Platelet-derived growth factor C plays a role in the branchial arch malformations induced by retinoic acid

BACKGROUND: All-trans-retinoic acid (RA) can produce branchial arch abnormalities in postimplantation rodent embryos cultured in vitro. Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Many members of the PDGF family are essential for branchial arch morphogenesis and can be regulated by RA. The roles of PDGF-C in branchial arch malformations induced by RA and possible mechanisms were investigated. METHODS: In whole embryo culture (WEC), mouse embryos were exposed to RA at 0, 0.1, 0.4, 1.0, or 10.0 microM, PDGF-C at 25, 50, or 75 ng/mL, or PDGF-C at 25, 50, or 75 ng/mL containing 0.4 microM RA. After 48 h of culture, mouse embryos were examined for dysmorphogenesis, and whole-mount immunohistochemistry was applied to PDGF-C. In explant cultures, explants were exposed to the same doses of RA and PDGF-C as WEC. Semiquantitative RT-PCR, zymography, and reverse zymography were used to evaluate the expressions and activities of matrix metalloproteinase (MMP)-2, MMP-14, and tissue inhibitor of metalloproteinase (TIMP)-2. RESULTS: PDGF-C was reduced by RA, and exogenous PDGF-C rescued the branchial arch malformations induced by RA. Moreover, PDGF-C prevented RA-induced inhibition of the migratory ability of mesenchymal cells in the first branchial arch, by regulating the expressions of MMP-2, MMP-14, and TIPM-2. CONCLUSIONS: Our results suggest that RA exposure reduces the expression of PDGF-C. The branchial arch malformations resulting from fetal RA exposure are caused at least partially by loss of PDGF-C and subsequent misregulations of the expressions of MMP-2, MMP-14, and TIMP-2.     

Craniofacial and axial skeletal defects induced by the fungicide triadimefon in the mouse

BACKGROUND: Triadimefon is an antifungal derived from triazole. In in vitro whole-rodent embryo cultures, triazole-derivatives showed specific teratogenic effects at the branchial apparatus. The aim of the present work was to test in vivo triadimefon (FON), in order to verify a relationship between triazole exposure, embryonic abnormalities, and/or fetal malformations. METHODS: Pregnant CD-1 mice were treated with 0-300 mg/kg FON by gavage on day 8 post coitum (p.c.) at 10:00 AM, and sacrificed on day 8 p.c. at 1:00 PM, on day 9 p.c. at 10:00 AM, on day 10 p.c. at 10:00 AM, and at term of gestation (day 18 p.c.). At midgestation, the embryos were processed for specific immunostainings to visualize the hindbrain segmentation (day 8 p.c.) and the neural crest cell migration (days 8 and 9 p.c.). Fetuses explanted at term were all processed for skeletal examination after double-staining of osseous and cartilaginous tissues. RESULTS: At midgestation, the immunostaining of rhombomeres 3 and 5 showed a light scattering of the immunostained areas; the neural crest cell migration was unaffected, but their localization at the branchial arch level was abnormal. At term, several severe malformations were observed at the craniofacial and at the axial skeletal level. Ectopic cartilage was observed at the upper jaw. CONCLUSIONS: Triadimefon is teratogenic. The observed craniofacial malformations could be explained by an alteration of the rhombomeric organization and neural crest migration to the branchial arches; the axial abnormalities could be explained by the abnormal segmental identity specification.     

Cyclopia-otocephaly association: a new case of the most severe variant of agnathia-holoprosencephaly complex

This report describes a new case of true cyclopia with otocephaly and additional brain malformations (alobar holoprosencephaly). This is a very rare occurrence involving lack of cleavage of the prosencephalon and disturbed development of the first branchial arch. An inductive defect of the prechordal mesoderm is considered as the cause for this malformation, which is a part of agnathia-holoprosencephaly complex.     

Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells

Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.     

Antifungal triazole derivative triadimefon induces ectopic maxillary cartilage by altering the morphogenesis of the first branchial arch

BACKGROUND: The triazole derivative, triadimefon (FON), induces branchial arch abnormalities in post-implantation rat embryos cultured in vitro, and cranio-facial malformations in mouse fetuses. Ectopic maxillary cartilage has been also described as a typical FON-related malformation. This work studies the morphogenesis of the ectopic cartilage in rat embryos and fetuses exposed in vivo to FON during the early postimplantation period. METHODS: Pregnant rats were treated with 0, 250, and 500 mg/kg FON on Day 9.5 of pregnancy (D9.5) and sacrificed at term (D20), during the early fetal period (D17) or at different embryogenetic periods (D10, D11, D12). The skeleton was examined after stain of bone and cartilage or of cartilage alone respectively at term or at D17. The neural crest cell (NCC) migration and compaction was investigated at D10 and D11 and the cranial nerve organization described at D12. RESULTS: Triadimefon is teratogenic in rats under the chosen experimental conditions. The malformations were at the level of the cranio-facial and axial skeleton at term and of the hindbrain nerves in embryos. A NCC abnormal migration and compaction was observed at the level of the first branchial arch: in FON-exposed embryos NCC were detected at the level of both maxillary and mandibular processes, whereas control embryos showed the immunostained tissue only at the level of the mandibular bud. CONCLUSIONS: The pathogenic pathway, proposed to explain the ectopic cartilage, is the displacement of part of the NCC-derived tissues at the maxillary region of the first branchial arch.     

Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy

Isotretinoin ingestion during the first trimester of human pregnancy can induce malformations of the skull, ears, face, central nervous system, eyes, palate, lungs, circulatory system, limbs, and digits. A single oral dose of isotretinoin on day 8 of gestation in hamsters induces a similar syndrome of congenital malformation. The present study concerned scanning electron microscopic (SEM) observation of embryonic and fetal hamster craniofacial structures at 4, 8, 12, 24, 48, and 72 hr after administration of an oral dose of 50 mg/kg isotretinoin or an equivalent volume of the vehicle. The variability in development among control embryos recovered 4 hr after treatment precluded objective assessment of pathologic change by SEM at very early time points. Craniofacial damage was obvious within 8-12 hr of isotretinoin treatment, and it included hypoplasia of the maxillary and mandibular processes of the first branchial arch, a rudimentary second arch, and apparent collapse of the forebrain. Equivalent fusion between the lateral nasal process and the maxillary process and between the medial nasal process and the maxillary process in treated and control embryos accounts for the very low incidence of cleft lip observed in fetuses. The terminal microstomia was not associated with excessive merging or overgrowth of the first arch components. Hypoplasia of the first arch can account for retinoid-induced macrostomia and microstomia.     

Dlx5 regulates regional development of the branchial arches and sensory capsules.

We report the generation and analysis of mice homozygous for a targeted deletion of the Dlx5 homeobox gene. Dlx5 mutant mice have multiple defects in craniofacial structures, including their ears, noses, mandibles and calvaria, and die shortly after birth. A subset (28%) exhibit exencephaly. Ectodermal expression of Dlx5 is required for the development of olfactory and otic placode-derived epithelia and surrounding capsules. The nasal capsules are hypoplastic (e.g. lacking turbinates) and, in most cases, the right side is more severely affected than the left. Dorsal otic vesicle derivatives (e. g. semicircular canals and endolymphatic duct) and the surrounding capsule, are more severely affected than ventral (cochlear) structures. Dlx5 is also required in mandibular arch ectomesenchyme, as the proximal mandibular arch skeleton is dysmorphic. Dlx5 may control craniofacial development in part through the regulation of the goosecoid homeobox gene. goosecoid expression is greatly reduced in Dlx5 mutants, and both goosecoid and Dlx5 mutants share a number of similar craniofacial malformations. Dlx5 may perform a general role in skeletal differentiation, as exemplified by hypomineralization within the calvaria. The distinct focal defects within the branchial arches of the Dlx1, Dlx2 and Dlx5 mutants, along with the nested expression of their RNAs, support a model in which these genes have both redundant and unique functions in the regulation of regional patterning of the craniofacial ectomesenchyme.     

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.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.     

Otocephaly, and pulmonary malformation association: two case reports

Otocephaly is a rare lethal malformation of the first and second branchial arches. We report two infants with otocephaly and failed resuscitation. Both infants had pulmonary malformations: pulmonary hypoplasia and two-lobed right lung. We report these rare associations with a brief review of the literature.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

Hox genes, neural crest cells and branchial arch patterning

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

Hox genes, neural crest cells and branchial arch patterning.

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

Diminished matrix metalloproteinase 2 (MMP-2) in ectomesenchyme-derived tissues of the Patch mutant mouse: regulation of MMP-2 by PDGF and effects on mesenchymal cell migration.

Platelet-derived growth factors (PDGF) regulate cell proliferation, survival, morphology, and migration, as well as deposition and turnover of the extracellular matrix. Important roles for the A form of PDGF (PDGF-A) during connective tissue morphogenesis have been highlighted by the murine Patch mutation, which includes a deletion of the alpha subunit of the PDGF receptor. Homozygous (Ph/Ph) embryos exhibit multiple connective tissue defects including cleft face (involving the first branchial arch and frontonasal processes), incomplete heart septation, and heart valve abnormalities before they die in utero. Analyses of the cell biology underlying the defects in Ph/Ph embryos have revealed a deficit in a matrix metalloproteinase (MMP-2) and one of its activators (MT-MMP) that are likely to be involved in cell migration and tissue remodeling, two processes necessary for normal cardiac and craniofacial development. Morphogenesis of these structures requires infiltration of ectomesenchymal precursors and their subsequent deposition and remodeling of extracellular matrix components. First branchial arch and heart tissue from E10.5 embryos were examined by gelatin zymography and RT-PCR in order to characterize the expression of MMPs in these tissues. Of the MMPs examined, only MMP-2 and one of its activators, MT-MMP, were expressed in the first arch and heart at this stage of development. Tissues from Ph/Ph embryos exhibited a significant decrease in both MMP-2 and MT-MMP compared to tissues from normal embryos of the same developmental stage. In order to assess whether this decrease affects the motile activity of mesenchymal cells, cell migration from Ph/Ph branchial arch explants was compared to migration from normal arch tissue and found to be significantly less. In addition, the migratory ability of branchial arch cells from normal explants could be reduced in a similar manner using a specific MMP inhibitor. Although it is still unclear whether the MMP-2 reduction is a direct result of the absence of response of Ph/Ph cells to PDGF-A treatment of normal branchial arch cells in vitro with recombinant PDGF-AA significantly upregulated MMP-2 protein. Together, these results suggest that PDGF-A regulates MMP-2 expression and activation during normal development and that faulty proteinase expression may be at least partially responsible for the developmental defects exhibited by Ph/Ph embryos.     

Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles

Recent knockout experiments in the mouse generated amazing craniofacial skeletal muscle phenotypes. Yet none of the genes could be placed into a molecular network, because the programme to control the development of muscles in the head is not known. Here we show that antagonistic signals from the neural tube and the branchial arches specify extraocular versus branchiomeric muscles. Moreover, we identified Fgf8 as the branchial arch derived signal. However, this molecule has an additional function in supporting the proliferative state of myoblasts, suppressing their differentiation, while a further branchial arch derived signal, namely Bmp7, is an overall negative regulator of head myogenesis.     

Mapping the mouse craniofacial mutation first arch (Far) to chromosome 2

First arch (Far) is a semidominant mutation that causes severe craniofacial defects in mice. Here we report the results of linkage studies with the chromosome 2 markers nonagouti, pallid, and Ulnaless. Far is loosely linked to nonagouti (24-37 cM), more closely linked to pallid (13-28 cM), and closely linked to Ulnaless (2.3 +/- 1.5 cM). The embryological defect in Far mutants is confined to one segmentally-derived region of the head, the anterior first branchial arch. It may therefore be significant that, in mapping near Ulnaless, Far also maps in the vicinity of the Hox-4 gene cluster.     

Smad4 is required to regulate the fate of cranial neural crest cells

Smad4 is the central mediator for TGF-β/BMP signals, which are involved in regulating cranial neural crest (CNC) cell formation, migration, proliferation and fate determination. It is unclear whether TGF-β/BMP signals utilize Smad-dependent or -independent pathways to control the development of CNC cells. To investigate the functional significance of Smad4 in regulating CNC cells, we generated mice with neural crest specific inactivation of the Smad4 gene. Our study shows that Smad4 is not required for the migration of CNC cells, but is required in neural crest cells for the development of the cardiac outflow tract. Smad4 is essential in mediating BMP signaling in the CNC-derived ectomesenchyme during early stages of tooth development because conditional inactivation of Smad4 in neural crest derived cells results in incisor and molar development arrested at the dental lamina stage. Furthermore, Smad-mediated TGF-β/BMP signaling controls the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. At the cellular level, a Smad4-mediated downstream target gene(s) is required for the survival of CNC cells in the proximal domain of the first branchial arch. Smad4 mutant mice show underdevelopment of the first branchial arch and midline fusion defects. Taken together, our data show that TGF-β/BMP signals rely on Smad-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis.     

A case of otocephaly with anencephaly and meningomyelocele

A case of otocephaly with anencephaly and meningomyelocele: Otocephaly is a rare lethal syndrome with microstomia, aglossia, agnathia, and synotia as major clinical features due to arrest in development of the first branchial arch. Some associated anomalies may be present as cyclopia, holoprosencephaly, cerebellar hypoplasia, situs inversus, and other visceral anomalies. We describe a case of fetus, spontaneously aborted in the 14th week of gestation with otocephaly complex (agnathia, synotia, microstomia) and associated anencephaly and meningomyelocele.