Pharyngeal Endoderm Research Articles

Ordered expression pattern of Hox and ParaHox genes along the alimentary canal in the ascidian juvenile.

The Hox and ParaHox genes of bilateria share a similar expression pattern along the body axis and are known to be associated with anterior-posterior patterning. In vertebrates, the Hox genes are also expressed in presomitic mesoderm and gut endoderm and the ParaHox genes show a restricted expression pattern in the gut-related derivatives. Regional expression patterns in the embryonic central nervous system of the basal chordates amphioxus and ascidian have been reported; however, little is known about their endodermal expression in the alimentary canal. We focus on the Hox and ParaHox genes in the ascidian Ciona intestinalis and investigate the gene expression patterns in the juvenile, which shows morphological regionality in the alimentary canal. Gene expression analyses by using whole-mount in situ hybridization reveal that all Hox genes have a regional expression pattern along the alimentary canal. Expression of Hox1 to Hox4 is restricted to the posterior region of pharyngeal derivatives. Hox5 to Hox13 show an ordered expression pattern correlated with each Hox gene number along the postpharyngeal digestive tract. This expression pattern along the anterior-posterior axis has also been observed in Ciona ParaHox genes. Our observations suggest that ascidian Hox and ParaHox clusters are dispersed; however, the ordered expression patterns along the alimentary canal appear to be conserved among chordates.

Thyroid bud morphogenesis requires CDC42- and SHROOM3-dependent apical constriction.

ABSTRACTEarly development of the gut endoderm and its subsequent remodeling for the formation of organ buds are accompanied by changes to epithelial cell shape and polarity. Members of the Rho-related family of small GTPases and their interacting proteins play multiple roles in regulating epithelial morphogenesis. In this study we examined the role of Cdc42 in foregut development and organ bud formation. Ablation of Cdc42 in post-gastrulation mouse embryos resulted in a loss of apical-basal cell polarity and columnar epithelial morphology in the ventral pharyngeal endoderm, in conjunction with a loss of apical localization of the known CDC42 effector protein PARD6B. Cell viability but not proliferation in the foregut endoderm was impaired. Outgrowth of the liver, lung and thyroid buds was severely curtailed in Cdc42-deficient embryos. In particular, the thyroid bud epithelium did not display the apical constriction that normally occurs concurrently with the outgrowth of the bud into the underlying mesenchyme. SHROOM3, a protein that interacts with Rho GTPases and promotes apical constriction, was strongly expressed in the thyroid bud and its sub-cellular localization was disrupted in Cdc42-deficient embryos. In Shroom3 gene trap mutant embryos, the thyroid bud epithelium showed no apical constriction, while the bud continued to grow and protruded into the foregut lumen. Our findings indicate that Cdc42 is required for epithelial polarity and organization in the endoderm and for apical constriction in the thyroid bud. It is possible that the function of CDC42 is partly mediated by SHROOM3.

Cellular and molecular events on the development of mammalian thyroid C cells

Thyroid C cells synthesize and secrete calcitonin, a serum calcium-lowering hormone. This review provides our current understanding of mammalian thyroid C cells from the molecular and morphological perspectives. Several transcription factors and signaling molecules involved in the development of C cells have been identified, and genes expressed in the pharyngeal pouch endoderm, neural crest-derived mesenchyme in the pharyngeal arches, and ultimobranchial body play critical roles for the development of C cells. It has been generally accepted, without much-supporting evidence, that mammalian C cells, as well as the avian cells, are derived from the neural crest. However, by fate mapping of neural crest cells in both Wnt1-Cre/R26R and Connexin(Cxn)43-lacZ transgenic mice, we showed that neural crest cells colonize neither the fourth pharyngeal pouch nor the ultimobranchial body. E-cadherin, an epithelial cell marker, is expressed in thyroid C cells and their precursors, the fourth pharyngeal pouch and ultimobranchial body. Furthermore, E-cadherin is colocalized with calcitonin in C cells. Recently, lineage tracing in Sox17-2A-iCre/R26R mice has clarified that the pharyngeal endoderm-derived cells give rise to C cells. Together, these findings indicate that mouse thyroid C cells are endodermal in origin.

Reiterative expression of pax1 directs pharyngeal pouch segmentation in medaka.

A striking characteristic of vertebrate development is the pharyngeal arches, which are a series of bulges on the lateral surface of the head of vertebrate embryos. Although each pharyngeal arch is segmented by the reiterative formation of endodermal outpocketings called pharyngeal pouches, the molecular network underlying the reiterative pattern remains unclear. Here, we show that pax1 plays crucial roles in pouch segmentation in medaka (Oryzias latipes) embryos. Importantly, pax1 expression in the endoderm prefigures the location of the next pouch before the cells bud from the epithelium. TALEN-generated pax1 mutants did not form pharyngeal pouches posterior to the second arch. Segmental expression of tbx1 and fgf3, which play essential roles in pouch development, was almost non-existent in the pharyngeal endoderm of pax1 mutants, with disturbance of the reiterative pattern of pax1 expression. These results suggest that pax1 plays a key role in generating the primary pattern for segmentation in the pharyngeal endoderm by regulating tbx1 and fgf3 expression. Our findings illustrate the crucial roles of pax1 in vertebrate pharyngeal segmentation and provide insights into the evolutionary origin of the deuterostome gill slit.

Extracellular cAMP drives gut morphogenesis

Pigment cells release cAMP to drive morphogenesis of the sea urchin gut.

Abstract 1427: Embigin is overexpressed in pancreatic ductal adenocarcinoma and regulates cell motility through epithelial to mesenchymal transition via the TGF-β pathway

Abstract Embigin is a member of the immunoglobulin superfamily and encodes a transmembrane glycoprotein. Strong expression of Embigin has been observed in the mouse endoderm during early postimplantation embryogenesis and in the gut and visceral endoderm at the somite stage. There have been reports of Embigin involvement in neuromuscular junction formation and plasticity; however, the molecular functions of Embigin in other organs are unknown. Pancreatic cancer is an extremely aggressive malignancy, and it is necessary to understand the biology of pancreatic cancer to better detect and treat this disease. Our aim was to investigate the possible role of Embigin in pancreatic cancer. In pancreatic ductal adenocarcinoma tissues, Embigin expression was higher than that in normal pancreatic tissues. Immunohistochemical analysis revealed expression of Embigin in pancreatic cancer cells, as well as expression of monocarboxylate transporter 2 (MCT2) in cancer tissues. To gain further insight, we transfected BxPC-3 and HPAC pancreatic cancer cells with siRNA or shRNA targeting Embigin and observed reductions in cell proliferation, migration, invasion, wound healing, and reduced levels of matrix metalloproteinases-2 and -9. Silencing of Embigin increased intracellular L-lactate concentration by 1.5-fold and decreased MCT2 levels at the plasma membrane. Furthermore, Embigin silencing led to a reduced expression of PI3K, GSK3-β, and Snail/Slug. Upon treating BxPC-3 cells with transforming growth factor-β (TGF-β), we observed elevated expression of Snail/Slug, Embigin, and Vimentin; meanwhile, when treating cells with SB-216763, a GSK3-β inhibitor, we noted decreases in GSK3-β, Snail/Slug, and Embigin expression, suggesting that the TGF-β signaling cascade, comprising PI3K, GSK3-β, Snail/Slug, and Embigin signals, mediates epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. These findings indicate the involvement of Embigin in EMT in pancreatic cancer progression and suggest Embigin as a putative target for the detection and/or treatment of pancreatic cancer. Citation Format: Dawoon E. Jung, Jeong Mi Kim, Chanyang Kim, Si Young Song. Embigin is overexpressed in pancreatic ductal adenocarcinoma and regulates cell motility through epithelial to mesenchymal transition via the TGF-β pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1427. doi:10.1158/1538-7445.AM2015-1427

Generation of stomach tissue from mouse embryonic stem cells.

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

An ultra-conserved element in Hoxa2 is required for the normal expression of Hoxa3 and normal thymus development (HEM7P.223)

Abstract We analyzed thymus development in a Hoxa2 mutant mouse (Hoxa21) that carries a deletion of an ultraconserved element in the protein coding sequence of Hoxa2. Our phenotypic analysis revealed that the Hoxa21 mutants exhibit hypoplastic and ectopic thymii. Further analysis demonstrated delayed Foxn1 expression within the developing thymus of the mutants. These phenotypes resemble the alterations in thymus development found in Hoxa3 mutant embryos. Intriguingly, analysis of thymus development in mutants carrying a different Hoxa2 null allele that contains an intact ultraconserved element (Hoxa22) revealed no alteration in thymus development. This findings suggested that the abnormal thymus development seen in the Hoxa21 mutants may be due to alterations in Hoxa3 expression. Consistent with this hypothesis Hoxa21 mutants exhibited greatly reduced Hoxa3 expression in neural crest-derived mesenchyme and pharyngeal endoderm, embryonic cell types required for normal thymus development. In addition analysis of Hoxa21+/- Hoxa3 null/+ compound heterozygotes revealed thymus phenotypes that were very similar to Hoxa3-deficient mutants. These data suggest that Hoxa2, although expressed in thymic primordium, does not play a direct role in thymic organogenesis. Our results suggest that the ultraconserved enhancer element located the first exon of Hoxa2 is necessary for normal Hoxa3 expression in cell types necessary for normal thymus development.

MicroRNA-17-92 regulates Tbx1 expression in third pharyngeal pouch endoderm (HEM7P.232)

Abstract The thymus and parathyroids originate from bilateral 3rd pharyngeal pouch (pp) endoderm. At E11.5, thymus- and parathyroid-fated domains express Foxn1 and Gcm2 respectively, transcription factors that regulate differentiation but do not specify organ fate. Tbx1, which encodes a T-box transcription factor required for segmentation of the pharyngeal endoderm is initially widely expressed in the 3rd pp, but by E10.5 is excluded from the ventral, thymus-fated domain. We recently reported that Tbx1 is a negative regulator of thymus development (Reeh et al. Development 141:2950, 2014). Ectopic expression of Tbx1 in the thymus-fated domain of the 3rd pp suppresses Foxn1 and inhibits thymic epithelial cell proliferation and differentiation. MicroRNAs (miRs) are important regulators of gene expression. We find that members of the miR-17-92 cluster are expressed in the ventral domain of the 3rd pp in wildtype embryos. Moreover, miR-17-92 downregulates Tbx1 in cardiac progenitor cells to promote their differentiation (Wang et al. Developmental Cell 19:903, 2010). Therefore, we proposed that miR-17-92 regulates Tbx1 expression in 3rd pp endoderm. In support of this hypothesis, we find that deletion of miR-17-92 enhances TBX1 and reduces FOXN1 in 3rd pp endoderm. The data support a model in which miR-17-92 plays an essential role in thymus development by regulating Tbx1 expression in the 3rd pp.

Eph-Pak2a signaling regulates branching of the pharyngeal endoderm by inhibiting late-stage epithelial dynamics

Branching morphogenesis depends on the precise temporal and spatial control of epithelial dynamics. In the vertebrate head, endodermal branches, called pharyngeal pouches, form through the transient stratification, collective migration and reorganization of epithelial cells into bilayers. Here, we report novel requirements for the EphrinB ligands B2a and B3b, the Ephb4a receptor and the Pak2a kinase in the development of pouches and the posterior facial skeleton that depends on pouches for its segmentation. Time-lapse imaging in zebrafish shows that EphB-Pak2a signaling is required to stabilize pouch epithelial cells at the end of branching morphogenesis. Transgenic rescue experiments further demonstrate that endodermal Eph-ephrin signaling promotes pouch integrity by targeting Pak2a to the plasma membrane, where subsequent activation by Wnt4a-Cdc42 signaling increases junctional E-cadherin in maturing pouches. Integration of Eph-ephrin and Wnt4a signaling through Pak2a thus signals the end of branching morphogenesis by increasing intercellular adhesion that blocks further epithelial rearrangements.

Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation.

Morphogenesis of vertebrate craniofacial skeletal elements is dependent on a key cell population, the cranial neural crest cells (NCC). Cranial NCC are formed dorsally in the cranial neural tube and migrate ventrally to form craniofacial skeletal elements as well as other tissues. Multiple extracellular signaling pathways regulate the migration, survival, proliferation, and differentiation of NCC. In this study, we demonstrate that Shh expression in the oral ectoderm and pharyngeal endoderm is essential for mandibular development. We show that a loss of Shh in these domains results in increased mesenchymal cell death in pharyngeal arch 1 (PA1) after NCC migration. This increased cell death can be rescued in utero by pharmacological inhibition of p53. Furthermore, we show that epithelial SHH is necessary for the early differentiation of mandibular cartilage condensations and, therefore, the subsequent development of Meckel's cartilage, around which the dentary bone forms. Nonetheless, a rescue of the cell death phenotype does not rescue the defect in cartilage condensation formation. Our results show that SHH produced by the PA1 epithelium is necessary for the survival of post-migratory NCC, and suggests a key role in the subsequent differentiation of chondrocytes to form Meckel's cartilage.

Hedgehog Expression During Development and Regeneration in the Hemichordate, Ptychodera flava.

Hedgehog is a toolkit gene conserved in metazoans. However, its function differs among taxa, and it shows versatile expression patterns in morphogenesis. We analyzed the expression pattern of hedgehog in the indirect development of the hemichordate, Ptychodera flava, during development and regeneration. Pf-Hh showed distinct enteropneust-specific expression at the anterior tip of the larvae, as well as deuterostome-conserved expression in the pharyngeal endoderm. In contrast, the gene is expressed only in the pharyngeal region during anterior regeneration, but not in the anterior tip of the proboscis. These data suggest that anterior regeneration is driven not only by conserved developmental mechanisms, but also by some regeneration-specific mechanism(s).

Neurogenic gene regulatory pathways in the sea urchin embryo.

During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo.

Revising the embryonic origin of thyroid C cells in mice and humans.

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail–chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin; both Foxa2 and E-cadherin were re-expressed at metastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification. The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development.

Eph-Pak2a signaling regulates branching of the pharyngeal endoderm by inhibiting late-stage epithelial dynamics.

Branching morphogenesis depends on the precise temporal and spatial control of epithelial dynamics. In the vertebrate head, endodermal branches, called pharyngeal pouches, form through the transient stratification, collective migration and reorganization of epithelial cells into bilayers. Here, we report novel requirements for the EphrinB ligands B2a and B3b, the Ephb4a receptor and the Pak2a kinase in the development of pouches and the posterior facial skeleton that depends on pouches for its segmentation. Time-lapse imaging in zebrafish shows that EphB-Pak2a signaling is required to stabilize pouch epithelial cells at the end of branching morphogenesis. Transgenic rescue experiments further demonstrate that endodermal Eph-ephrin signaling promotes pouch integrity by targeting Pak2a to the plasma membrane, where subsequent activation by Wnt4a-Cdc42 signaling increases junctional E-cadherin in maturing pouches. Integration of Eph-ephrin and Wnt4a signaling through Pak2a thus signals the end of branching morphogenesis by increasing intercellular adhesion that blocks further epithelial rearrangements.

ABCC5 is required for cAMP-mediated hindgut invagination in sea urchin embryos.

ATP-binding cassette (ABC) transporters are evolutionarily conserved proteins that pump diverse substrates across membranes. Many are known to efflux signaling molecules and are extensively expressed during development. However, the role of transporters in moving extracellular signals that regulate embryogenesis is largely unexplored. Here, we show that a mesodermal ABCC (MRP) transporter is necessary for endodermal gut morphogenesis in sea urchin embryos. This transporter, Sp-ABCC5a (C5a), is expressed in pigment cells and their precursors, which are a subset of the non-skeletogenic mesoderm (NSM) cells. C5a expression depends on Delta/Notch signaling from skeletogenic mesoderm and is downstream of Gcm in the aboral NSM gene regulatory network. Long-term imaging of development reveals that C5a knockdown embryos gastrulate, but ∼90% develop a prolapse of the hindgut by the late prism stage (∼8 h after C5a protein expression normally peaks). Since C5a orthologs efflux cyclic nucleotides, and cAMP-dependent protein kinase (Sp-CAPK/PKA) is expressed in pigment cells, we examined whether C5a could be involved in gastrulation through cAMP transport. Consistent with this hypothesis, membrane-permeable pCPT-cAMP rescues the prolapse phenotype in C5a knockdown embryos, and causes archenteron hyper-invagination in control embryos. In addition, the cAMP-producing enzyme soluble adenylyl cyclase (sAC) is expressed in pigment cells, and its inhibition impairs gastrulation. Together, our data support a model in which C5a transports sAC-derived cAMP from pigment cells to control late invagination of the hindgut. Little is known about the ancestral functions of ABCC5/MRP5 transporters, and this study reveals a novel role for these proteins in mesoderm-endoderm signaling during embryogenesis.

The role of the Pax1/9 gene in the early development of amphioxus pharyngeal gill slits.

The pharynx is a major characteristic of chordates. Compared with vertebrates, amphioxus has an advantage for the study of pharynx development, as embryos lack neural crest, and the pharynx is mainly derived from endoderm cells. The Pax1/9 subfamily genes have essential roles in vertebrate pharyngeal patterning, but it is not known if the Pax1/9 gene has similar functions in amphioxus pharynx development. To answer this question, we examined the Pax1/9 gene expression pattern in amphioxus embryos at different developmental stages, and observed morphological changes following Pax1/9 knockdown. RT-qPCR analysis indicated that Pax1/9 expression was initiated during early neurula stage and rapidly peaked during mid-neurula stage. Furthermore, in situ hybridization analysis showed that Pax1/9 transcripts were localized exclusively in the most endodermal region of the developing pharynx in early neurula stage embryos; however, Pax1/9 expression was strikingly down-regulated in the region where gill slits would form from the fusion of endoderm and ectoderm in subsequent developmental stages and was maintained in the border regions between adjacent gill slits. Knockdown of Pax1/9 function using both morpholino and siRNA approaches led to embryonic defects in the first three gill slits, and fusion of the first two gill slits. Moreover, the expression levels of the pharyngeal marker genes Six1/2 and Tbx1/10 were reduced in Pax1/9 knockdown embryos. From these observations, we concluded that the Pax1/9 gene has an important role in the initial differentiation of amphioxus pharyngeal endoderm and in the formation of gill slits, most likely via modulation of Six1/2 and Tbx1/10 expression.

Gut endoderm takes flight from the wings of mesoderm

The endoderm layer destined to be primitive gut is a mosaic of earlier visceral endoderm and definitive endoderm that arises later, during gastrulation. Live imaging now reveals that in mouse embryos, definitive endoderm cells egress from underlying mesoderm and intercalate into the overlying cell layer. This process requires SOX17-mediated control of basement membrane organization.

SOX17 links gut endoderm morphogenesis and germ layer segregation.

Gastrulation leads to three germ layers, ectoderm, mesoderm and endoderm that are separated by two basement membranes. In the mouse embryo, the emergent gut endoderm results from the widespread intercalation of cells of two distinct origins: pluripotent epiblast-derived definitive endoderm (DE) and extra-embryonic visceral endoderm (VE). Here we image the trajectory of prospective DE cells prior to intercalating into the VE epithelium. We show that the transcription factor SOX17, which is activated in prospective DE cells prior to intercalation, is necessary for gut endoderm morphogenesis and the assembly of the basement membrane that separates gut endoderm from mesoderm. Our results mechanistically link gut endoderm morphogenesis and germ layer segregation, two central and conserved features of gastrulation.

Wnt signaling interacts with bmp and edn1 to regulate dorsal-ventral patterning and growth of the craniofacial skeleton.

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.