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.     

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.     

Agnathia and associated malformations in a male rhesus monkey

BACKGROUND: Agnathia is a rare malformation characterized by the absence of the mandible. METHODS: A male rhesus monkey with malformations was found dead and studied by internal examination, radiographs and histopathology. RESULTS: A case of a rare first branchial arch anomaly with agenesis of the mandible and tongue is presented. The animal also had visceral deformities. However, ears were normal in shape and only slightly low in position. The craniofacial malformations may reflect incomplete separation of the first branchial arch into its maxillary and mandibular processes. CONCLUSIONS: The association between the craniofacial and other corporal anomalies is unclear.     

Is Far a Hox mutation?

The mouse First arch mutation, Far, causes a severe syndrome of craniofacial defects described previously. All of the known defects are derived from the anterior first arch, and to a very small extent, the dorsal second arch. Recently Far has been shown to be closely linked to Ulnaless on chromosome 2, and therefore in the vicinity of the Hox-4 gene cluster. This paper reports the results of several studies focused on the development origin of the most consistently expressed dominant effect caused by Far, an abnormal major bifurcation of the maxillary nerve. Nerve-stained whole-mount preparations of day 12 embryos showed that in Far mutants the maxillary nerve appears to have a central wedge missing from the normal single-stalked fan shape, and that the nerve defect in Far/Far and +/Far may be equally severe. The effect of retinoic acid on the development of the maxillary nerve was tested. Maternal treatment with 5 mg/kg retinoic acid on day 9 of gestation had no detectable effect on the maxillary nerve of +/Far embryos, and similar treatment with a teratogenic dosage (20 mg/kg) on day 8 or 9 produced no Far-like maxillary nerve defects in genetically normal embryos. The neural crest cells that give rise to nerves and mesenchyme of the first arch originate from specific rhombomeres, discrete segments of the developing head. The rhombomeres of 15 embryos at the 14-23 somite stages, of which 75% are expected to be +/Far or Far/Far, were examined. There was no detectable defect in segmentation or morphology of the rhombomeres compared with controls. The significance of ectopic cartilage in the palate of Far/Far mutants in relation to nerve bifurcation was explored. In histological studies, five out of six Far/Far day-15 fetuses had a rod of ectopic cartilage lateral to the posterior palate, running parallel to, and morphologically similar to, Meckel's cartilage, and lying between the two trunks of the abnormally bifurcated maxillary nerve. None of six +/Far day-15 fetuses examined had detectable ectopic cartilage in this region. We hypothesize that the maxillary nerve defects in Far mutants may be explained by the presence of an ectopic precartilaginous blastema that does not always further develop into detectable cartilage. The ectopic cartilage found in Far/Far resembles the epibranchial cartilage expressed in more posterior branchial arches and in the first arch of lower organisms, and therefore may represent an atavistic posteriorization of the anterior first arch in Far mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon

BACKGROUND: Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON‐induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS: Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250 µ M) at different stages and examined after 48 hr of culture. The sensitive window for FON‐induced abnormalities was during the hours E9 h8.00 PM–E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS: FON was able to affect the expression of some genes in a stage‐specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON‐induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA‐related signals. Birth Defects Res (Part B) 92:77‐81, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Locally released retinoic acid repatterns the first branchial arch cartilages in vivo

The fates of cranial neural crest cells are unique compared to trunk neural crest. Cranial neural crest cells form bone and cartilage and ultimately these cells make up the entire facial skeleton. Previous studies had established that exogenous retinoic acid has effects on neurogenic derivatives of cranial neural crest cells and on segmentation of the hindbrain. In the present study we investigated the role of retinoic acid on the skeletal derivatives of migrating cranial neural crest cells. We wanted to test whether low doses of locally applied retinoic acid could respecify the neural crest-derived, skeletal components of the beak in a reproducible manner. Retinoic acid-soaked beads were positioned at the presumptive mid-hindbrain junction in stage 9 chicken embryos. Two ectopic cartilage elements were induced, the first a sheet of cartilage ventral and lateral to the quadrate and the second an accessory cartilage rod branching from Meckel's cartilage. The accessory rod resembled a retroarticular process that had formed within the first branchial arch domain. In addition the quadrate was often displaced laterally and fused to the retroarticular process. The next day following bead implantation, expression domains of Hoxa2 and Hoxb1 were shifted in an anterior direction up to the mesencephalon and Msx-2 was slightly down-regulated in the hindbrain. Despite down-regulation in neural crest cells, the onset of Msx-2 expression in the facial prominences at stage 18-20 was normal. This correlates with normal distal beak morphology. Focal labeling of neural crest with DiI showed that instead of migrating in a neat group toward the second branchial arch, a cohort of labeled cells from r4 spread anteriorly toward the proximal first arch region. AP-2 expression data confirmed the uninterrupted presence of AP-2-expressing cells from the anterior mesencephalon to r4. The morphological changes can be explained by mismigration of r4 neural crest into the first arch, but at the same time maintenance of their identity. Up-regulation of the Hoxa2 gene in the first branchial arch may have encouraged r4 cells to move in the anterior direction. This combination of events leads to the first branchial arch assuming some of the characteristics of the second branchial arch.     

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.     

Role of the early epithelium in the patterning of the teeth and Meckel's cartilage.

Embryonic epithelium from the mandibular branchial arch organizes the dentition and the deposition of Meckel's cartilage. During 9-11 days of gestation mandibular arch epithelium can induce teeth in nondental ectomesenchyme in both mice and birds. In addition, the deposition of Meckel's cartilage as a rod of cartilage in the middle of the first branchial arch is under the control of the epithelium. The epithelium inhibits chondrogenesis; if it is removed, large amorphous masses of cartilage are found instead of the narrow rod typical of Meckel's cartilage.     

Study of the mechanisms of BUdR-induced cleft palate in the mouse

This study was designed to examine the pathogenesis of bromodeoxyuridine (BUdR)-induced clefts of the secondary palate in the LACA mouse. Intraperitoneal injections of BUdR (500 mg/kg body weight) were given at various days and combinations of days between E11 and E15 (plug day = E1). Treatment on E11 alone resulted in approximately 22% of fetuses with cleft palate when the latter were examined either on E16 or E19. Treatment on E11 and E12 approximately doubled the above incidence, and treatment on E11, 12 and 13 raised it to 100%. However, no treatment, either single or multiple, caused cleft palate when given later than E11. This suggests that the cellular changes caused by BUdR that lead to cleft palate must be inflicted during E11 and that such damage can be repaired in about 80% of embryos. All fetuses with cleft palate had severe micrognathia on E16 and E19, which skeletal staining showed to be the result of a bilateral sigmoid buckling of Meckel's cartilage. Studies with the scanning electron microscope (SEM) on E15, 16, and 19 suggested strongly that the micrognathia caused a relative macroglossia and hence mechanical interference with palatal shelf reorientation. Histological studies with the light microscope showed that BUdR caused cellular necrosis in many embryonic tissues during the 24 hours after its administration. This necrosis was strikingly more severe in the mandibular rudiment of the first branchial arch than in the maxillary. The latter observation accords well with findings by other workers that cell proliferation is more rapid in the mandibular blastema than in the maxillary. Transmission electron microscope (TEM) studies of the buckled region of Meckel's cartilage failed to reveal any ultrastructural differences from control Meckel's cartilage. Hence BUdR had only interfered with the shape of the cartilage but not with its histiogenesis. We conclude that BUdR, by its cytotoxicity or antidifferentiative effects, interfered with the formation of the anterior end of Meckel's cartilage, initiating a chain of events leading through micrognathia and relative macroglossia to failure of palatal shelf reorientation and cleft palate.     

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.     

Malformations in offspring of diabetic rats: morphometric analysis of neural crest-derived organs and effects of maternal vitamin E treatment

BACKGROUND: We have previously reported on a malformation-prone Sprague-Dawley rat substrain (U), which presents a high frequency of micrognathia in the offspring of diabetic mothers. This malformation is related to impaired development of the cranial neural crest cells (NCC); the defect may be prevented by antioxidative treatment of the mother. METHODS: We have therefore investigated whether fetuses of diabetic rats display other malformations associated with altered cranial NCC development and whether maternal vitamin E supplementation may affect such malformations. RESULTS: Fetuses of diabetic rats showed low-set external ears, severely malformed Meckel's cartilage, small thyroid and thymus, and absence of parathyroid glands. Cardiac anomalies were frequently observed, including rightward displacement of the aorta, double outlet right ventricle (DORV), persistent truncus arteriosus (PTA) combined with ventricular septal defects due to a malaligned outlet septum. The malformations in the outflow tract included abnormalities of the great arteries; right-sided aortic arch/descending aorta, and double aortic arches. These defects tended to occur together within individual fetuses. Maternal dietary treatment with 2% vitamin E markedly reduced the severity of the malformations. CONCLUSIONS: The phenotypic appearance of these defects is strikingly similar to the DiGeorge anomaly in humans, which has been found in children of diabetic mothers together with an overrepresentation of PTA and DORV. The malformations associated with defective NCC development in the offspring of diabetic U rats show several morphological similarities to those in humans; hence the teratogenic mechanisms may be similar and accessible for study.     

Developmental anomalies induced by all-trans retinoic acid in fetal mice: I. Macroscopic findings

The administration of a single dose of all-trans retinoic acid on day 8 of gestation to pregnant mice, ICR strain, led to malformed fetuses in all of the litters. All-trans retinoic acid (RA) was dissolved in olive oil and given in doses of 60 or 40 mg/kg of body weight. The control mice were given vehicle alone. Examination on day 18 of gestation of the fetuses exposed to 60 mg/kg showed various malformations, such as exencephaly, exophthalmus, micrognathia, agnathia, cleft palate, cleft lower lip, spina bifida, atresia ani, tail anomalies, agenesis of the kidneys, or hydronephrosis. In the fetuses exposed to 40 mg/kg, isolated cleft palate was much more common than in those exposed to 60 mg/kg. Double-stained preparations of bone and cartilage showed cranio-facial anomalies and axial skeletal anomalies: a- or hypogenesis of palatine or maxillary bones, tympanic ring, squamosal temporal bone or otic ossicles in cartilage, and fusion of basioccipital to basisphenoid and maxilla, zygomatic and mandibular bones; a- or hypogenesis of caudal vertebrae and supernumerary thoracic and lumbar vertebrae. These results indicate that anomalies comparable to those seen in the infants of mothers treated with isotretinoin, 13-cis retinoic acid, during pregnancy can also be induced in mice and suggest that the site affected by RA may be neural crest cells, including those in the cephalic and caudal regions, and cells committed to somitic mesoderm in the trunk region.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.     

Temporospatial cell interactions regulating mandibular and maxillary arch patterning

The cellular origin of the instructive information for hard tissue patterning of the jaws has been the subject of a long-standing controversy. Are the cranial neural crest cells prepatterned or does the epithelium pattern a developmentally uncommitted population of ectomesenchymal cells? In order to understand more about how orofacial patterning is controlled we have investigated the temporal signalling interactions and responses between epithelium and mesenchymal cells in the mandibular and maxillary primordia. We show that within the mandibular arch, homeobox genes that are expressed in different proximodistal spatial domains corresponding to presumptive molar and incisor ectomesenchymal cells are induced by signals from the oral epithelium. In mouse, prior to E10, all ectomesenchyme cells in the mandibular arch are equally responsive to epithelial signals such as Fgf8, indicating that there is no pre-specification of these cells into different populations and suggesting that patterning of the hard tissues of the mandible is instructed by the epithelium. By E10.5, ectomesenchymal cell gene expression domains are still dependent on epithelial signals but have become fixed and ectopic expression cannot be induced. At E11 expression becomes independent of epithelial signals such that removal of the epithelium does not affect spatial ectomesenchymal expression. Significantly, however, the response of ectomesenchyme cells to epithelial regulatory signals was found to be different in the mandibular and maxillary primordium. Thus, whereas both mandibular and maxillary arch epithelia could induce Dlx2 and Dlx5 expression in the mandible and Dlx2 expression in the maxilla, neither could induce Dlx5 expression in the maxilla. Reciprocal cell transplantations between mandibular and maxillary arch ectomesenchymal cells revealed intrinsic differences between these populations of cranial neural crest-derived cells. Research in odontogenesis has shown that the oral epithelium of the mandibular and maxillary primordia has unique instructive signaling properties required to direct odontogenesis, which are not found in other branchial arch epithelia. As a consequence, development of jaw-specific skeletal structures may require some prespecification of maxillary ectomesenchyme to restrict the instructive influence of the epithelial signals and allow development of maxillary structures distinct from mandibular structures.     

Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.     

The fate of cranial neural crest cells in the Australian lungfish, Neoceratodus forsteri

The cranial neural crest has been shown to give rise to a diversity of cells and tissues, including cartilage, bone and connective tissue, in a variety of tetrapods and in the zebrafish. It has been claimed, however, that in the Australian lungfish these tissues are not derived from the cranial neural crest, and even that no migrating cranial neural crest cells exist in this species. We have earlier documented that cranial neural crest cells do migrate, although they emerge late, in the Australian lungfish. Here, we have used the lipophilic fluorescent dye, DiI, to label premigratory cranial neural crest cells and follow their fate until stage 43, when several cranial skeletal elements have started to differentiate. The timing and extent of their migration was investigated, and formation of mandibular, hyoid and branchial streams documented. Cranial neural crest was shown to contribute cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g., Meckel's cartilage, quadrate), the hyoid arch (e.g., the ceratohyal) and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofibers in cranial muscles. We conclude that cranial neural crest migration and fate in the Australian lungfish follow the stereotyped pattern documented in other vertebrates.