Certain aggressive melanoma cell lines exhibit a dedifferentiated phenotype, expressing genes that are characteristic of various cell types including endothelial, neural, and stem cells. Moreover, we have shown that aggressive melanoma cells can participate in neovascularization in vivo and vasculogenic mimicry in vitro, demonstrating that these cells respond to microenvironmental cues and manifest developmental plasticity. To explore this plasticity further, we transplanted human metastatic melanoma cells into zebrafish blastula‐stage embryos and monitored their behavior post‐transplantation. The data show that human metastatic melanoma cells placed in the zebrafish embryo survive, exhibit motility, and divide. The melanoma cells do not form tumors nor integrate into host organs, but instead become scattered throughout the embryo in interstitial spaces, reflecting the dedifferentiated state of the cancer cells. In contrast to the fate of melanoma cells, human melanocytes transplanted into zebrafish embryos most frequently become distributed to their normal microenvironment of the skin, revealing that the zebrafish embryo contains possible homing cues that can be interpreted by normal human cells. Finally, we show that within the zebrafish embryo, metastatic melanoma cells retain their dedifferentiated phenotype. These results demonstrate the utility of the zebrafish embryonic model for the study of tumor cell plasticity and suggest that this experimental paradigm can be a powerful one in which to investigate tumor‐microenvironment interactions. Developmental Dynamics 233:1560–1570, 2005.

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Preplacodal ectoderm arises near the end of gastrulation as a narrow band of cells surrounding the anterior neural plate. This domain later resolves into discrete cranial placodes that, together with neural crest, produce paired sensory structures of the head. Unlike the better-characterized neural crest, little is known about early regulation of preplacodal development. Classical models of ectodermal patterning posit that preplacodal identity is specified by readout of a discrete level of Bmp signaling along a DV gradient. More recent studies indicate that Bmp-antagonists are critical for promoting preplacodal development. However, it is unclear whether Bmp-antagonists establish the proper level of Bmp signaling within a morphogen gradient or, alternatively, block Bmp altogether. To begin addressing these issues, we treated zebrafish embryos with a pharmacological inhibitor of Bmp, sometimes combined with heat shock-induction of Chordin and dominant-negative Bmp receptor, to fully block Bmp signaling at various developmental stages. We find that preplacodal development occurs in two phases with opposing Bmp requirements. Initially, Bmp is required before gastrulation to co-induce four transcription factors, Tfap2a, Tfap2c, Foxi1, and Gata3, which establish preplacodal competence throughout the nonneural ectoderm. Subsequently, Bmp must be fully blocked in late gastrulation by dorsally expressed Bmp-antagonists, together with dorsally expressed Fgf and Pdgf, to specify preplacodal identity within competent cells abutting the neural plate. Localized ventral misexpression of Fgf8 and Chordin can activate ectopic preplacodal development anywhere within the zone of competence, whereas dorsal misexpression of one or more competence factors can activate ectopic preplacodal development in the neural plate. Conversely, morpholino-knockdown of competence factors specifically ablates preplacodal development. Our work supports a relatively simple two-step model that traces regulation of preplacodal development to late blastula stage, resolves two distinct phases of Bmp dependence, and identifies the main factors required for preplacodal competence and specification. Cranial placodes, which produce sensory structures in the head, arise from a contiguous band of preplacodal ectoderm surrounding the anterior neural plate during gastrulation. Little is known about early regulation of preplacodal ectoderm, but modulation of signaling through Bone Morphogenetic Protein (Bmp) is clearly involved. Recent studies show that dorsally expressed Bmp-antagonists help establish preplacodal ectoderm, but it is not clear whether antagonists titrate Bmp to a discrete low level that actively induces preplacodal fate or, alternatively, whether Bmp must be fully blocked to permit preplacodal development. We show that in zebrafish preplacodal development occurs in distinct phases with differing Bmp requirements. Initially, Bmp is required before gastrulation to render all ventral ectoderm competent to form preplacodal tissue. We further show that four transcription factors, Foxi1, Gata3, Tfap2a, and Tfap2c, specifically mediate preplacodal competence. Once induced, these factors no longer require Bmp. Thereafter, Bmp must be fully blocked by dorsally expressed Bmp-antagonists to permit preplacodal development. In addition, dorsally expressed Fgf and/or Pdgf are also required, activating preplacodal development in competent cells abutting the neural plate. Thus, we have resolved the role of Bmp and traced the regulation of preplacodal development to pre-gastrula stage.