Identification and functional analysis of novel facial patterning genes in the duplicated beak chicken embryo.

Cranial neural crest cells form the majority of the facial skeleton. However exactly when the pattering information and hence jaw identity is established is not clear. We know that premigratory neural crest cells contain a limited amount of information about the lower jaw but the upper jaw and facial midline are specified later by local tissue interactions. The environmental signals leading to frontonasal identity have been explored by our group in the past. Altering the levels of two signaling pathways (Bone Morphogenetic Protein) and retinoic acid (RA) in the chicken embryo creates a duplicated midline on the side of the upper beak complete with egg tooth in place of maxillary derivatives (Lee et al., 2001). Here we analyze the transcriptome 16 h after bead placement in order to identify potential mediators of the identity change in the maxillary prominence. The gene list included RA, BMP and WNT signaling pathway genes as well as transcription factors expressed in craniofacial development. There was also cross talk between Noggin and RA such that Noggin activated the RA pathway. We also observed expression changes in several poorly characterized genes including the upregulation of Peptidase Inhibitor-15 (PI15). We tested the functional effects of PI15 overexpression with a retroviral misexpression strategy. PI15 virus induced a cleft beak analogous to human cleft lip. We next asked whether PI15 effects were mediated by changes in expression of major clefting genes and genes in the retinoid signaling pathway. Expression of TP63, TBX22, BMP4 and FOXE1, all human clefting genes, were upregulated. In addition, ALDH1A2, ALDH1A3 and RA target, RARß were increased while the degradation enzyme CYP26A1 was decreased. Together these changes were consistent with activation of the RA pathway. Furthermore, PI15 retrovirus injected into the face was able to replace RA and synergize with Noggin to induce beak transformations. We conclude that the microarrays have generated a rich dataset containing genes with important roles in facial morphogenesis. Moreover, one of these facial genes, PI15 is a putative clefting gene and is in a positive feedback loop with RA.

Avian facial morphogenesis is regulated by c-Jun N-terminal kinase/planar cell polarity (JNK/PCP) wingless-related (WNT) signaling.

Wingless-related proteins (WNTs) regulate extension of the central axis of the vertebrate embryo (convergent extension) as well as morphogenesis of organs such as limbs and kidneys. Here, we asked whether WNT signaling directs facial morphogenesis using a targeted approach in chicken embryos. WNT11 is thought to mainly act via ß-catenin-independent pathways, and little is known about its role in craniofacial development. RCAS::WNT11 retrovirus was injected into the maxillary prominence, and the majority of embryos developed notches in the upper beak or the equivalent of cleft lip. Three-dimensional morphometric analysis revealed that WNT11 prevented lengthening of the maxillary prominence, which was due in part to decreased proliferation. We next determined, using a series of luciferase reporters, that WNT11 strongly induced JNK/planar cell polarity signaling while repressing the ß-catenin-mediated pathway. The activation of the JNK-ATF2 reporter was mediated by the DEP domain of Dishevelled. The impacts of altered signaling on the mesenchyme were assessed by implanted Wnt11- or Wnt3a-expressing cells (activates ß-catenin pathway) into the maxillary prominence or by knocking down endogenous WNT11 with RNAi. Host cells were attracted to Wnt11 donor cells. In contrast, cells exposed to Wnt3a or the control cells did not migrate. Cells in which endogenous WNT11 was knocked down were more oriented and shorter than those exposed to exogenous WNT11. The data suggest that JNK/planar cell polarity WNT signaling operates in the face to regulate several morphogenetic events leading to lip fusion.

Dual functions for WNT5A during cartilage development and in disease.

Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the canonical pathway from mediating the matrix loss phenotype. The JNK antagonist partially rescued the Wnt5a phenotype supporting this non-canonical pathway as the main mediator of the cartilage matrix degradation. Our study reveals two new roles for WNT5A in development and disease: 1) to repress canonical Wnt signaling in cartilage blastema in order to promote normal differentiation and 2) in conditions of excess to stimulate degradation of mature cartilage matrix via non-canonical pathways.

Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos.

Here we report that highly concentrated cationic lipid/helper lipid-nucleic acid complexes (lipoplexes) can facilitate reproducible delivery of a variety of oligonucleotides and plasmids to chicken embryos or to mouse embryonic mesenchyme. Specifically, liposomes composed of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)/1,2 dioleoyl glycero-3-phosphorylethanolamine (DOPE) prepared at 18-mM concentrations produced high levels of transfection of exogenous genes in vivo and in vitro. Furthermore, we report sufficient uptake of plasmids expressing interference RNA to decrease expression of both exogenous and endogenous genes. The simplicity of preparation, implementation, and relatively low toxicity of this transfection reagent make it an attractive alternative for developmental studies in post-gastrulation vertebrate embryos.

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo.

BACKGROUND: Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis. RESULTS: In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II. CONCLUSION: Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

Whole genome microarray analysis of chicken embryo facial prominences.

The face is one of the three regions most frequently affected by congenital defects in humans. To understand the molecular mechanisms involved, it is necessary to have a more complete picture of gene expression in the embryo. Here, we use microarrays to profile expression in chicken facial prominences, post neural crest migration and before differentiation of mesenchymal cells. Chip-wide analysis revealed that maxillary and mandibular prominences had similar expression profiles while the frontonasal mass chips were distinct. Of the 3094 genes that were differentially expressed in one or more regions of the face, a group of 56 genes was subsequently validated with quantitative polymerase chain reaction (QPCR) and a subset examined with in situ hybridization. Microarrays trends were consistent with the QPCR data for the majority of genes (81%). On the basis of QPCR and microarray data, groups of genes that characterize each of the facial prominences can be determined.

Expression of WNT signalling pathway genes during chicken craniofacial development.

A comprehensive expression analysis of WNT signalling pathway genes during several stages of chicken facial development was performed. Thirty genes were surveyed including: WNT1, 2B, 3A, 4, 5A, 5B, 6, 7A, 7B, 8B, 8C, 9A, 9B, 11, 11B, 16, CTNNB1, LEF1, FRZB1, DKK1, DKK2, FZD1-8, FZD10. The strictly canonical WNTs (2B, 7A, 9B, and 16) in addition to WNT4 WNT6 (both canonical and non-canonical) are epithelially expressed, whereas WNT5A, 5B, 11 are limited to the mesenchyme. WNT16 is limited to the invaginating nasal pit, respiratory epithelium, and lip fusion zone. Antagonists DKK1 and FRZB1 are expressed in the fusing primary palate but then are decreased at stage 28 when fusion is beginning. This suggests that canonical WNT signalling may be active during lip fusion. Mediators of canonical signalling, CTNNB1, LEF1, and the majority of the FZD genes are expressed ubiquitously. These data show that activation of the canonical WNT pathway is feasible in all regions of the face; however, the localization of ligands and antagonists confers specificity.

Novel skeletogenic patterning roles for the olfactory pit.

The position of the olfactory placodes suggests that these epithelial thickenings might provide morphogenetic information to the adjacent facial mesenchyme. To test this, we performed in ovo manipulations of the nasal placode in the avian embryo. Extirpation of placodal epithelium or placement of barriers on the lateral side of the placode revealed that the main influence is on the lateral nasal, not the frontonasal, mesenchyme. These early effects were consistent with the subsequent deletion of lateral nasal skeletal derivatives. We then showed in rescue experiments that FGFs are required for nasal capsule morphogenesis. The instructive capacity of the nasal pit epithelium was tested in a series of grafts to the face and trunk. Here, we showed for the first time that nasal pits are capable of inducing bone, cartilage and ectopic PAX7 expression, but these effects were only observed in the facial grafts. Facial mesenchyme also supported the initial projection of the olfactory nerve and differentiation of the olfactory epithelium. Thus, the nasal placode has two roles: as a signaling center for the lateral nasal skeleton and as a source of olfactory neurons and sensory epithelium.

FGF signals from the nasal pit are necessary for normal facial morphogenesis.

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.

Wnt signaling in somite development.

During vertebrate embryogenesis, specialized mesodermal structures, called somites, give rise to a variety of mesodermal tissues including skeletal muscles, vertebrae and dermis. Development of the somites is a rhythmic process that involves a series of steps including segmentation of the paraxial mesoderm, epithelialization, somite formation, somite maturation, somite patterning and differentiation of somitic cells into different lineages. Wnt signaling has been found to play crucial roles in multiple steps of somite development. In this review, we present a brief overview of current knowledge on Wnt signaling events during the development of somites and their derivatives.

Wnt signaling in limb organogenesis.

Secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development of a wide range of taxa from Hydra to humans. The most extensively studied Wnt signaling pathway is the canonical Wnt pathway, which controls gene expression by stabilizing beta-catenin, and regulates a multitude of developmental processes. More recently, noncanonical Wnt pathways, which are beta-catenin-independent, have been found to be important developmental regulators. Understanding the mechanisms of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. Limb development is a paradigm to study the principles of Wnt signaling in various developmental contexts. In the developing vertebrate limb, Wnt signaling has been shown to have important functions during limb bud initiation, limb outgrowth, early limb patterning, and later limb morphogenesis events. This review provides a brief overview on the diversity of Wnt-dependent signaling events during embryonic development of the vertebrate limb.

Expression pattern of Dll4 during chick embryogenesis.

Notch and Delta signaling regulates cell-fate decisions in a variety of tissues in diverse organisms through cell-to-cell interactions. In this study we isolated a 696 bp fragment of chick Delta-like 4 (Dll4) cDNA and analyzed its expression pattern during chick development by in situ hybridization. We report a detailed description of cDll4 expression from HH-stage 8-30. Expression is seen in extraembryonic tissues and in the dorsal aorta throughout development but is absent from venules. Dll4 is expressed in the embryonic blood vessels, heart, somites, neural tube, limb, pharyngeal arches, esophagus, and in the developing eye. In accordance with the report from mice, cDll4 is a marker of the arterial type of endothelial cells. These analyses show that Dll4 is expressed in a wide range of tissues and organs suggesting its role in vascular development during chick embryogenesis.

FGFs, Wnts and BMPs mediate induction of VEGFR-2 (Quek-1) expression during avian somite development.

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By implanting FGF 8b/8c or SU 5402 beads into the paraxial mesoderm, we show that FGF8 in addition to BMP4 from the intermediate mesoderm (IM) is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. The expression of Quek1 in the medial somite half is normally repressed by the notochord and Sfrps-expression in the neural tube. Over-expression of Wnt 1/3a also results in an up-regulation of Quek1 expression in the somites. We also show that up-regulation of FGF8/Wnt 1/3a leads to an increase in the number of endothelial cells, whereas inhibition of FGF and Wnt signaling by SU 5402 and Sfrp-2 results in a loss of endothelial cells. Our results demonstrate that the regulation of Quek1 expression in the somites is mediated by the cooperative actions of BMP4, FGF8 and Wnt-signaling pathways.

Expression pattern of Vasohibin during chick development.

Vasohibin is an angiogenesis inhibitor that is induced in endothelial cells in an autocrine manner. In this study, we cloned a 500-bp fragment of chick Vasohibin cDNA and analyzed its expression pattern by in situ hybridization during chick development. From HH-stage 3, expression of Vasohibin is observed in the area opaca and it is expressed throughout the primitive streak during later stages. At HH-stage 11, Vasohibin is expressed in head paraxial mesoderm, in the vitelline vein, dorsal neural tube, intermediate and lateral plate mesoderm, Wolffian duct, and blood islands at the caudal part of the embryo. In epithelial somites, expression is seen in the region around the somitocoel, and after somite maturation, expression is observed in the myotome, which becomes stronger with development. Expression is detected in fore and hind brain, also in the retina and lens vesicle of the developing eye. In the early limb bud, expression is initiated in the mesenchyme and becomes stronger during later stages. Expression in the limb mesoderm remains strong at the margins but decreases in the central mesenchyme. At day 7, expression is seen in interdigital grooves of the digits and digit-demarcating regions. During organogenesis, expression is seen in the anlagen of the esophagus, trachea, duodenum, lungs, liver, heart, and gut. Our analysis shows that Vasohibin is expressed in a wide range of tissues and organs suggesting that Vasohibin acts as a physiological regulator of vascular development during chick embryogenesis.

Regulation of ectodermal Wnt6 expression by the neural tube is transduced by dermomyotomal Wnt11: a mechanism of dermomyotomal lip sustainment.

Ectodermal Wnt6 plays an important role during development of the somites and the lateral plate mesoderm. In the course of development, Wnt6 expression shows a dynamic pattern. At the level of the segmental plate and the epithelial somites, Wnt6 is expressed in the entire ectoderm overlying the neural tube, the paraxial mesoderm and the lateral plate mesoderm. With somite maturation, expression becomes restricted to the lateral ectoderm covering the ventrolateral lip of the dermomyotome and the lateral plate mesoderm. To study the regulation of Wnt6 expression, we have interfered with neighboring signaling pathways. We show that Wnt1 and Wnt3a signaling from the neural tube inhibit Wnt6 expression in the medial surface ectoderm via dermomyotomal Wnt11. We demonstrate that Wnt11 is an epithelialization factor acting on the medial dermomyotome, and present a model suggesting Wnt11 and Wnt6 as factors maintaining the epithelial nature of the dorsomedial and ventrolateral lips of the dermomyotome, respectively, during dermomyotomal growth.

Role of Wnt-6 in limb myogenesis.

Cells from the ventrolateral lip of the dermomyotome at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The precursor cells are under the influence of various signaling factors in the limb. Dorsal and ventral ectoderm influences various aspects of limb development. In addition to our previous studies, we investigated the influence of ectoderm and Wnt-6 on somitic cells in the limb bud. We show that in the absence of ectoderm the precursor cells never form muscle cells but differentiate into endothelial cells. In addition, we show that Wnt-6 that is secreted from the ectoderm influences the precursor cells to form muscle even in the absence of ectoderm. This indicates that Wnt-6 is an ectodermal signal that induces somite-derived progenitor cells to form muscle cells during limb development.

Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis.

Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.

The formation of the avian scapula blade takes place in the hypaxial domain of the somites and requires somatopleure-derived BMP signals.

The avian scapula is a long bone located dorsally on the thorax. The cranial part that articulates with the upper limb is derived from the somatopleure of the forelimb field, while the caudal part, the scapula blade, originates from the dermomyotomes of brachial and thoracic somites. In previous studies, we have shown that scapula blade formation is intrinsically controlled by segment-specific information as well as extrinsically by ectoderm-derived signals. Here, we addressed the role of signals derived from the lateral plate mesoderm on scapula development. Chick-quail chimera experiments revealed that scapula precursor cells are located within the hypaxial domain of the dermomyotome adjacent to somatopleural cells. Barrier implantation between these two cell populations inhibited scapula blade formation. Furthermore, we identified BMPs as scapula-inducing signals from the somatopleure using injection of Noggin-producing cells into the hypaxial domain of scapula-forming dermomyotomes. We found that inhibition of BMP activity interfered with scapula-specific Pax1 expression and scapula blade formation. Taken together, we demonstrate that the scapula-forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure for their specification and differentiation.

Comparative analysis of the expression patterns of Wnts during chick limb development.

Wnts control a number of processes during limb development-from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. We analyzed the expression pattern of various Wnts (4, 5a, 5b, 6, 11, and 14) in whole mount in situ hybridization during chick wing development. From HH stage 26, expression of Wnt 4 is observed in the central elbow region and wrist-forming regions, and during later stages, expression is seen in the joint-forming regions of the whole limb. Wnt 5a is expressed throughout the limb mesenchyme during early limb developmental stages, and later, at HH stage 23, it becomes predominantly confined to the distal tip, leaving low expression levels proximally. At HH stage 29, expression at the distal tip is restricted to the interdigital regions, and at day 8, expression is seen in the region surrounding the phalanges. Wnt 5b expression is first observed in the AER at HH stage 20 and later in the dorsal and ventral mesenchyme surrounding the cartilage elements of the limb. Expression of Wnt 6 is observed from HH stage 17 until day 8 in the dorsal and ventral ectoderm and also in the dorsoventral limb boundaries. Expression of Wnt 11 is observed in the proximal dorsal mesenchyme of the limb from HH stage 23 onward and later in the dorsal and ventral subectodermal mesenchyme and in the regions adjacent to the digits at day 8. Weak expression of Wnt 14 is observed at the proximal mesenchyme of the limb at HH stage 23; later, it extends as a transverse strip surrounding the cartilage elements as well as in the interdigital mesenchyme.

BMP4 and noggin control embryonic blood vessel formation by antagonistic regulation of VEGFR-2 (Quek1) expression.

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By grafting of either intermediate mesoderm or BMP4 beads into the paraxial mesoderm, we show that BMP4 is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. Separation of somites from intermediate mesoderm leads to down-regulation of Quek1 expression. The expression of Quek1 in the medial somite half is normally repressed by the notochord and becomes up-regulated and lateromedially expanded after separation of the notochord. Our results show that up-regulation of BMP4 leads to an increase of the number of blood vessels, whereas inhibition of BMP4 by noggin results in a reduction of blood vessels.