Frontonasal Ectodermal Zone Research Articles

PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary mechanisms.

Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.

Wnt Signaling Drives Correlated Changes in Facial Morphology and Brain Shape.

Canonical Wnt signaling plays multiple roles critical to normal craniofacial development while its dysregulation is known to be involved in structural birth defects of the face. However, when and how Wnt signaling influences phenotypic variation, including those associated with disease, remains unclear. One potential mechanism is via Wnt signaling’s role in the patterning of an early facial signaling center, the frontonasal ectodermal zone (FEZ), and its subsequent regulation of early facial morphogenesis. For example, Wnt signaling may directly alter the shape and/or magnitude of expression of the sonic hedgehog (SHH) domain in the FEZ. To test this idea, we used a replication-competent avian sarcoma retrovirus (RCAS) encoding Wnt3a to modulate its expression in the facial mesenchyme. We then quantified and compared ontogenetic changes in treated to untreated embryos in the three-dimensional (3D) shape of both the SHH expression domain of the FEZ, and the morphology of the facial primordia and brain using iodine-contrast microcomputed tomography imaging and 3D geometric morphometrics (3DGM). We found that increased Wnt3a expression in early stages of head development produces correlated variation in shape between both structural and signaling levels of analysis. In addition, altered Wnt3a activation disrupted the integration between the forebrain and other neural tube derivatives. These results show that activation of Wnt signaling influences facial shape through its impact on the forebrain and SHH expression in the FEZ, and highlights the close relationship between morphogenesis of the forebrain and midface.

Creating Avian Forebrain Chimeras to assess Facial Development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

MiR-199 family contributes to regulation of sonic hedgehog expression during craniofacial development.

The frontonasal ectodermal zone (FEZ) is a signaling center that regulates patterned development of the upper jaw, and Sonic hedgehog (SHH) mediates FEZ activity. Induction of SHH expression in the FEZ results from SHH-dependent signals from the brain and neural crest cells. Given the role of miRNAs in modulating gene expression, we investigated the extent to which miRNAs regulate SHH expression and FEZ signaling. In the FEZ, the miR-199 family appears to be regulated by SHH-dependent signals from the brain; expression of this family increased from HH18 to HH22, and upon activation of SHH signaling in the brain. However, the miR-199 family is more broadly expressed in the mesenchyme of the frontonasal process and adjacent neuroepithelium. Downregulating the miR-199 genes expanded SHH expression in the FEZ, resulting in wider faces, while upregulating miR-199 genes resulted in decreased SHH expression and narrow faces. Hypoxia inducible factor 1 alpha (HIF1A) and mitogen-activated protein kinase kinase kinase 4 (MAP3K4) appear to be potential targets of miR-199b. Reduction of MAP3K4 altered beak development but increased apoptosis, while reducing HIF1A reduced expression of SHH in the FEZ and produced malformations independent of apoptosis. Our results demonstrate that this miRNA family appears to participate in regulating SHH expression in the FEZ; however, specific molecular mechanisms remain unknown.

Mapping the Multi‐Modal Distribution of Craniofacial Phenotypes in NOSIP Mutants

Nitric Oxide Synthase Interacting Protein (NOSIP) is a E3 mono‐ubiquitin ligase that regulates the activity of protein phosphatase 2A. Deletion of NOSIP leads to severe and variable malformations of the craniofacial complex. Our goal was to use NOSIP mutant mice to understand of mechanisms of phenotypic variation. The phenotype of an individual embryo is the product of the developmental trajectory that it previously traversed. We propose that this concept of canalization is essential for understanding the etiology of complex phenotypic outcomes. We have previously shown that the brain patterns the face, so sequential perturbations affecting the brain and face may create unique developmental trajectories that each embryo transits. We tested this by creating a 3D morphospace of NOSIP heterozygous and homozygous mutants to determine if embryos occupy unique morphospace regions based on affected craniofacial structures. Embryos between embryonic day 10.5 (E10.5) to E17.5 were collected, fixed, and imaged (mCT). Anatomical features were reconstructed using Avizo. NOISP expression appears to be restricted to the neuroepithelium, lower jaw mesenchyme, and mesenchyme and epithelium in tissue adjacent to the frontonasal ectodermal zone between stage E10.5‐11.5. Embryos with a single telencephalic lobe had more severe facial malformations, including micrognathia and obstructed airway without significant changes in early cell apoptosis (via TUNEL staining). Embryos with normal telencephalic vesicle separation had less severe facial phenotypes, including left‐right mild asymmetry and clefting. Overall, it appears that NOSIP‐KO creates a multi‐modal distribution of craniofacial phenotypes along the developmental timeline by affecting early brain development, suggesting that understanding canalization may provide insight into the complexity of genotype‐phenotype map (Fig. 1).Support or Funding InformationFunding was provided by NIH F32DE08476 and R01DE019638 grants.Schematic of the changing phenotypic landscape describing the multi‐modal distribution of craniofacial phenotypes within NOSIP+/− and NOSIP−/− embryos compared to wildtype (light blue).Figure 1

The Silent Treatment: miR199 Family Silences Shh during Craniofacial Development

Early craniofacial development requires precise timing and spatial regulation of key signaling pathways in order for proper facial structures to form. Perturbations, particularly in the sonic hedgehog (Shh) signaling pathway, can result in craniofacial birth defects that cause significant harm and financial costs. microRNAs (miRNA) play many roles in developing systems by repressing gene expression, but despite the enormous number of miRNAs that have been identified, few have been related to biological processes through direct targets. Here we identified the miR199 family as Shh‐regulated miRNAs during development of the upper jaw in a chick model.We performed a miRNA microarray analysis on chick embryonic ectoderm before (stage HH18) and after (stage HH22) formation of the Frontonasal Ectodermal Zone (FEZ) and after activating or blocking SHH signaling in the brain. We identified a family of miRNAs (miR199b, mir199* and miR199) that appeared to be regulated by SHH signaling in the brain. These are increased between stages HH18 and HH22, and increase upon activation of Shh signaling and decrease upon blockade of Shh signaling, and these data were confirmed by qPCR. To assess the function of this family of miRNAs during facial development, we performed miR199 knock‐down and overexpression experiments and examined the phenotypic and molecular changes. Up‐regulation of each miRNA led to decreased Shh expression in the FEZ and facial malformations that are consistent with decreased Shh signaling in the face. Using antagomirs to knockdown each miRNA, we observed that Shh expression was expanded and the faces were wider, reminiscent of SHH gain‐of‐function experiments. Hence, it appears that expression of these miRNAs in the FEZ are regulated by SHH signaling from the brain, and these miRNAs appear to regulate SHH signaling from the FEZ. We then identified Smurf2, Map3k4, Hif1alpha, and Dyrk1a as targets of the miR‐199 family of miRNAs via mir‐base. We confirmed that each of these genes behaved like targets in miR‐gain and loss‐of‐function experiments described above. Using pmirGLO luciferase, we identified MAP3K4 and HIF as direct targets of miR199. Finally, we generated a miRNA knockdown cassette to reduce expression of Hif1α and Map3k4. Blocking HIF1α resulted in a large reduction of Shh expression in the FEZ, and resulted in severe craniofacial malformations, while blocking Map3k4 altered facial morphology, resulting in altered mediolateral beak development at later stages. Overall, our results demonstrate that Shh signaling within the brain contributes to facial development by regulating a family of miRNAs that then regulate the expression of Shh in the FEZ via targeting the transcription factor Hif1α.Support or Funding InformationThis work was supported by the National Institutes of Health (R01 DE018234‐A1)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development and growth of the facial skeleton in individual skeletal classes in the human population. Literature review

Studies regarding development of the facial skeleton and understanding of morphogenetic background of various types of skeletal classes are important not only from a cognitive point of view, but also from a clinical one, in relation to treatment of patients according to a personal phenotype. Aim. This paper aimed to present a review of literature regarding development and growth of the facial skeleton in the aspect of skeletal classes. Material and methods. A review of the literature from the years 2001– 2016 was conducted using the PubMed database. Key words used to search for articles included: skeletal class development, skeletal class malocclusion development. Inclusion criteria were based on selection of original papers regarding development and growth of facial skeleton in the prenatal and postnatal period. Exclusion criteria included review papers and case reports. Results. Articles selected based on inclusion criteria indicate there are many reasons affecting a direction of morphogenesis of the facial skeleton and mutual correlations during development. It is possible to understand these processes as a result of distinguishing between growth and physical development in the prenatal and postnatal period. Important factors present in the prenatal period include, among others, the following: interactions of the Frontonasal Ectodermal Zone, shh proteins, fgf8 proteins and BMP signalling. Factors present in the postnatal period depend on structural, functional, anatomical, genetic and growth patterns interactions. The following factors affecting development of skeletal classes were analysed: sexual dimorphism, effects of masticatory muscles, permanent tooth eruption, and quality of the respiratory tract. Conclusions. Studies regarding development of the facial skeleton and morphogenetic background of various types of skeletal classes contribute to improvement of clinical aspects of treatment, according to a personal phenotype and any dysfunctions associated with the skeletal structure. (Hovhannisyan A, Kostrzewa-Janicka J, Zadurska M, Mierzwińska-Nastalska E. Development and growth of the facial skeleton in individual skeletal classes in the human population. Literature review. Orthod Forum 2018; 14: 48-60)

Signals from the brain induce variation in avian facial shape.

How developmental mechanisms generate the phenotypic variation that is the raw material for evolution is largely unknown. Here, we explore whether variation in a conserved signaling axis between the brain and face contributes to differences in morphogenesis of the avian upper jaw. In amniotes, including both mice and avians, signals from the brain establish a signaling center in the ectoderm (the Frontonasal ectodermal zone or "FEZ") that directs outgrowth of the facial primordia. Here we show that the spatial organization of this signaling center differs among avians, and these correspond to Sonic hedgehog (Shh) expression in the basal forebrain and embryonic facial shape. In ducks this basal forebrain domain is present almost the entire width, while in chickens it is restricted to the midline. When the duck forebrain is unilaterally transplanted into stage matched chicken embryos the face on the treated side resembles that of the donor. Combined with previous findings, these results demonstrate that variation in a highly conserved developmental pathway has the potential to contribute to evolutionary differences in avian upper jaw morphology. Developmental Dynamics 244:1133-1143, 2015. © 2015 Wiley Periodicals, Inc.

A molecular mechanism for the origin of a key evolutionary innovation, the bird beak and palate, revealed by an integrative approach to major transitions in vertebrate history.

The avian beak is a key evolutionary innovation whose flexibility has permitted birds to diversify into a range of disparate ecological niches. We approached the problem of the mechanism behind this innovation using an approach bridging paleontology, comparative anatomy, and experimental developmental biology. First, we used fossil and extant data to show the beak is distinctive in consisting of fused premaxillae that are geometrically distinct from those of ancestral archosaurs. To elucidate underlying developmental mechanisms, we examined candidate gene expression domains in the embryonic face: the earlier frontonasal ectodermal zone (FEZ) and the later midfacial WNT-responsive region, in birds and several reptiles. This permitted the identification of an autapomorphic median gene expression region in Aves. To test the mechanism, we used inhibitors of both pathways to replicate in chicken the ancestral amniote expression. Altering the FEZ altered later WNT responsiveness to the ancestral pattern. Skeletal phenotypes from both types of experiments had premaxillae that clustered geometrically with ancestral fossil forms instead of beaked birds. The palatal region was also altered to a more ancestral phenotype. This is consistent with the fossil record and with the tight functional association of avian premaxillae and palate in forming a kinetic beak.

Correlations Between the Morphology of Sonic Hedgehog Expression Domains and Embryonic Craniofacial Shape.

Quantitative analysis of gene expression domains and investigation of relationships between gene expression and developmental and phenotypic outcomes are central to advancing our understanding of the genotype-phenotype map. Gene expression domains typically have smooth but irregular shapes lacking homologous landmarks, making it difficult to analyze shape variation with the tools of landmark-based geometric morphometrics. In addition, 3D image acquisition and processing introduce many artifacts that further exacerbate the problem. To overcome these difficulties, this paper presents a method that combines optical projection tomography scanning, a shape regularization technique and a landmark-free approach to quantify variation in the morphology of Sonic hedgehog expression domains in the frontonasal ectodermal zone (FEZ) of avians and investigate relationships with embryonic craniofacial shape. The model reveals axes in FEZ and embryonic-head morphospaces along which variation exhibits a sharp linear relationship at high statistical significance. The technique should be applicable to analyses of other 3D biological structures that can be modeled as smooth surfaces and have ill-defined shape.

Facial Morphogenesis: Physical and Molecular Interactions Between the Brain and the Face.

Morphogenesis of the brain and face is intrinsically linked by a number of factors. These include: origins of tissues, adjacency allowing their physical interactions, and molecular cross talk controlling growth. Neural crest cells that form the facial primordia originate on the dorsal neural tube. In the caudal pharyngeal arches, a Homeobox code regulates arch identity. In anterior regions, positional information is acquired locally. Second, the brain is a structural platform that influences positioning of the facial primordia, and brain growth influences the timing of primordia fusion. Third, the brain helps induce a signaling center, the frontonasal ectodermal zone, in the ectoderm, which participates in patterned growth of the upper jaw. Similarly, signals from neural crest cells regulate expression of fibroblast growth factor 8 in the anterior neural ridge, which controls growth of the anterior forebrain. Disruptions to these interactions have significant consequences for normal development of the craniofacial complex, leading to structural malformations and birth defects.

Modulation of Shh signaling by microRNAs

Sonic hedgehog (Shh) exhibits a dynamic expression sequence in the head. First, Shh is detected in the forebrain and then Shh is induced in the facial ectoderm where it forms a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates morphogenesis of the upper jaw. Induction of Shh in the FEZ is regulated by SHH from the brain, but the mechanisms underlying this induction pattern is unknown. Here, we examine the extent to which the SHH pathway is regulated by miRNAs. Microarray analysis revealed that miR199b was expressed at lower levels in the FEZ at HH18 compared to HH22, and was up‐regulated in response to exogenous SHH. In situ hybridization at HH18 revealed that miR199b was expressed broadly in the future Shh domain of the FEZ, and by HH21 became restricted to the edge of the Shh domain in the FEZ. A similar complementary expression pattern between Shh and miR199b was found in the floorplate of the neural tube. Over‐expressing mir199b in embryos caused narrowing of the face that was associated with decreased Shh expression. There was no obvious increase in apoptosis, but we observed reduced cell proliferation in the mesenchyme. To assess how miR199b affects SHH signaling, we determined that infection of RCAS::mir199b in DF‐1 cells inhibited SHH signal activation and cell proliferation. Taken together, these results suggest that miR199b acts a negative regulator of SHH signaling in embryos and cultured cells.Grant Funding Source : This work was funded by grant from the National Institutes of Health (5R01‐DE018234 to R.M.).MicroRNA array was supported from the Sandler Asthma Basic Research (SABRE) Center Functional Genomics Core Facility.

Tissue Interactions that Regulate Facial Morphogenesis

Molecular signaling among the forebrain, neural crest mesenchyme, and facial ectoderm regulate morphogenesis of the upper jaw. The forebrain and neural crest mesenchyme regulate expression of Sonic Hedgehog in the Frontonasal Ectodermal Zone (FEZ), which controls proximodistal extension and dorsoventral polarity of the upper jaw. The FEZ is highly conserved among vertebrates, but the spatial pattern of Shh expression is varied and highly correlated with the shape of the facial primordia in the developing upper jaw. In birds Shh expression spans the entire Frontonasal Process, while in mammals (human and mouse) Shh is restricted to lateral domains associated with the Median Nasal Processes. These expression patterns appear to result from molecular signals, including SHH itself, from the forebrain. From these observations we predicted that the signaling axis among the brain, mesenchyme, and ectoderm could be used to generate variation during evolutionary and disease processes. We have tested this by modulating SHH signaling in the brain of chick embryos to varying degrees and assessing facial shape using 3‐dimensional morphometrics and multivariate statistical analyses. This perturbation produced embryos with continuous phenotypic variation. The shape outcomes were highly correlated with SHH signaling, cell proliferation rates, and the organization of the FEZ in treated embryos. Further, these results revealed that phenotypic variation may result from non‐linear mechanisms by which signaling pathways operate. For instance, when the SHH pathway was blocked maximally, a highly penetrant phenotype resulted, while at lower levels of blockade the phenotypes rapidly returned to more normal shapes. Thus, large phenotypic variation was generated by small changes in receptor activation over certain ligand concentrations.

Neural crest cells pattern the surface cephalic ectoderm during FEZ formation

Multiple fibroblast growth factor (Fgf) ligands are expressed in the forebrain and facial ectoderm, and vascular endothelial growth factor (VEGF) is expressed in the facial ectoderm. Both pathways activate the MAP kinase cascade and can be suppressed by SU5402. We placed a bead soaked in SU5402 into the brain after emigration of neural crest cells was complete. Within 24 hr we observed reduced pMEK and pERK staining that persisted for at least 48 hr. This was accompanied by significant apoptosis in the face. By day 15, the upper beaks were truncated. Molecular changes in the FNP were also apparent. Normally, Shh is expressed in the frontonasal ectodermal zone and controls patterned growth of the upper jaw. In treated embryos, Shh expression was reduced. Both the structural and molecular deficits were mitigated after transplantation of FNP-derived mesenchymal cells. Thus, mesenchymal cells actively participate in signaling interactions of the face, and the absence of neural crest cells in neurocristopathies may not be merely structural.

Signaling by SHH rescues facial defects following blockade in the brain

The Frontonasal Ectodermal Zone (FEZ) is a signaling center in the face that expresses Sonic hedgehog (Shh) and regulates patterned growth of the upper jaw. Blocking SHH in the forebrain blocks Shh expression in the FEZ and creates malformations resembling holoprosencephaly (HPE), while inhibition of BMP signaling in the mesenchyme blocks FEZ formation and causes similar dysmorphology. Thus, the brain could regulate FEZ formation by SHH or BMP signaling, and if so, activating one of these pathways in the face might alleviate the effects of repression of SHH in the brain. We blocked SHH signaling in the brain while adding SHH or BMP between the neural and facial ectoderm of the frontonasal process. When applied early, SHH restored Shh expression in the FEZ and significantly improved shape outcomes, which contrasts with our previous experiments that showed later SHH treatments have no effect. BMP-soaked beads introduced early and late caused apoptosis that exacerbated malformations. Finally, removal of Smoothened from neural crest cells did not inhibit Shh expression in the FEZ. Collectively, this work suggests that a direct, time-sensitive SHH signal from the brain is required for the later induction of Shh in the FEZ. We propose a testable model of FEZ activation and discuss signaling mediators that may regulate these interactions.

Ectodermal WNT/β-catenin signaling shapes the mouse face

The canonical Wnt/β-catenin pathway is an essential component of multiple developmental processes. To investigate the role of this pathway in the ectoderm during facial morphogenesis, we generated conditional β-catenin mouse mutants using a novel ectoderm-specific Cre recombinase transgenic line. Our results demonstrate that ablating or stabilizing β-catenin in the embryonic ectoderm causes dramatic changes in facial morphology. There are accompanying alterations in the expression of Fgf8 and Shh, key molecules that establish a signaling center critical for facial patterning, the frontonasal ectodermal zone (FEZ). These data indicate that Wnt/β-catenin signaling within the ectoderm is critical for facial development and further suggest that this pathway is an important mechanism for generating the diverse facial shapes of vertebrates during evolution.

A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm

Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these interactions, we activated the Sonic hedgehog (SHH) pathway in the brain. Beginning 72 hours after activation of the SHH pathway, growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, we observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered. Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to hypoplasia of the skeleton, and this phenotype was reproduced when we blocked the FGF pathway. Thus, the brain establishes multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as a result of disease can alter these centers and thereby generate morphological variation.

Unique organization of the frontonasal ectodermal zone in birds and mammals

The faces of birds and mammals exhibit remarkable morphologic diversity, but how variation arises is not well-understood. We have previously demonstrated that a region of facial ectoderm, which we named the frontonasal ectodermal zone (FEZ), regulates proximo-distal extension and dorso-ventral polarity of the upper jaw in birds. In this work, we examined the equivalent ectoderm in murine embryos and determined that the FEZ is conserved in mice. However, our results revealed that fundamental differences in the organization and constituents of the FEZ in mice and chicks may underlie the distinct growth characteristics that distinguish mammalian and avian embryos during the earliest stages of development. Finally, current models suggest that neural crest cells regulate size and shape of the upper jaw, and that signaling by Bone morphogenetic proteins (Bmps) within avian neural crest helps direct this process. Here we show that Bmp expression patterns in neural crest cells are regulated in part by signals from the FEZ. The results of our work reconcile how a conserved signaling center that patterns growth of developing face may generate morphologic diversity among different animals. Subtle changes in the organization of gene expression patterns in the FEZ could underlie morphologic variation observed among and within species, and at extremes, variation could produce disease phenotypes.

Signaling by bone morphogenetic proteins directs formation of an ectodermal signaling center that regulates craniofacial development

We previously described a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates growth and patterning of the frontonasal process (FNP). The FEZ is comprised of FNP ectoderm flanking a boundary between Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. Our objective was to examine BMP signaling during formation of the FEZ. We blocked BMP signaling throughout the FNP prior to FEZ formation by infecting chick embryos at stage 10 (HH10) with a replication-competent avian retrovirus encoding the BMP antagonist Noggin. We assessed gene expression patterns in the FNP 72 h after infection (∼ HH22) and observed that Shh expression was reduced or absent. In the mesenchyme, we observed that Bmp2 transcripts were absent while the Bmp4 expression domain was expanded proximally. In addition to the molecular changes, infected embryos also exhibited facial malformations at 72 and 96 h after infection suggesting that the FEZ did not form. Our data indicate that reduced cell proliferation, but not apoptosis, in the mesenchyme contributed to the phenotype that we observed. Additionally, adding exogenous SHH into the mesenchyme of RCAS- Noggin-infected embryos did not restore Bmp2 and Bmp4 to a normal pattern of expression. These data indicate that BMP signaling mediates interactions between tissues in the FNP that regulate FEZ formation; and that the correct pattern of Bmp2 and Bmp4, but not Bmp7, expression in the FNP mesenchyme requires signaling by the BMP pathway .