually given. Moreover, they did not extend to the tapered end of the trunk, which is not a (vertebrated) 'tail'. At stages 17-23 the maximum number of centra was 38-39, including coccygeal vertebrae 4-5. Although most of the somites appear during primary development, all of the spinal ganglia develop during secondary development (stages 13-18). The number of ganglia was at a maximum of 35 at stage 18, but was reduced to 32 already by stage 23. Important points confirmed in this study are that the number of occipital somites in the human is four, and that the level of final closure of the caudal neuropore is future somite 31, which represents approximately future sacral vertebra 2. The interpretation of relevant neural tube defects is discussed in the light of the findings. The ascensus of the conus medullaris during the fetal period is well established, but a concomitant ascent of the situs neuroporicus is proposed here, and has implications for defects that involve tethering of the spinal cord. The main results are integrated in comprehensive graphic representations of the levels and the interrelationships of (a) somites and centra, and (b) somites, neural crest, and spinal ganglia. These may aid in the elucidation of some frequently occurring anomalous conditions., (Copyright 2003 S. Karger AG, Basel)

A loss-of-function mutation in the mouse delta-like3 (Dll3) gene has been generated following gene targeting, and results in severe axial skeletal defects. These defects, which consist of highly disorganised vertebrae and costal defects, are similar to those associated with the Dll3-dependent pudgy mutant in mouse and with spondylocostal dysplasia (MIM 277300) in humans. This study demonstrates that Dll3(neo) and Dll3(pu) are functionally equivalent alleles with respect to the skeletal dysplasia, and we suggest that the three human DLL3 mutations associated with spondylocostal dysplasia are also functionally equivalent to the Dll3(neo) null allele. Our phenotypic analysis of Dll3(neo)/Dll3(neo) mutants shows that the developmental origins of the skeletal defects lie in delayed and irregular somite formation, which results in the perturbation of anteroposterior somite polarity. As the expression of Lfng, Hes1, Hes5 and Hey1 is disrupted in the presomitic mesoderm, we suggest that the somitic aberrations are founded in the disruption of the segmentation clock that intrinsically oscillates within presomitic mesoderm.

It has been known for some time that ablation of the neural tube and/or the notochord in the chick embryo leads to a massive wave of cell death in the adjacent somites. It is postulated that in the normal embryo, survival signals emanate from the neural tube and/or notochord that suppress apoptosis in the cells of the somites, except for a small population of sclerotome cells that are programmed to die naturally. In this study we show that axial ablation results in the death of sclerotome and not somitic neural crest cells, and we have examined the apoptotic response of these cells to the ablation. We show that several elements of the apoptotic cascade become detectable in somite cells in response to the withdrawal of survival signals. We demonstrate the down-regulation of bcl-2 protein in the somites adjacent to, and caudal to, the site of ablation, corresponding to the region that displays an elevated level of cell death. Although caspase-9 appeared to be activated in somites at all levels of the trunk, caspase-2 showed a clear response to the ablation of the axial structures. Removal of the neural tube and notochord produced an up-regulation of caspase-2 activity in somites in the region of the operation. Cleavage of two down-stream substrates of these caspases was examined. The cleavage of poly (ADP-ribose) polymerase (PARP) was apparent in somites at all levels of the trunk, and showed only a modest up-regulation after ablation. By contrast, the cleavage of DNA fragmentation factor (DFF45) showed a marked up-regulation in response to ablation, suggesting that this is a primary substrate for a caspase-dependent apoptotic mechanism. Evidence was also found for a caspase-independent mechanism, since the expression of apoptosis-inducing factor (AIF) was found to be very sensitive to, and up-regulated in somites by, axial ablation. Because the wave of apoptosis that is precipitated in somites by removal of the axial structures may be mediated by BMP-4, we examined the levels of BMP-4 in somites in response to axial ablation. BMP-4 expression was clearly up-regulated in somites adjacent to, or close to, the site of operation.

Cromakalim is a K(+) channel opener that causes smooth muscle relaxation by activating ATP-sensitive K(+) (K(ATP)) channels and producing membrane hyperpolarization. Cromakalim counteracts sulfonylurea-induced K(ATP) channel inhibition in adult cells, but little is known regarding its embryonic effects, alone or in combination with sulfonylureas. K(ATP) channels have been demonstrated in the embryo, but their role in normal and abnormal development is unknown. Early-somite mouse embryos were exposed for 24 hr in vitro to cromakalim at concentrations of 0 (Cntl), 1, 10, 100, 200, or 500 microM in 0.125% DMSO. Embryos were also exposed for 24 hr in vitro to a dysmorphogenic tolbutamide concentration (110 microg/ml) combined with a subdysmorphogenic concentration of cromakalim (1 microM). Embryos were evaluated for somite number, heart rate, malformations, and embryonic and yolk sac protein content. Embryos exposed to 1 microM cromakalim were similar to controls. Cromakalim exposure increased malformation rates at concentrations >/=200 microM, decreased heart rates at >/=10 microM, and decreased somite and protein values at 500 microM. Defects involved cranial neural tube, optic vesicle, heart, and somites. A malformation rate of 59% in embryos exposed to 110 microg/ml tolbutamide was reduced to 13% by adding 1 microM cromakalim to the culture medium. Heart rate, somite number, and protein values were also improved by combined exposure to cromakalim and tolbutamide compared with exposure to tolbutamide alone. These results support previous findings with diazoxide (K(+) channel opener) and chlorpropamide (sulfonylurea) and further suggest a potential role for K(ATP) channel effects in sulfonylurea-induced dysmorphogenesis., (Copyright 1999 Wiley-Liss, Inc.)

Environmental influences are known to produce segmental defects in a variety of organisms. In this paper we report upon segmental aberrations produced by brief heat shocks delivered to developing zebrafish embryos. The initial defects in the segmental pattern of somitic boundaries and motoneuron axon outgrowth were usually observed five somites caudal to the somite which was forming at the time of heat shock application. Segmental defects in zebrafish embryos exposed to a single heat shock treatment can occur in a periodic pattern similar to the multiple disturbances observed to occur in chick embryos. These data are discussed with regard to models involving cell cycle synchrony or 'clock and wavefront' schemes in the process of somitogenesis.

Gene knock-out and knock-in strategies are employed to investigate the function of MesP1. MesP1 belongs to the same family of bHLH transcription factors as MesP2. The early expression pattern observed in the early mesoderm at the onset of gastrulation is restricted to Mesp1, while the later expression pattern in the anterior presomitic mesoderm during somitogenesis is almost the same for Mesp1 as for Mesp2. Homozygous Mesp1 null mice exhibited growth retardation after 7.5 dpc and died before 10.5 dpc with many developmental defects. The function of MesP1 during somitogenesis was not clearly revealed because of their early death and the possible compensation by MesP2. In order to examine the functions of MesP1 during somitogenesis, we replaced the Mesp2 gene with Mesp1 cDNA, using a gene knock-in strategy. The introduced Mesp1 cDNA could rescue the defects caused by Mesp2 deficiency in a dosage-dependent manner. Mice which lacked Mesp2 expression but had four copies of the Mesp1 gene survived into the adulthood and were fertile. The skeletal defects and the reduction in expression of Notch1, Notch2 and FGFR-1 previously observed in Mesp2 null mice were almost completely rescued by the introduced MesP1. Thus, it is concluded that the functions of MesP1 during somitogenesis, like MesP2, are also mediated via notch-delta and FGF signaling systems., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved.)

Mouse embryos homozygous for the loop-tail (Lp) mutation fail to initiate neural tube closure at E8.5, leading to a severe malformation in which the neural tube remains open from midbrain to tail. During initiation of closure, the normal mouse neural plate bends sharply in the midline, at the site of the future floor plate. In contrast, Lp/Lp embryos exhibit a broad region of flat neural plate in the midline, displacing the sites of neuroepithelial bending to more lateral positions. Sonic hedgehog (Shh) and Netrin1 are expressed in abnormally broad domains in the ventral midline of the E9.5 Lp/Lp neural tube, suggesting over-abundant differentiation of the floor plate. The notochord is also abnormally broad in Lp/Lp embryos with enlarged domains of Shh and Brachyury expression. The paraxial mesoderm shows evidence of ventralisation, with increased expression of the sclerotomal marker Pax1, and diminished expression of the dermomyotomal marker Pax3. While the expression domain of Pax3 does not differ markedly from wild-type, there is a dorsal shift in the domain of Pax6 expression in the neural tube at caudal levels of Lp/Lp embryos. We suggest that the Lp mutation causes excessive differentiation of floor-plate and notochord, with over-production of Shh from these midline structures causing ventralisation of the paraxial mesoderm and, to a lesser extent, the neural tube. Comparison with other mouse mutants suggests that the enlarged floor plate may be responsible for the failure of neural tube closure in Lp/Lp embryos., (Copyright 1998 Elsevier Science Ireland Ltd.)

In open brain (opb) mutant embryos, developmental defects of the trunk spinal cord were spatially correlated with severe defects of the epaxial somite derivatives including sclerotomes, whereas hypaxial somite derivatives are much less affected. Later in development, the neural arches (epaxial sclerotome derivatives) formed but were severely disorganized, and also the distal ribs (hypaxial sclerotome derivatives) were malformed. Adjacent neural arches and vertebral bodies were often fused where joints should have formed suggesting defects of the intrasomitic borderlines. Moreover, neural arches frequently and ribs sometimes were split into halves at distinct levels along the dorso-ventral body axis. This suggests that 'resegmentation' of sclerotomes across the somite borders did not completely occur. These prominent skeletal defects were preceded by reduced expression of Pax1 along the intrasomitic borderlines, and incomplete maintenance of somite borders between central sclerotome moieties. The defects of the axial skeleton were accompanied by segmentation defects of the myotomes which were split distally, and also partly fused from adjacent segments across somite borders. The segmentation defects observed suggest that in opb mutants both segmental borderlines, the somite borders and the intrasomitic borderlines (fissures), were affected and behaved paradoxically.

Zebrafish embryos and larvae have stage-specific patterns of motility or locomotion. Two embryonic structures accomplish this behavior: the central nervous system (CNS) and skeletal muscles. To identify genes that are functionally involved in mediating and controlling different patterns of embryonic and larval motility, we included a simple touch response test in our zebrafish large-scale genetic screen. In total we identified 166 mutants with specific defects in embryonic motility. These mutants fall into 14 phenotypically distinct groups comprising at least 48 genes. Here we describe the various phenotypic groups including mutants with no or reduced motility, mechanosensory defective mutants, 'spastic' mutants, circling mutants and motor circuit defective mutants. In 63 mutants, defining 18 genes, striation of somitic muscles is reduced. Phenotypic analysis provides evidence that these 18 genes have distinct and consecutive functions during somitic muscle development. The genes sloth (slo) and frozen (fro) already act during myoblast differentiation, while 13 genes appear to function later, in the formation of myofibers and the organization of sarcomeres. Mutations in four other genes result in muscle-specific degeneration. 103 mutations, defining at least 30 genes, cause no obvious defects in muscle formation and may instead affect neuronal development. Analysis of the behavioral defects suggests that these genes participate in the diverse locomotion patterns observed, such as touch response, rhythmic tail movements, equilibrium control, or that they simply confer general motility to the animal. In some of these mutants specific defects in the developing nervous system are detected. Mutations in two genes, nevermind (nev) and macho (mao), affect axonal projection in the optic tectum, whereas axon formation and elongation of motorneurons are disrupted by mutations in the diwanka (diw) and the unplugged (unp) genes.

Using the benzothiazolium-4-quinolium dye, TO-PRO-1, to detect cell death in live embryos, we labeled a developmental series of Wnt-1 null mutant and wild type embryos to determine if cell death contributed to the absence of the midbrain and rostral metencephalon observed in Wnt-1 mutant embryos. We found that there is no detectable cell death at early somite stages in Wnt-1 mutant embryos. However, we detected a significant, but transient, population of dying cells within the anterior dorsal metencephalon in 20-29 somite stage embryos. These cells located in the anterior dorsal metencephalon also stain positive using the TUNEL technique that utilizes terminal transferase to label DNA fragments that are typical in the nuclei of apoptotic cells. Thus, programmed cell death plays a role in the loss of the metencephalon, but apparently does not contribute to the earliest aspect of the mutant phenotype, namely the loss of the midbrain.