Katherine Fu

Initiation and patterning of the snake dentition are dependent on Sonic Hedgehog signaling

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.

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. Developmental Dynamics 238:1150–1165, 2009.

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.

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 Meckels 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 Meckels 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.

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. Developmental Dynamics 239:574–591, 2010.

Avian Facial Morphogenesis Is Regulated by c-Jun N-terminal Kinase/Planar Cell Polarity (JNK/PCP) Wingless-related (WNT) Signaling

Background: Numerous genes contribute to the increased risk of facial clefting, but the cellular mechanisms involved are not well understood. Results: JNK/PCP wingless-related signaling is demonstrated as a major regulator of face shape. Conclusion: WNT11 induces clefting via inhibiting proliferation, compressing facial prominences, and changing cell behavior. Significance: JNK/PCP WNT signaling is implicated in the pathophysiology underlying craniofacial abnormalities. 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 β-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 β-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 β-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.

Spatiotemporal Localization of Periostin and Its Potential Role in Epithelial-Mesenchymal Transition during Palatal Fusion

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

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. Developmental Dynamics 240:2108–2119, 2011.

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, RARβ 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.

MORN5 Expression during Craniofacial Development and Its Interaction with the BMP and TGFβ Pathways

MORN5 (MORN repeat containing 5) is encoded by a locus positioned on chromosome 17 in the chicken genome. The MORN motif is found in multiple copies in several proteins including junctophilins or phosphatidylinositol phosphate kinase family and the MORN proteins themselves are found across the animal and plant kingdoms. MORN5 protein has a characteristic punctate pattern in the cytoplasm in immunofluorescence imaging. Previously, MORN5 was found among differentially expressed genes in a microarray profiling experiment of the chicken embryo head. Here, we provided in situ hybridization to analyse, in detail, the MORN5 expression in chick craniofacial structures. The expression of MORN5 was first observed at stage HH17-18 (E2.5). MORN5 expression gradually appeared on either side of the primitive oral cavity, within the maxillary region. At stage HH20 (E3), prominent expression was localized in the mandibular prominences lateral to the midline. From stage HH20 up to HH29 (E6), there was strong expression in restricted regions of the maxillary and mandibular prominences. The frontonasal mass (in the midline of the face) expressed MORN5, starting at HH27 (E5). The expression was concentrated in the corners or globular processes, which will ultimately fuse with the cranial edges of the maxillary prominences. MORN5 expression was maintained in the fusion zone up to stage HH29. In sections MORN5 expression was localized preferentially in the mesenchyme. Previously, we examined signals that regulate MORN5 expression in the face based on a previous microarray study. Here, we validated the array results with in situ hybridization and QPCR. MORN5 was downregulated 24 h after Noggin and/or RA treatment. We also determined that BMP pathway genes are downstream of MORN5 following siRNA knockdown. Based on these results, we conclude that MORN5 is both regulated by and required for BMP signaling. The restricted expression of MORN5 in the lip fusion zone shown here supports the human genetic data in which MORN5 variants were associated with increased risk of non-syndromic cleft lip with or without cleft palate.