Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling

1-Phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 μM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling.     

Alterations in migrating cranial neural crest cells in embryos of mice fed retinoic acid

Alterations in migrating neural crest cells induced by all-trans retinoic acid (RA) were studied morphologically and immunohistochemically in the cranial portion of 8-day-old mouse embryos which were derived from dams given 60, 40 or 0 mg kg of RA and killed 2 to 8 h later. Additionally, the embryos exposed to 4 mg/kg of actinomycin D (AD) on day 8 of gestation for 5 h were examined similarly. Light microscopy revealed that RA was cytotoxic and caused the appearance of pleomorphic nuclei, extra-large nucleoli and cytoplasmic budding which replaced lamellipodia and spike-like projections. Electron microscopy revealed pleomorphic nuclei containing nucleoli with major granular portions frequently surrounded with heterochromatin, monosomes, and phagosomes. A monosomal distribution pattern was different from that seen in the neural crest cells exposed to AD. The latter showed incomplete polyribosomal dispersion with fewer nucleolar components. Fewer neural crest cells with choline acetyltransferase-like immunoreactivity were detected in RA- and AD-exposed embryos than in the controls. These findings suggest that excess RA inhibits acetylcholine synthesis of the migrating neural crest cells, in a manner different from AD, and that it enhances phagocytosis. These phenomena modify the characteristics of neural crest cells resulting in craniofacial malformations.     

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.     

Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development

Craniofacial and ocular morphogenesis require proper regulation of cranial neural crest migration, proliferation, survival and differentiation. Although alterations in maternal thyroid hormone (TH) are associated with congenital craniofacial anomalies, the role of TH on the neural crest has not been previously described. Using zebrafish, we demonstrate that pharmacologic and genetic alterations in TH signaling disrupt cranial neural crest migration, proliferation, and survival, leading to craniofacial, extraocular muscle, and ocular developmental abnormalities. In the rostral cranial neural crest that gives rise to the periocular mesenchyme and the frontonasal process, retinoic acid (RA) rescued migratory defects induced by decreased TH signaling. In the caudal cranial neural crest, TH and RA had reciprocal effects on anterior and posterior pharyngeal arch development. The interactions between TH and RA signaling were partially mediated by the retinoid X receptor. We conclude that TH regulates both rostral and caudal cranial neural crest. Further, coordinated interactions of TH and RA are required for proper craniofacial and ocular development.     

Expression of the retinoic acid catabolising enzyme CYP26B1 in the chick embryo and its regulation by retinoic acid

We have cloned a fragment of Cyp26B1, a novel retinoic acid (RA) catabolising enzyme, and examined its expression pattern during early stages of chick embryogenesis. It is expressed from stage 7 in the tail bud, an anterior patch of mesenchyme, the heart, the endothelium of the vasculature, the eye, the limb bud, the hindgut and in a complex pattern in the rhombomeres of the hindbrain. As such it has a non-overlapping expression with chick Cyp26A1, the other RA catabolising enzyme, but shows a combination of features of mouse Cyp26A1 and Cyp26B1. We have also examined its expression in the quail embryo and in the RA-free quail embryo. In the absence of RA, Cyp26B1 is only expressed in the hindbrain and fails to be expressed in all the other regions of the embryo, most dramatically in the trunk. Adding back RA rescues Cyp26B1 expression.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.     

Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest

The anterior-posterior identities of cells in the hindbrain and cranial neural crest are thought to be determined by their Hox gene expression status, but how and when cells become committed to these identities remain unclear. Here we address this in zebrafish by cell transplantation, to test plasticity in hox expression in single cells. We transplanted cells alone, or in small groups, between hindbrain rhombomeres or between the neural crest primordia of pharyngeal arches. We found that transplanted cells regulated hox expression according to their new environments. The degree of plasticity, however, depended on both the timing and the size of the transplant. At later stages transplanted cells were more likely to be irreversibly committed and maintain their hox expression, demonstrating a progressive loss of responsiveness to the environmental signals that specify segmental identities. Individual transplanted cells also showed greater plasticity than those lying within the center of larger groups, suggesting that a community effect normally maintains hox expression within segments. We also raised experimental embryos to larval stages to analyze transplanted cells after differentiation and found that neural crest cells contributed to pharyngeal cartilages appropriate to the anterior-posterior level of the new cellular environment. Thus, consistent with models implicating hox expression in control of segmental identity, plasticity in hox expression correlates with plasticity in final cell fate.     

A CYP26B1 polymorphism enhances retinoic acid catabolism and may aggravate atherosclerosis

All-trans retinoic acid, controlled by cytochrome P450, family 26 (CYP26) enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26 subfamily B, polypeptide 1 (CYP26B1) in atherosclerosis and the effects of a genetic polymorphism in CYP26B1 on retinoid catabolism. We found that CYP26B1 mRNA was induced by retinoic acid in human atherosclerotic arteries, and CYP26B1 and the macrophage marker CD68 were colocalized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic arteries than in normal arteries. Databases were queried for nonsynonymous CYP26B1 single nucleotide polymorphisms (SNPs) and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophagelike cells. The minor variant catabolized retinoic acid with significantly higher efficiency, indicating that rs2241057 is functional and suggesting reduced retinoid availability in tissues with the minor variant. rs2241057 was investigated in a Stockholm Coronary Atherosclerosis Risk Factor (SCARF) subgroup. The minor allele was associated with slightly larger lesions, as determined by angiography. In summary, this study identifies the first CYP26B1 polymorphism that alters CYP26B1 capacity to metabolize retinoic acid. CYP26B1 was expressed in macrophage-rich areas of human atherosclerotic lesions, induced by retinoic acid and increased in murine atherosclerosis. Taken together, the results indicate that CYP26B1 capacity is genetically regulated and suggest that local CYP26B1 activity may influence atherosclerosis.     

Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding

Homology models of cytochrome P450 RA1 (CYP26A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using MOE software the lowest energy CYP26A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP26A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP26A1 model. The natural substrate, all-trans-retinoic acid (atRA), and inhibitor R 15866, were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure. Multiple hydrophobic interactions, including the side chains of TRP112, PHE299, PHE222, PHE84, PHE374 and PRO371, are important for binding of atRA and R115866. Additional hydrogen bonding interactions were noted as follows: atRA-- C==O of the atRA carboxylate group and ARG86; R115866--benzothiazole nitrogen and the backbone NH of SER115.     

The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives

Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung's disease.     

Irreversible effects of retinoic acid pulse on Xenopus jaw morphogenesis: new insight into cranial neural crest specification

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1–15 min) pulses of low doses of retinoic acid (RA: 0.25–2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6‐hr window (developmental stages NF15–NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA‐dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes. Birth Defects Res (Part B) 89:493–503, 2010. © 2010 Wiley‐Liss, Inc.     

Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish

Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)^pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.     

Effects of retinoic acid on the development of the facial skeleton in hamsters: early changes involving cranial neural crest cells

Treatment of gravid hamsters with 60/mg of retinoic acid on the 8th day of pregnancy resulted in facial skeleton defects in 100% of the survivors examined by alizarin staining at term. An investigation of the early stages in the development of these malformations indicated that the teratogen induced delayed and disorganized patterns of cranial neural crest cell migration as well as extensive death and damage of crest cells. The results demonstrate that retinoic acid provides a useful tool for studies in the pathogenesis of facial skeletal abnormalities in vivo. Moreover, the extensive defects seen in the teratogen-treated litters at term, together with the results of the microscopical analyses, support the hypothesis that cranial neural crest cells make an important contribution to the development of the mammalian facial skeleton.     

The mych gene is required for neural crest survival during zebrafish development

Background

Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes.

Principal Findings

We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain.

Significance

Mych is a novel factor required for neural crest cell survival in zebrafish.     

An intermediate level of BMP signaling directly specifies cranial neural crest progenitor cells in zebrafish

The specification of the neural crest progenitor cell (NCPC) population in the early vertebrate embryo requires an elaborate network of signaling pathways, one of which is the Bone Morphogenetic Protein (BMP) pathway. Based on alterations in neural crest gene expression in zebrafish BMP pathway component mutants, we previously proposed a model in which the gastrula BMP morphogen gradient establishes an intermediate level of BMP activity establishing the future NCPC domain. Here, we tested this model and show that an intermediate level of BMP signaling acts directly to specify the NCPC. We quantified the effects of reducing BMP signaling on the number of neural crest cells and show that neural crest cells are significantly increased when BMP signaling is reduced and that this increase is not due to an increase in cell proliferation. In contrast, when BMP signaling is eliminated, NCPC fail to be specified. We modulated BMP signaling levels in BMP pathway mutants with expanded or no NCPCs to demonstrate that an intermediate level of BMP signaling specifies the NCPC. We further investigated the ability of Smad5 to act in a graded fashion by injecting smad5 antisense morpholinos and show that increasing doses first expand the NCPCs and then cause a loss of NCPCs, consistent with Smad5 acting directly in neural crest progenitor specification. Using Western blot analysis, we show that P-Smad5 levels are dose-dependently reduced in smad5 morphants, consistent with an intermediate level of BMP signaling acting through Smad5 to specify the neural crest progenitors. Finally, we performed chimeric analysis to demonstrate for the first time that BMP signal reception is required directly by NCPCs for their specification. Together these results add substantial evidence to a model in which graded BMP signaling acts as a morphogen to pattern the ectoderm, with an intermediate level acting in neural crest specification.     

RALDH-independent generation of retinoic acid during vertebrate embryogenesis by CYP1B1

Several independent lines of evidence have revealed an instructive role for retinoic acid (RA) signalling in the establishment of normal pattern and cellular specification of the vertebrate embryo. Molecular analyses have previously identified the major RA-synthesising (RALDH1-3) and RA-degrading (CYP26A-C1) enzymes as well as other components involved in RA processing (e.g. CRABP). Although the majority of the early effects of RA can be attributed to the activity of RALDH2, many other effects are suggestive of the presence of an as yet unidentified RA source. Here we describe the identification, expression, biochemistry and functional analysis of CYP1B1, a member of the cytochrome p450 family of mono-oxygenases, and provide evidence that it contributes to RA synthesis during embryonic patterning. We present in vitro biochemical data demonstrating that this enzyme can generate both all-trans-retinal (t-RAL) and all-trans-retinoic acid (t-RA) from the precursor all-trans-retinol (t-ROH), but unlike the CYP26s, CYP1B1 cannot degrade t-RA. In particular, we focussed on the capacity of CYP1B1 to regulate the molecular mechanisms associated with dorsoventral patterning of the neural tube and acquisition of motor neuron progenitor domain identity. Concordant with its sites of expression and biochemistry, data are presented demonstrating that CYP1B1 is capable of eliciting responses that are consistent with the production of RA. Taken together, we propose that these data provide strong support for CYP1B1 being one of the RALDH-independent components by which embryos direct RA-mediated patterning.