The functions of the Snail family of zinc-finger transcription factors are essential during embryonic development. One of their best-known functions is to induce epithelial to mesenchymal transitions (EMTs), which convert epithelial cells into migratory mesenchymal cells. In recent years, many orthologues of the Snail family have been identified throughout the animal kingdom, and their study is providing new clues about the EMTdependent and -independent functions of Snail proteins. Here, we discuss these functions and how they influence cell behaviour during development and during diseases such as metastatic cancer. From these findings, we propose that Snail genes act primarily as survival factors and inducers of cell movement, rather than as inducers of EMT or cell fate. Summary

The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Neural crest progenitor cells are the main contributors to craniofacial cartilage and connective tissue of the vertebrate head. These progenitor cells also give rise to the pigment, neuronal and glial cell lineages. To study the molecular basis of neural crest differentiation, we have cloned the gene disrupted in the mont blanc (mobm610) mutation,which affects all neural crest derivatives. Using a positional candidate cloning approach we identified an A to G transition within the 3′ splice site of the sixth intron of the tfap2a gene that abolishes the last exon encoding the crucial protein dimerization and DNA-binding domains. Neural crest induction and specification are not hindered in mobm610 mutant embryos, as revealed by normal expression of early neural crest specific genes such as snail2, foxd3and sox10. In addition, the initial stages of cranial neural crest migration appear undisturbed, while at a later phase the craniofacial primordia in pharyngeal arches two to seven fail to express their typical set of genes (sox9a, wnt5a, dlx2, hoxa2/b2). In mobm610 mutant embryos, the cell number of neuronal and glial derivatives of neural crest is greatly reduced, suggesting that tfap2a is required for their normal development. By tracing the fate of neural crest progenitors in live mont blanc(mobm610) embryos, we found that at 24 hpf neural crest cells migrate normally in the first pharyngeal arch while the preotic and postotic neural crest cells begin migration but fail to descend to the pharyngeal region of the head. TUNEL assay and Acridine Orange staining revealed that in the absence of tfap2a a subset of neural crest cells are unable to undergo terminal differentiation and die by apoptosis. Furthermore, surviving neural crest cells in tfap2a/mobm610 mutant embryos proliferate normally and later differentiate to individual derivatives. Our results indicate that tfap2a is essential to turn on the normal developmental program in arches 2-7 and in trunk neural crest. Thus, tfap2a does not appear to be involved in early specification and cell proliferation of neural crest, but it is a key regulator of an early differentiation phase and is required for cell survival in neural crest derived cell lineages.

Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

SummaryNoradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid and require tfap2a for expression of the neurotransmitter phenotype

The Snail genes are implicated in processes that involve cell movement, both during embryonic development and tumour progression. In teleosts, the vertebrate Snail1 gene is represented by two distinct genes, snail1a and snail1b (previously snail1 and snail2). These genes are expressed in complementary mesodermal domains and their combined expression matches that of their mammalian counterpart. By analysing their loss and gain of function, we found that the most-anterior axial mesendodermal cells, the precursors of the polster, move in a cohesive manner directed by the activity of snail1a-and snail1b-expressing cells surrounding these precursors. The cell-autonomous function of Snail1 proteins regulates cell motility and influences the behaviour of Snailnegative neighbouring cells. Snail1a is required by the prechordal plate for it to reach its normal position, whereas Snail1b controls the acquisition of its normal shape. These non-redundant functions of Snail1a and Snail1b in controlling axial mesendoderm migration comply with the duplication-degeneration-complementation model, and indicate that Snail genes not only act as inducers of epithelial-to-mesenchymal transition, but also as more general regulators of cell adhesion and movement.

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