Neural Crest Epithelial-to-mesenchymal Transition Research Articles

SMPD3 expression is spatially regulated in the developing embryo by SOXE factors

During epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression of SMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide and necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of the SMPD3 locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenous SMPD3 within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SOXE-family transcription factors SOX9 and SOX10 regulate the expression of SMPD3 in migrating neural crest cells. Further, ChIP-seq and nascent transcription analysis reveal that SOX10 directly regulates expression of an SMPD3 enhancer specific to migratory neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.

"Beyond transcription: How post-transcriptional mechanisms drive neural crest EMT".

The neural crest is a stem cell population that originates from the ectoderm during the initial steps of nervous system development. Neural crest cells delaminate from the neuroepithelium by undergoing a spatiotemporally regulated epithelial-mesenchymal transition (EMT) that proceeds in a coordinated wave head-to-tail to exit from the neural tube. While much is known about the transcriptional programs and membrane changes that promote EMT, there are additional levels of gene expression control that neural crest cells exert at the level of RNA to control EMT and migration. Yet, the role of post-transcriptional regulation, and how it drives and contributes to neural crest EMT, is not well understood. In this mini-review, we explore recent advances in our understanding of the role of post-transcriptional regulation during neural crest EMT.

Paracrine regulation of neural crest EMT by placodal MMP28.

Epithelial-mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for neural crest EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of neural crest cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing.

Temporal changes in plasma membrane lipid content induce endocytosis to regulate developmental epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is a dramatic change in cellular physiology during development and metastasis, which requires coordination between cell signaling, adhesion, and membrane protrusions. These processes all involve dynamic changes in the plasma membrane; yet, how membrane lipid content regulates membrane function during EMT remains incompletely understood. By screening for differential expression of lipid-modifying genes over the course of EMT in the avian neural crest, we have identified the ceramide-producing enzyme neutral sphingomyelinase 2 (nSMase2) as a critical regulator of a developmental EMT. nSMase2 expression begins at the onset of EMT, and in vivo knockdown experiments demonstrate that nSMase2 is necessary for neural crest migration. We find that nSMase2 promotes Wnt and BMP signaling and is required to activate the mesenchymal gene expression program. Mechanistically, we show that nSMase2-dependent ceramide production is necessary for and sufficient to up-regulate endocytosis and is required for Wnt co-receptor internalization. Finally, inhibition of endocytosis in the neural crest mimics the loss of migration and Wnt signaling observed following nSMase2 knockdown. Our results support a model in which nSMase2 is expressed at the onset of neural crest EMT to produce ceramide and facilitate receptor-mediated endocytosis of Wnt and BMP signaling complexes, thereby activating promigratory gene expression. These results highlight the critical role of plasma membrane lipid metabolism in regulating transcriptional changes during developmental EMT programs.

LATS1/2 control TGFB-directed epithelial-to-mesenchymal transition in the murine dorsal cranial neuroepithelium through YAP regulation.

Hippo signaling, an evolutionarily conserved kinase cascade involved in organ size control, plays key roles in various tissue developmental processes, but its role in craniofacial development remains poorly understood. Using the transgenic Wnt1-Cre2 driver, we inactivated the Hippo signaling components Lats1 and Lats2 in the cranial neuroepithelium of mouse embryos and found that the double conditional knockout (DCKO) of Lats1/2 resulted in neural tube and craniofacial defects. Lats1/2 DCKO mutant embryos had microcephaly with delayed and defective neural tube closure. Furthermore, neuroepithelial cell shape and architecture were disrupted within the cranial neural tube in Lats1/2 DCKO mutants. RNA sequencing of embryonic neural tubes revealed increased TGFB signaling in Lats1/2 DCKO mutants. Moreover, markers of epithelial-to-mesenchymal transition (EMT) were upregulated in the cranial neural tube. Inactivation of Hippo signaling downstream effectors, Yap and Taz, suppressed neuroepithelial defects, aberrant EMT and TGFB upregulation in Lats1/2 DCKO embryos, indicating that LATS1/2 function via YAP and TAZ. Our findings reveal important roles for Hippo signaling in modulating TGFB signaling during neural crest EMT.

The Role of Connexin‐43 in Cranial Neural Crest Cell Differentiation

BackgroundMost features of the craniofacial complex that are necessary for feeding derive from cranial neural crest cells (cNCCs). These pluripotent stem cells originate from the dorsal neural folds and undergo a series of coordinated processes such as induction, epithelial‐to‐mesenchymal transition (EMT), migration, and differentiation. While most of these processes have been well‐studied for their roles in development, much remains to be known about the final step cNCCs must undergo, differentiation. Connexin‐43 (Cx43) is a gap junctional protein that is widely expressed, evolutionarily conserved, and has been well‐studied for its impacts on neural crest and bone. Previous research has shown that loss of Cx43 function impacts neural crest EMT and migration and can delay early osteoblast and chondrocyte differentiation; however, it is unknown how Cx43 deficiency in cNCCs impacts cNCC differentiation into osteochondrogenic lineages and the resultant impacts on craniofacial morphology. Therefore, the purpose of this project is to determine the effects of a neural crest‐specific loss of Cx43 on osteogenic and chondrogenic differentiation, and how these effects may alter skull phenotype.MethodsWe have developed a mouse model wherein the gene encoding for Cx43 has been conditionally knocked out in neural crest cells and their derivatives using the Wnt1‐Cre2 driver (Wnt1‐Cre2+/‐;mTmG+/+;Cx43fl/fl, herein Cx43cKO). To compare the developing skull phenotype between Cx43cKO mice and littermates in which the Cx43 gene is not floxed (Wnt1‐Cre2+/‐; mTmG+/+, herein Cx43WT), we will collect homologous landmark data from microCT images of newborn skulls and use geometric morphometric analyses to quantify and visualize shape differences in neural crest‐ and mesoderm‐derived portions of the skull between genotypes.To determine the localized effects of a neural‐crest specific loss of Cx43 function on osteoblast and chondrocyte differentiation, we will use immunofluorescence staining. Expression of chondrocyte, osteoblast, and osteocyte differentiation markers will be localized and semi‐quantitatively compared between genotypes and between neural crest‐ and mesoderm‐derived cells. With these assays, we aim to understand the relationship between Cx43, cNCC differentiation, and the subsequent impacts on skull morphology.Preliminary ResultsCx43cKO neonates show decreased ossification compared to Cx43WT littermates, which is most prominent in facial bones such as the nasal bones, and cranial vault bones such as the frontal and parietal bones. Potential driving forces for this decreased ossification in neural crest‐ and mesoderm‐derived bones are delayed differentiation of the osteoblast‐osteocyte lineage and disrupted signaling between cells in neighbouring bones.Significance & Future DirectionsThis study will help elucidate the role of cNCC differentiation in skull development, and how loss of Cx43 function impacts skull development. We will add embryonic and post‐natal timepoints to further understand the impacts of neural crest‐specific deletion of Cx43 throughout development; pilot data with Cx43cKO adult mice show that alterations to skull shape persist and overtly affect the face and mandible.

Transcriptomic Identification of Draxin-Responsive Targets During Cranial Neural Crest EMT.

Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest – RHOB – and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

MID1 and MID2 regulate cell migration and epithelial-mesenchymal transition via modulating Wnt/β-catenin signaling.

BackgroundThe ubiquitin E3 ligase activity has been ascribed to MID1, the causative gene of X-linked OS, and its homologue, MID2. Both alpha4, the common MID protein partner, and PP2Ac in MID-alpha4-PP2Ac complexes can be ubiquitylated. Ubiquitylation of alpha4 converted its function toward PP2Ac from protective to destructive, while PP2A also affected MID protein phosphorylation and their subsequent trafficking on microtubules. It was believed that disruption of the function of MID1-alpha4-PP2A complex was vital to the pathogenesis of craniofacial malformation, the most prominent clinical manifestation of OS, although the detailed molecular mechanisms was not unravelled.MethodsThe cellular level of PP2A and phosphor-PP2A in cells overexpressing MID1/MID2 or in cells with siRNA mediated MID1/MID2 gene silencing was analyzed using Western blot. The Wnt signaling in these cells was further monitored using TCF/LEF luciferase reporter assay and the cellular level of β-catenin was also verified using western blot. Given the crosstalk of E-cadherin and Wnt via the common effector β-catenin, the potential influences of MID1/MID2 on the cell migration and epithelial-mesenchymal transition (EMT) were investigated using wound healing assay and immunofluorescence for E-cadherin and vimentin, respectively.ResultsHere, we presented the increased phosphorylation of PP2Ac in cells overexpressing MID1/MID2, and vice versa, in vitro, while the cellular level of total PP2Ac was unaffected. In addition, β-catenin, the effector of canonical Wnt signaling, was downregulated in cells overexpressing MID1/MID2 and upregulated in cells with siRNA mediated MID1/MID2 gene silencing. Down-regulated Wnt/β-catenin signaling by Okadaic acid, a specific inhibitor of PP2A, was partially rescued by siRNA mediated MID1/MID2 gene silencing. In consistent, an activated EMT and accelerated cell migration in cells with MID1/MID2 gene silencing were observed, and vice versa.ConclusionsThe results in this study indicated roles for MID1 and MID2 in regulating cell migration/EMT via modulating Wnt/β-catenin signaling, which might help to understand the molecular etiology of the facial abnormalities that are usually the consequences of defective neural crest cells migration and EMT at the early stage of craniofacial development.

Protein glycosylation in neural crest epithelial‐to‐mesenchymal transition and migration

Neural crest epithelial‐to‐mesenchymal transition (EMT) and migration involves coordinated remodeling of cell‐cell and cell‐matrix adhesions. During this cell phase transition event, interactions between adhesive molecules need to be dynamically regulated. We recently explored the involvement of protein glycosylation in this process. Protein glycosylation is one of the most complexed, yet least studied types of post‐translational modification. By attaching various forms of oligosaccharide (glycan) on different sites of a protein, glycosylation can regulate protein function by modulating its interaction with other molecules. Through the employment of chemical inhibitors for protein glycosylation, we demonstrated that glycosylation is required for neural crest EMT and migration in frog embryos. To gain a comprehensive understanding of how different proteins are glycosylated during this process, we performed a glycoproteomic analysis at different stages of neural crest development. Through this approach, we identified numerous membrane and secreted proteins that are differentially glycosylated, which may contribute to the dynamic changes in cell‐cell and cell‐matrix interactions during neural crest EMT and migration. We focused on E‐cadherin, a well‐known player in EMT and cell migration, to determine the function of specific protein glycosylation in protein activity and consequently in neural crest cell behavior. Our analysis identified two glycosylation linkage sites on E‐cadherin. The two glycosylation sites were mutated individually or in combination, and the function of mutant E‐cadherin was compared with that of wild type E‐cadherin. Our results showed that mutant E‐cadherins were less effective in promoting neural crest cell migration and this was partly due to an increase in the strength of cell‐cell adhesion mediated by the mutan t proteins. Therefore, our findings suggest a new mechanism to fine‐tune the activity of cell adhesive molecules during EMT and migration.Support or Funding InformationNIH R00 DE022796, Georgia Tech Startup Fund

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

Draxin alters laminin organization during basement membrane remodeling to control cranial neural crest EMT

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.

MMP14 Regulates Cranial Neural Crest Epithelial-to-Mesenchymal Transition and Migration.

Neural crest is a vertebrate specific cell population. Induced at lateral borders of the neural plate, neural crest cells (NCCs) subsequently undergo epithelial-to-mesenchymal transition (EMT) to detach from the neuroepithelium before migrating into various locations in the embryo. Despite the wealth of knowledge of transcription factors involved in this process, little is known about the effectors that directly regulate neural crest EMT and migration. Here, we examined the activity of matrix metalloproteinase MMP14 in NCCs and found that MMP14 is expressed in both premigratory and migrating NCCs. Overexpression of MMP14 led to premature migration of NCCs, while down-regulation of MMP14 resulted in reduced neural crest migration. Transplantation experiment further showed that MMP14 is required in NCCs, whereas MMP2, which can be activated by MMP14, is required in the surrounding mesenchyme. in vitro explant culture showed that MMP14 is required for neural crest EMT but not for spreading. This is possibly mediated by the changes in cadherin levels, as decreasing MMP14 level led to increased cadherin expression and increasing MMP14 level led to reduced cadherin expression. The results demonstrate that MMP14 is critical for neural crest EMT and migration, partially through regulating the levels of cadherins. Developmental Dynamics 247:1083-1092, 2018. © 2018 Wiley Periodicals, Inc.

Draxin acts as a molecular rheostat of canonical Wnt signaling to control cranial neural crest EMT.

Neural crest cells undergo a spatiotemporally regulated epithelial-to-mesenchymal transition (EMT) that proceeds head to tailward to exit from the neural tube. In this study, we show that the secreted molecule Draxin is expressed in a transient rostrocaudal wave that mirrors this emigration pattern, initiating after neural crest specification and being down-regulated just before delamination. Functional experiments reveal that Draxin regulates the timing of cranial neural crest EMT by transiently inhibiting canonical Wnt signaling. Ectopic maintenance of Draxin in the cranial neural tube blocks full EMT; while cells delaminate, they fail to become mesenchymal and migratory. Loss of Draxin results in premature delamination but also in failure to mesenchymalize. These results suggest that a pulse of intermediate Wnt signaling triggers EMT and is necessary for its completion. Taken together, these data show that transient secreted Draxin mediates proper levels of canonical Wnt signaling required to regulate the precise timing of initiation and completion of cranial neural crest EMT.

Lamprey neural crest migration is Snail-dependent and occurs without a differential shift in cadherin expression

The acquisition of neural crest cells was a key step in the origin of the vertebrate body plan. An outstanding question is how neural crest cells acquired their ability to undergo an epithelial-mesenchymal transition (EMT) and migrate extensively throughout the vertebrate embryo. We tested if differential regulation of classical cadherins—a highly conserved feature of neural crest EMT and migration in jawed vertebrates—mediates these cellular behaviors in lamprey, a basal jawless vertebrate. Lamprey has single copies of the type I and type II classical cadherins (CadIA and CadIIA). CadIIA is expressed in premigratory neural crest, and requires the transcription factor Snail for proper expression, yet CadIA is never expressed in the neural tube during neural crest development, suggesting that differential regulation of classical cadherin expression is not required to initiate neural crest migration in basal vertebrates. We hypothesize that neural crest cells evolved by retention of regulatory programs linking distinct mesenchymal and multipotency properties, and emigrated from the neural tube without differentially regulating type I/type II cadherins. Our results point to the coupling of mesenchymal state and multipotency as a key event facilitating the origin of migratory neural crest cells.

Should I stay or should I go? Cadherin function and regulation in the neural crest.

Our increasing comprehension of neural crest cell development has reciprocally advanced our understanding of cadherin expression, regulation, and function. As a transient population of multipotent stem cells that significantly contribute to the vertebrate body plan, neural crest cells undergo a variety of transformative processes and exhibit many cellular behaviors, including epithelial-to-mesenchymal transition (EMT), motility, collective cell migration, and differentiation. Multiple studies have elucidated regulatory and mechanistic details of specific cadherins during neural crest cell development in a highly contextual manner. Collectively, these results reveal that gradual changes within neural crest cells are accompanied by often times subtle, yet important, alterations in cadherin expression and function. The primary focus of this review is to coalesce recent data on cadherins in neural crest cells, from their specification to their emergence as motile cells soon after EMT, and to highlight the complexities of cadherin expression beyond our current perceptions, including the hypothesis that the neural crest EMT is a transition involving a predominantly singular cadherin switch. Further advancements in genetic approaches and molecular techniques will provide greater opportunities to integrate data from various model systems in order to distinguish unique or overlapping functions of cadherins expressed at any point throughout the ontogeny of the neural crest.

Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis

The epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, calledTg(snai1b:GFP), which labels epithelial cells undergoing EMT to producesox10-positive neural crest (NC) cells. Time-lapse and lineage analysis ofTg(snai1b:GFP)embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. TreatingTg(snai1b:GFP)embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RAin vivoand raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells.

Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis.

ABSTRACTThe epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP), which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC) cells. Time-lapse and lineage analysis of Tg(snai1b:GFP) embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP) embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells.

Abstract 4096: Identification of EMT inhibitors using novel zebrafish reporter lines

Abstract The Epithelial to Mesenchymal Transition (EMT) plays a crucial role during development and aberrant reactivation of EMT in tumor cells promotes metastasis and drug resistance. Mechanisms that control EMT remain incompletely understood, hampering efforts to target EMT in cancer patients. To identify upstream inducers and downstream effectors of EMT we have developed a novel zebrafish neural crest (NC) EMT reporter line, Tg(snail1b:GFP), for in vivo screening of EMT and cell migration inhibitors. After screening a number of candidate kinase inhibitors for their ability to block NC migration in live Tg(snail1b:GFP) embryos, we found that the AXL receptor tyrosine kinase inhibitor, TP-0903 (HCI-2084), potently blocks hindbrain morphogenesis and cranial NC cell migration. Treatment of embryos at the 3-somite stage with TP-0903 significantly decreases twist1a expression, a key regulator of EMT, blocks differentiation of dlx2a-expressing chondrogenic precursors, and instead promotes the differentiation of mitfa-expressing melanophore precursors that form adjacent to the neural tube. RNA-Seq analysis revealed that TP-0903 treated embryos have a significant upregulation of retinoic acid (RA) signaling. Co-treatment of TP-0903 with DEAB, an RA inhibitor, rescues the hindbrain and NC migration and differentiation defects caused by TP-0903 treatment. We are currently investigating the potential role of AXL in NC cell migration and differentiation, and evaluating TP-0903 as a possible therapy for neuroblastoma, by using the zebrafish neuroblastoma pre-clinical model and human neuroblastoma cells in vitro. Citation Format: Laura Jimenez, Jindong Wang, Cicely Jette, Steven L. Warner, David Bearss, Rodney Stewart. Identification of EMT inhibitors using novel zebrafish reporter lines. [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 4096. doi:10.1158/1538-7445.AM2015-4096

FoxD3 regulates cranial neural crest EMT via downregulation of tetraspanin18 independent of its functions during neural crest formation

The scaffolding protein tetraspanin18 (Tspan18) maintains epithelial cadherin-6B (Cad6B) to antagonize chick cranial neural crest epithelial-to-mesenchymal transition (EMT). For migration to take place, Tspan18 must be downregulated. Here, we characterize the role of the winged-helix transcription factor FoxD3 in the control of Tspan18 expression. Although we previously found that Tspan18 mRNA persists several hours past the stage it would normally be downregulated in FoxD3-deficient neural folds, we now show that Tspan18 expression eventually declines. This indicates that while FoxD3 is crucial for initial downregulation of Tspan18, other factors subsequently impact Tspan18 expression. Remarkably, the classical EMT transcription factor Snail2 is not one of these factors. As in other vertebrates, FoxD3 is required for chick cranial neural crest specification and migration, however, FoxD3 has surprisingly little impact on chick cranial neural crest cell survival. Strikingly, Tspan18 knockdown rescues FoxD3-dependent neural crest migration defects, although neural crest specification is still deficient. This indicates that FoxD3 promotes cranial neural crest EMT by eliciting Tspan18 downregulation separable from its Tspan18-independent activity during neural crest specification and survival.