Generating gradients of retinoic acid in the chick embryo: Cyp26C1 expression and a comparative analysis of the Cyp26 enzymes.

We have cloned a novel retinoic acid (RA) catabolizing enzyme, Cyp26C1, in the chick and describe here its distribution during early stages of chick embryogenesis. It is expressed from stage 4 in the presumptive anterior (cephalic) mesoderm, in a subset of cephalic neural crest cells, the ventral otic vesicle, mesenchyme adjacent to the otic vesicle, the branchial pouches and grooves, a part of the neural retina, and the anterior telencephalon, and shows a dynamic expression in the hindbrain rhombomeres and neuronal populations within them. By examining the distribution of Cyp26C1 in the RA-free quail embryo, we can determine which of these expression domains is dependent on RA, and it is only the rhombomeric sites that do not appear, suggesting a role for RA in this location. The most striking domain of Cyp26C1 distribution is in the anterior cephalic mesoderm, which is adjacent to the domain of Raldh2 in the trunk mesoderm, but separated from it by a gap dorsal to which the posterior hindbrain will develop. We suggest that a gradient of RA within the mesoderm generated by Raldh2 and catabolized by Cyp26C1 could be responsible for patterning the hindbrain. We have compared this distribution of Cyp26C1 with that of Cyp26A1 and Cyp26B1 in the chick and shown that they generally occupy nonoverlapping sites of expression in the embryo, and as a result, we suggest individual roles for each of the Cyp enzymes in the developing embryo.     

The regulation of endogenous retinoic acid level through CYP26B1 is required for elevation of palatal shelves

Background: In previous studies, we investigated the effects of excess retinoic acid (RA) during palatogenesis by RA administration to pregnant mice. In the present study, we deleted Cyp26b1, one of the RA‐degrading enzymes, to further study the effects of excess RA in the normal developing palate and to understand how endogenous levels of RA are regulated. Results: Excess RA, due to the absence of Cyp26b1, targets cells in the bend region of the palatal shelves and inhibits their horizontal elevation, leading to cleft palate. An organ culture of Cyp26b1?/? palatal shelves after tongue removal did not rescue the impaired elevation of the palatal shelves. The expression of Fgf10, Bmp2, and Tbx1, important molecules in palatal development, was down‐regulated. Cell proliferation was decreased in the bend region of palatal shelves. Tongue muscles were hypoplastic and/or missing in Cyp26b1?/? mice. Conclusions: We demonstrated that CYP26B1 is essential during palatogenesis. Excess RA due to the lack of Cyp26b1 suppresses the expression of key regulators of palate development in the bend region, resulting in a failure in the horizontal elevation of the palatal shelves. The regulation of RA signaling through CYP26B1 is also necessary for the development of tongue musculature and for tongue depression. Developmental Dynamics 241:1744–1756, 2012. © 2012 Wiley Periodicals, Inc.     

Plasticity of neural crest–placode interaction in the developing visceral nervous system

The reciprocal relationship between rhombomere (r)‐derived cranial neural crest (NC) and epibranchial placodal cells derived from the adjacent branchial arch is critical for visceral motor and sensory gangliogenesis, respectively. However, it is unknown whether the positional match between these neurogenic precursors is hard‐wired along the anterior–posterior (A/P) axis. Here, we use the interaction between r4‐derived NC and epibranchial placode‐derived geniculate ganglion as a model to address this issue. In Hoxa1^?/?b1^?/? embryos, r2 NC compensates for the loss of r4 NC. Specifically, a population of r2 NC cells is redirected toward the geniculate ganglion, where they differentiate into postganglionic (motor) neurons. Reciprocally, the inward migration of the geniculate ganglion is associated with r2 NC. The ability of NC and placodal cells to, respectively, differentiate and migrate despite a positional mismatch along the A/P axis reflects the plasticity in the relationship between the two neurogenic precursors of the vertebrate head. Developmental Dynamics 240:1880–1888, 2011. © 2011 Wiley‐Liss, Inc.     

The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures

The active derivative of vitamin A, retinoic acid (RA), is essential for normal embryonic development. The spatio-temporal distribution of embryonic RA results from regulated expression of RA-synthesizing retinaldehyde dehydrogenases and RA-metabolizing cytochrome P450s (CYP26). Excess RA administration or RA deficiency results in a complex spectrum of embryonic abnormalities. As a first step in understanding the developmental function of RA-metabolizing enzymes, we have disrupted the murine Cyp26A1 gene. We report that Cyp26A1-null mutants die during mid-late gestation and show a number of major morphogenetic defects. Spina bifida and truncation of the tail and lumbosacral region (including abnormalities of the kidneys, urogenital tract, and hindgut) are the most conspicuous defects, leading in extreme cases to a sirenomelia ("mermaid tail") phenotype. Cyp26A1 mutants also show posterior transformations of cervical vertebrae and abnormal patterning of the rostral hindbrain, which appears to be partially posteriorly transformed. These defects correlate with two major sites of Cyp26A1 expression in the rostral neural plate and embryonic tail bud. Because all of the Cyp26A1(-/-) abnormalities closely resemble RA teratogenic effects, we postulate that the key function of CYP26A1 is to maintain specific embryonic areas in a RA-depleted state, to protect them against the deleterious effect of ectopic RA signaling.     

The oxidizing enzyme CYP26a1 tightly regulates the availability of retinoic acid in the gastrulating mouse embryo to ensure proper head development and vasculogenesis.

Retinoic acid (RA) has been implicated as one of the signals providing a posterior character to the developing vertebrate central nervous system. Embryonic RA first appears in the posterior region of the gastrulating embryo up to the node level, where it may signal within the adjacent epiblast and/or newly induced neural plate to induce a hindbrain and spinal cord fate. Conversely, rostral head development requires forebrain-inducing signals produced by the anterior visceral endoderm and/or prechordal mesoderm, and there is evidence that RA receptors must be in an unliganded state to ensure proper head development. As RA is a diffusible lipophilic molecule, some mechanism(s) must therefore have evolved to prevent activation of RA targets in anterior regions of the embryo. This might result from RA catabolism mediated by the CYP26A1 oxidizing enzyme, which is transiently expressed in anteriormost embryonic tissues; however, previous analysis of Cyp26a1(-/-) mouse mutants did not clearly support this hypothesis. Here we show that Cyp26a1(-/-) null mutants undergo head truncations when exposed to maternally-derived RA, at doses that do not affect wild-type head development. These anomalies are linked to a widespread ectopic RA signaling activity in rostral head tissues of CYP26A1-deficient embryos. Thus, CYP26A1 is required in the anterior region of the gastrulating mouse embryo to prevent teratological effects that may result from RA signaling. We also report a novel role of CYP26A1 during early development of the intra- and extra-embryonic vascular networks.     

Evolutionarily conserved function of Gbx2 in anterior hindbrain development

The amino acid sequence across the DNA‐binding homeodomain of Gbx2 is highly conserved across multiple species. In mice, Gbx2 is essential for establishment of the midbrain–hindbrain boundary (MHB), and in development of anterior hindbrain structures, rhombomeres (r) 1–r3, and the r2/r3‐derived cranial nerve V. In contrast, studies in zebrafish have implicated gbx1 in establishment of the MHB. Therefore, we tested potential roles for gbx2 in anterior hindbrain development in zebrafish. gbx2 knockdown with antisense morpholino results in increased cell death in r2, r3, and r5 and a truncation of the anterior hindbrain, similar to the defect in Gbx2^?/? mice. Moreover, there is abnormal clustering of cranial nerve V cell bodies in r2 and r3 indicative of defects in aspects of anterior hindbrain patterning. These phenotypes can be rescued by expression of the mouse GBX2 protein. These results suggest that gbx2/Gbx2 has an evolutionarily conserved role in anterior hindbrain development. Developmental Dynamics 240:828–838, 2011. © 2011 Wiley‐Liss, Inc.     

Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit–interdigit junction

Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA‐generating enzyme retinaldehyde dehydrogenase‐2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2?/? mouse embryos lose expression of the RARE‐lacZ RA‐reporter transgene and matrix metalloproteinase‐11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit–interdigit junction. Raldh2?/? autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down‐regulated in Raldh2?/? autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit–interdigit junction suggests RA controls tissue remodeling as well as apoptosis. Developmental Dynamics 239:665–671, 2010. © 2009 Wiley‐Liss, Inc.     

Investigation of retinoic acid function during embryonic brain development using retinaldehyde‐rescued Rdh10 knockout mice

Background: Retinoic acid (RA) signaling controls patterning and neuronal differentiation within the hindbrain, but forebrain RA function remains controversial. RA is produced from metabolism of retinol to retinaldehyde by retinol dehydrogenase (RDH), followed by metabolism of retinaldehyde to RA by retinaldehyde dehydrogenase (RALDH). Previous studies on Raldh2?/? and Raldh3?/? mice demonstrated an RA requirement for γ‐aminobutyric acid (GABA)ergic and dopaminergic differentiation in forebrain basal ganglia, but no RA requirement was observed during early forebrain patterning or subsequent forebrain cortical expansion. However, other studies suggested that RA controls forebrain patterning, and analysis of ethylnitrosourea‐induced Rdh10 mutants suggested that RA synthesized in the meninges stimulates forebrain cortical expansion. Results: We generated Rdh10?/? mouse embryos that lack RA activity early in the head and later in the meninges. We observed defects in hindbrain patterning and eye RA signaling, but early forebrain patterning was unaffected. Retinaldehyde treatment of Rdh10?/? embryos from E7–E9 rescues a cranial skeletal defect, resulting in E14.5 embryos lacking meningeal RA activity but maintaining normal forebrain shape and cortical expansion. Conclusions: Rdh10?/? embryos demonstrate that RA controls hindbrain but not early forebrain patterning, while studies on retinaldehyde‐rescued Rdh10?/? embryos show that meningeal RA synthesis is unnecessary to stimulate forebrain cortical expansion. Developmental Dynamics 242:1056–1065, 2013. © 2013 Wiley Periodicals, Inc.     

Tulp3 is a critical repressor of mouse hedgehog signaling

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby‐like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3^?/? embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3^?/?/Shh^?/? compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh^?/? embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140–1149, 2009. © 2009 Wiley‐Liss, Inc.     

Notch pathway regulation of neural crest cell development in vivo

Background : The function of Notch signaling in murine neural crest–derived cell lineages in vivo was examined. Results : Conditional gain (Wnt1Cre;Rosa^Notch) or loss (Wnt1Cre;RBP‐J^f/f) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa^Notch embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP‐J^f/f mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa^Notch and Wnt1Cre;RBP‐J^f/f embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa^Notch embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP‐J^f/f embryos. Conclusions : Together, these studies demonstrate essential cell‐autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease. Developmental Dynamics 241:376–389, 2012. © 2011 Wiley Periodicals, Inc.     

Analysis of hindbrain neural crest migration in the long-tailed monkey (Macaca fascicularis)

Neural crest cells make a substantial contribution to normal craniofacial development. Despite advances made in identifying migrating neural crest cells in avian embryos and, more recently, rodent embryos, knowledge of crest cell migration in primates has been limited to what was obtained by conventional morphological techniques. In order to determine the degree to which the nonhuman primate fits the mammalian pattern, we studied the features of putative neural crest cell migration in the hindbrain of the long-tailed monkey (Macaca fascicularis) embryo. Cranial crest cells were identified on the basis of reported distributional and morphological criteria as well as by immunocytochemical detection of the neural cell adhesion molecule (N-CAM) that labels a subpopulation of these cells. The persistent labeling of a sufficient number of crest cells with antibodies to N-CAM following their exit from the rostral, preotic and post-otic regions of the hindbrain facilitated tracking them along subectodermal pathways to their respective destinations in the first, second and third pharyngeal arches. Peroxidase immunocytochemistry was also employed to localize laminin and collagen-IV in neuroepithelial basement membranes. At stage 10 (8–11 somites), crest emigration occurred in areas of unfused neural folds through focal disruptions in the neuroepithelial basement membrane in both the rostral and pre-otic regions, although there was little evidence of crest migration in the post-otic hindbrain. By stage 11 (16–17 somites), the neural folds were fused (pre- and post-otic hindbrain) or in the process of fusing (rostral hindbrain), yet crest cell emigration was apparent in all three areas through discontinuities in the basement membrane. Emigration was essentially complete at stage 12 (21 somites) as indicated by nearly continuous cranial neural tube basement membranes. At this stage the pre-ganglia (trigeminal, facioacoustic and glossopharyngeal) were consistently stained with N-CAM. The current study has provided new information on mammalian neural crest in a well-established experimental model for normal and abnormal human development, including its use as a model for the retinoic acid syndrome. In this regard, the current results provide the basis for probing the mechanisms of retinoid embryopathy which may involve perturbation of hindbrain neural crest development.     

Zebrafish acvr2a and acvr2b exhibit distinct roles in craniofacial development.

To examine the roles of activin type II receptor signaling in craniofacial development, full-length zebrafish acvr2a and acvr2b clones were isolated. Although ubiquitously expressed as maternal mRNAs and in early embryogenesis, by 24 hr postfertilization (hpf), acvr2a and acvr2b exhibit restricted expression in neural, hindbrain, and neural crest cells (NCCs). A morpholino-based targeted protein depletion approach was used to reveal discrete functions for each acvr2 gene product. The acvr2a morphants exhibited defects in the development of most cranial NCC-derived cartilage, bone, and pharyngeal tooth structures, whereas acvr2b morphant defects were largely restricted to posterior arch structures and included the absence and/or aberrant migration of posterior NCC streams, defects in NCC-derived posterior arch cartilages, and dysmorphic pharyngeal tooth development. These studies revealed previously uncharacterized roles for acvr2a and acvr2b in hindbrain and NCC patterning, in NCC derived pharyngeal arch cartilage and joint formation, and in tooth development.     

Neural stem cell self‐renewal requires the Mrj co‐chaperone

The Mrj co‐chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj^?/? embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin‐walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down‐regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj^?/? neuroepithelial cells. Furthermore, Mrj^?/? neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self‐renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi‐1, a self‐renewal protein. Furthermore, unlike in Mrj^?/? placentas, intermediate filament‐containing aggregates do not accumulate in Mrj^?/? neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self‐renewal. Developmental Dynamics 238:2564–2574, 2009. © 2009 Wiley‐Liss, Inc.     

Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the chick.

Within the hindbrain region, neural crest cell migration is organized into three streams that follow the segmentation of the neuroepithelium into distinct rhombomeric compartments. Although the streaming of neural crest cells is known to involve signals derived from the neuroepithelium, the molecular properties underlying this process are poorly understood. Here, we have mapped the expression of the signaling component of two secreted class III Semaphorins, Semaphorin (Sema) 3A and Sema 3F, at time points that correspond to neural crest cell migration within the hindbrain region of the chick. Both Semaphorins are expressed within rhombomeres at levels adjacent to crest-free mesenchyme and expression of the receptor components essential for Semaphorin activity by neural crest cells suggests a function in restricting neural crest cell migration. By using bead implantation and electroporation in ovo, we define a role for both Semaphorins in the maintenance of neural crest cell streams in proximity to the neural tube. Attenuation of Semaphorin signaling by expression of soluble Neuropilin-Fc resulted in neural crest cells invading adjacent mesenchymal territories that are normally crest-free. The loss or misguidance of specific neural crest cell populations after changes in Semaphorin signaling also affects the integration of the cranial sensory ganglia. Thus, Sema 3A and 3F, expressed and secreted by the hindbrain neuroepithelium contributes to the appropriate positioning of neural crest cells in proximity to the neural tube, a process crucial for the subsequent establishment of neuronal connectivity within the hindbrain region.     

Sema3E is required for migration of cranial neural crest cells in zebrafish: Implications for the pathogenesis of CHARGE syndrome

CHARGE syndrome is a congenital disorder with multiple malformations in the craniofacial structures, and cardiovascular and genital systems, which are mainly affected by neural crest defects caused by loss‐of‐function mutations within chromodomain helicase DNA‐binding protein 7 (CHD7). However, many patients with CHARGE syndrome test negative for CHD7. Semaphorin 3E (sema3E) is a gene reported to be mutated in patients with CHARGE syndrome. However, its role in the pathogenesis of CHARGE syndrome has not been verified experimentally. Here, we report that the knockdown of sema3E results in severe craniofacial malformations, including small eyes, defective cartilage and an abnormal number of otoliths in zebrafish embryos, which resemble the major features of CHARGE syndrome. Further analysis reveals that the migratory cranial neural crest cells are scattered in the region of the hindbrain, and the postmigratory neural crest cells are reduced in the pharyngeal arches upon sema3E knockdown. Notably, immunostaining and time‐lapse imaging analyses of a neural crest cell‐labelled transgenic fish line, sox10:EGFP, show that the migration of cranial neural crest cells is severely impaired, and many of these cells are misrouted upon sema3E knockdown. Furthermore, the sox10‐expressing cranial neural crest cells are scattered in chd7 homozygous mutants, which phenocopied the phenotype in sema3E morphants. Overexpression of sema3E rescues the phenotype of scattered cranial neural crest cells in chd7 homozygotes, indicating that chd7 may control the expression of sema3E to regulate cranial neural crest cell migration. Collectively, our data demonstrate that sema3E is involved in the pathogenesis of CHARGE syndrome by modulating cranial neural crest cell migration.     

Synectin‐dependent regulation of arterial maturation

Synectin, a ubiquitously expressed PDZ scaffold protein, has been shown to be a key regulator in the formation of arterial vasculature. Examination of the retinal vasculature in synectin^?/? mice demonstrated poor mural cell coverage of and attachment to the forming arterial tree, a defect reminiscent of retinal abnormalities observed in platelet derived growth factor (PDGF) ‐B^?/? mice. Primary cultures of synectin^?/? smooth muscle cells had normal expression of PDGFR‐β and migrated normally in response to PDGF‐BB. However, expression of PDGF‐BB protein, but not mRNA, was reduced in lysates from arterial, but not venous, primary synectin^?/? endothelial cells (EC), that was restored by inhibition of proteosomal degradation. Transduction of synectin^?/? and ^+/+ EC with a bicistronic Pdgfb/gfp construct, resulted in comparable expression of green fluorescent protein in both EC populations while PDGF‐BB expression was severely reduced in synectin^?/? EC. Finally, synectin expression in synectin^?/? arterial EC restored PDGF‐BB protein levels. These results suggest that synectin deficiency results in increased degradation of PDGF‐BB protein in arterial EC and, consequently, reduced recruitment of mural cells to newly forming arteries. This observation may explain the selective reduction in arterial morphogenesis observed in synectin knockout mice. Developmental Dynamics 238:604–610, 2009. © 2009 Wiley‐Liss, Inc.     

Loss of the small GTPase Arl8b results in abnormal development of the roof plate in mouse embryos

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene‐trapped mutant (Arl8b^?/?) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b^?/? embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b^?/? embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b^?/? embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.     

Myosin‐X is required for cranial neural crest cell migration in Xenopus laevis

Myosin‐X (MyoX) belongs to a large family of unconventional, nonmuscle, actin‐dependent motor proteins. We show that MyoX is predominantly expressed in cranial neural crest (CNC) cells in embryos of Xenopus laevis and is required for head and jaw cartilage development. Knockdown of MyoX expression using antisense morpholino oligonucleotides resulted in retarded migration of CNC cells into the pharyngeal arches, leading to subsequent hypoplasia of cartilage and inhibited outgrowth of the CNC‐derived trigeminal nerve. In vitro migration assays on fibronectin using explanted CNC cells showed significant inhibition of filopodia formation, cell attachment, spreading and migration, accompanied by disruption of the actin cytoskeleton. These data support the conclusion that MyoX has an essential function in CNC migration in the vertebrate embryo. Developmental Dynamics 238:2522–2529, 2009. © Published 2009 Wiley‐Liss, Inc.^?