Spatio‐temporal dynamics of gene expression of the Edn1‐Dlx5/6 pathway during development of the lower jaw

The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin‐1 (Edn1)‐dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo‐mandibular identity. Here, to better analyze the spatio‐temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1‐dependent and ‐independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1→Dlx5/6→Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events. genesis 48:362–373, 2010. © 2010 Wiley‐Liss, Inc.     

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish

During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.     

Using Xenopus to analyze neurocristopathies like Kabuki syndrome

Neurocristopathies are human congenital syndromes that arise from defects in neural crest (NC) development and are typically associated with malformations of the craniofacial skeleton. Genetic analyses have been very successful in identifying pathogenic mutations, however, model organisms are required to characterize how these mutations affect embryonic development thereby leading to complex clinical conditions. The African clawed frog Xenopus laevis provides a broad range of in vivo and in vitro tools allowing for a detailed characterization of NC development. Due to the conserved nature of craniofacial morphogenesis in vertebrates, Xenopus is an efficient and versatile system to dissect the morphological and cellular phenotypes as well as the signaling events leading to NC defects. Here, we review a set of techniques and resources how Xenopus can be used as a disease model to investigate the pathogenesis of Kabuki syndrome and neurocristopathies in a wider sense.     

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon‐exposed embryos: is the amphibian Xenopus laevis a suitable model for gene‐based comparative teratology?

BACKGROUND: The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch‐1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene‐based comparative teratology, this work evaluates the expression of Shh, Ptch‐1, Gsc, and Msx2 in FON‐exposed X. laevis embryos. METHODS: Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT‐PCR. RESULTS: The developmental rate was never affected by FON at any considered stage. FON‐exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON‐exposed stage 17 embryos. Shh, Ptch‐1, Msx2 showed a high fluctuation of expression both in control and in FON‐exposed samples both at stages 17 and 27. CONCLUSION: The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene‐based comparative teratology. Birth Defects Res (Part B) 92:189–194, 2011. © 2011 Wiley‐Liss, Inc.     

Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling

Background: Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial–mesenchymal interactions. METHODS: This study investigates the role of Dlx5 in the epithelial–mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high‐density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial–mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium. RESULTS: Dlx5 deletion alters expression of TGFβ₁, important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA‐treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects. CONCLUSIONS : Disruption in an Fgf3, ‐10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA. Birth Defects Res (Part B) 2008. ©2008 Wiley‐Liss, Inc.     

Msx1 and Dlx5 function synergistically to regulate frontal bone development

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1^?/?;Dlx5^?/? mice, the frontal bones defect was more severe than that of either Msx1^?/? or Dlx5^?/? mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development. genesis 48:645–655, 2010. © 2010 Wiley‐Liss, Inc.     

Retinoic Acid Signaling Regulates Sonic Hedgehog and Bone Morphogenetic Protein Signalings During Genital Tubercle Development

Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA‐induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA‐synthesizing enzyme, Raldh2, and for the RA‐catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1^?/? mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss–of‐function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development.     

Experimental studies on reproductive toxicologic effects of lamotrigine in mice

BACKGROUND: Virtually all antiepileptic drugs (AED) tested so far have been found to be teratogenic. The second generation AED possess a number of therapeutic advantages over the older ones. There are, however, very little data on their effects on embryonic development. A recent report suggests that lamotrigine (LTG) can be teratogenic to human fetuses. With only a few cases of prenatal exposure to LTG in the record, however, it has not been possible to establish a recognizable pattern of malformations in the infants of LTG‐treated mothers. OBJECTIVES: The objectives of the present study were to evaluate the reproductive toxic effects of LTG RESULTS: Single (50–200 mg/kg) or multiple doses (25, 50, 75 mg/kg) of LTG were administered by intraperitoneal (i.p.) injection (note that the therapeutic administration is oral) to groups of TO mice on gestation day (GD) 7 or 8. Fetuses were collected on GD 18. Maternal toxic effects including a dose‐related mortality, a high incidence of abortion, embryo lethality, congenital malformations and intrauterine growth retardation (IUGR) were observed in the LTG‐treated group. Administration of LTG in multiple low doses resulted in a better maternal survival and increased incidence of embryonic resorption and malformations with increasing dose; IUGR was significant but not dose‐dependent. The malformations characteristic of the LTG multiple low dose group fetuses included maxillary‐mandibular hypoplasia, exencephaly, cleft palate, median facial cleft, urogenital anomalies and varying degrees of caudal regression. Skeletal malformations and developmental delay of the skeleton were observed both in single and multiple dose groups. CONCLUSIONS: The results of this study indicate that LTG administered i.p. at high doses can induce intrauterine growth retardation and at low multiple doses causes a dose‐dependent increase in embryonic resorption, craniofacial and caudal malformations as well as maternal toxicity in the mouse. Previous studies in other laboratories have used oral route of exposure and concluded that there are no teratogenic effects of LTG at dose levels that are not maternally toxic. Birth Defects Res B 68:428–438, 2003. © 2003 Wiley‐Liss, Inc.     

Expanding EMC foldopathies: Topogenesis deficits alter the neural crest

The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.     

Xenopus leads the way: Frogs as a pioneering model to understand the human brain

From its long history in the field of embryology to its recent advances in genetics, Xenopus has been an indispensable model for understanding the human brain. Foundational studies that gave us our first insights into major embryonic patterning events serve as a crucial backdrop for newer avenues of investigation into organogenesis and organ function. The vast array of tools available in Xenopus laevis and Xenopus tropicalis allows interrogation of developmental phenomena at all levels, from the molecular to the behavioral, and the application of CRISPR technology has enabled the investigation of human disorder risk genes in a higher‐throughput manner. As the only major tetrapod model in which all developmental stages are easily manipulated and observed, frogs provide the unique opportunity to study organ development from the earliest stages. All of these features make Xenopus a premier model for studying the development of the brain, a notoriously complex process that demands an understanding of all stages from fertilization to organogenesis and beyond. Importantly, core processes of brain development are conserved between Xenopus and human, underlining the advantages of this model. This review begins by summarizing discoveries made in amphibians that form the cornerstones of vertebrate neurodevelopmental biology and goes on to discuss recent advances that have catapulted our understanding of brain development in Xenopus and in relation to human development and disease. As we engage in a new era of patient‐driven gene discovery, Xenopus offers exceptional potential to uncover conserved biology underlying human brain disorders and move towards rational drug design.     

Gene expression in Xenopus laevis embryos after Triadimefon exposure

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.     

ALK5-Mediated Transforming Growth Factor β Signaling in Neural Crest Cells Controls Craniofacial Muscle Development via Tissue-Tissue Interactions

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5^fl/fl mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5^fl/fl mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5^fl/fl mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.     

A conserved role for retinoid signaling in vertebrate pancreas development

Retinoic acid (RA) signaling plays critical roles in the regionalization of the central nervous system and mesoderm of all vertebrates that have been examined. However, to date, a role for RA in pancreas and liver development has only been demonstrated for the teleost zebrafish. Here, we demonstrate that RA signaling is required for development of the pancreas but not the liver in the amphibian Xenopus laevis and the avian quail. We disrupted RA signaling in Xenopus tadpoles, using both a pharmacological and a dominant-negative strategy. RA-deficient quail embryos were obtained from hens with a dietary deficiency in vitamin A. In both species we found that pancreas development was dependent on RA signaling. Furthermore, treatment of Xenopus tadpoles with exogenous RA led to an expansion of the pancreatic field. By contrast, liver development was not perturbed by manipulation of RA signaling. Taken together with our previous finding that RA signaling is necessary and sufficient for zebrafish pancreas development, these data support the hypothesis that a critical role for RA signaling in pancreas development is a conserved feature of the vertebrates.     

Investigation of the teratogenic potential of ochratoxin A and B using the FETAX system

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by certain Aspergillus and Penicillium species. It has been observed to be teratogenic in a number of animal models including rat, mouse, hamster, and chick, with reduced birth weight and craniofacial abnormalities being the most commonly observed malformations. Neither the potential of OTA to cause malformations in humans nor its teratogenic mode of action is known. The FETAX system is an embryotoxicity assay system, with a high correlation to animal models and epidemiological data. Analysis of OTA-mediated teratogenesis using this system could provide a useful tool for the generation of high numbers of samples for mechanistic studies. METHODS: Using the standard ASTM 96-hr exposure protocol, the effect of OTA and its structural analogue OTB on the development of Xenopus laevis embryos in vitro was assessed. The accumulation of both substances in Xenopus embryos was also examined using tritiated OTA and OTB. RESULTS: Both OTA and OTB caused craniofacial malformations, while OTA also caused reduced embryo growth. As expected, OTA was far more potent in inducing these effects than OTB. This could at least in part be due to greater levels of OTA being accumulated within the embryos. CONCLUSIONS: The ability of FETAX to differentiate between close structural analogues indicates the assay has great potential for the elucidation of the embryotoxic and teratogenic mechanisms of action. Hence, the model could provide a suitable system for the investigation of other known teratogens or for the pre-screening of new agents for teratogenic potential.     

Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development

Background information. pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. Results. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)‐based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross‐rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. Conclusion. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.     

An ontology for <Emphasis Type="Italic">Xenopus</Emphasis> anatomy and development

Background  

The frogs Xenopus laevis and Xenopus (Silurana) tropicalis are model systems that have produced a wealth of genetic, genomic, and developmental information. Xenbase is a model organism database that provides centralized access to this information, including gene function data from high-throughput screens and the scientific literature. A controlled, structured vocabulary for Xenopus anatomy and development is essential for organizing these data.     

Different embryo-fetal toxicity effects for three VLA-4 antagonists

BACKGROUND: VLA‐4 (Very late antigen 4, integrin α₄β₁) plays an important role in cell‐cell interactions that are critical for development. Homozygous null knockouts of the α₄subunit of VLA‐4 or VCAM‐1 (cell surface ligand to VLA‐4) in mice result in abnormal placental and cardiac development and embryo lethality. Objectives of the current study were to assess and compare the teratogenic potential of three VLA‐4 antagonists. METHODS: IVL745, HMR1031, and IVL984 were each evaluated by the subcutaneous route in standard embryo‐fetal developmental toxicity studies in rats and rabbits. IVL984 was also evaluated in mice. Fetuses were examined externally, viscerally, and skeletally. RESULTS: IVL745 did not cause significant maternal or fetal effects at doses up to 100 or 250 mg/kg/day in rats or rabbits, respectively. HMR1031 treatment resulted in marked maternal toxicity and slight fetal toxicity at the highest tested doses of 200 and 75 mg/kg/day in rats and rabbits, respectively. HMR1031 embryo‐fetal effects consisted of slightly lower body weight and crown‐rump length in rats and minor sternebral defects in rabbits. IVL984 treatment resulted in minimal maternal effects at doses up to 40, 15, and 100 mg/kg/day in rats, rabbits, and mice, respectively (excluding abortions in rabbits). However, marked developmental effects were observed at the lowest tested IVL984 doses, 1, 0.2, and 3 mg/kg/day in rats, rabbits, and mice, respectively. IVL984 embryo‐fetal effects consisted of increased total post‐implantation loss due to early resorptions and high incidences of cardiac malformations and skeletal malformations and/or variations. Notably, spiral septal defects were observed in up to 76% of rat fetuses and up to 58% of rabbit fetuses. CONCLUSIONS: Dramatic differences in teratogenic potential were observed: IVL745 was not teratogenic, HMR1031 caused slight embryo‐fetal effects at maternally‐toxic doses, and IVL984 was a potent teratogen at doses where direct maternal toxicity was limited to abortions in rabbits. Prominent effects of IVL984 included embryo lethality and cardiac malformations including spiral septal defects in three species. Birth Defects Res B 71:55–68, 2004. © 2004 Wiley‐Liss, Inc.