Transcriptionally regulated cell adhesion network dictates distal tip cell directionality.

BACKGROUND: The mechanisms that govern directional changes in cell migration are poorly understood. The migratory paths of two distal tip cells (DTC) determine the U-shape of the C. elegans hermaphroditic gonad. The morphogenesis of this organ provides a model system to identify genes necessary for the DTCs to execute two stereotyped turns. RESULTS: Using candidate genes for RNAi knockdown in a DTC-specific strain, we identified two transcriptional regulators required for DTC turning: cbp-1, the CBP/p300 transcriptional coactivator homologue, and let-607, a CREBH transcription factor homologue. Further screening of potential target genes uncovered a network of integrin adhesion-related genes that have roles in turning and are dependent on cbp-1 and let-607 for expression. These genes include src-1/Src kinase, tln-1/talin, pat-2/α integrin and nmy-2, a nonmuscle myosin heavy chain. CONCLUSIONS: Transcriptional regulation by means of cbp-1 and let-607 is crucial for determining directional changes during DTC migration. These regulators coordinate a gene network that is necessary for integrin-mediated adhesion. Overall, these results suggest that directional changes in cell migration rely on the precise gene regulation of adhesion.

mig-38, a novel gene that regulates distal tip cell turning during gonadogenesis in C. elegans hermaphrodites.

In Caenorhabditis elegans gonad morphogenesis, the final U-shapes of the two hermaphrodite gonad arms are determined by migration of the distal tip cells (DTCs). These somatic cells migrate in opposite directions on the ventral basement membrane until specific extracellular cues induce turning from ventral to dorsal and then centripetally toward the midbody region on the dorsal basement membrane. To dissect the mechanism of DTC turning, we examined the role of a novel gene, F40F11.2/mig-38, whose depletion by RNAi results in failure of DTC turning so that DTCs continue their migration away from the midbody region. mig-38 is expressed in the gonad primordium, and expression continues throughout DTC migration where it acts cell-autonomously to control DTC turning. RNAi depletion of both mig-38 and ina-1, which encodes an integrin adhesion receptor, enhanced the loss of turning phenotype indicating a genetic interaction between these genes. Furthermore, the integrin-associated protein MIG-15/Nck-interacting kinase (NIK) works with MIG-38 to direct DTC turning as shown by mig-38 RNAi with the mig-15(rh80) hypomorph. These results indicate that MIG-38 enhances the role of MIG-15 in integrin-dependent DTC turning. Knockdown of talin, a protein that is important for integrin activation, causes the DTCs to stop migration prematurely. When both talin and MIG-38 were depleted by RNAi treatment, the premature stop phenotype was suppressed. This suppression effect was reversed upon additional depletion of MIG-15 or its binding partner NCK-1. These results suggest that both talin and the MIG-15/NCK-1 complex promote DTC motility and that MIG-38 may act as a negative regulator of the complex. We propose a model to explain the dual role of MIG-38 in motility and turning.

Daughterless dictates Twist activity in a context-dependent manner during somatic myogenesis.

Somatic myogenesis in Drosophila relies on the reiterative activity of the basic helix-loop-helix transcriptional regulator, Twist (Twi). How Twi directs multiple cell fate decisions over the course of mesoderm and muscle development is unclear. Previous work has shown that Twi is regulated by its dimerization partner: Twi homodimers activate genes necessary for somatic myogenesis, whereas Twi/Daughterless (Da) heterodimers lead to the repression of these genes. Here, we examine the nature of Twi/Da heterodimer repressive activity. Analysis of the Da protein structure revealed a Da repression (REP) domain, which is required for Twi/Da-mediated repression of myogenic genes, such as Dmef2, both in tissue culture and in vivo. This domain is crucial for the allocation of mesodermal cells to distinct fates, such as heart, gut and body wall muscle. By contrast, the REP domain is not required in vivo during later stages of myogenesis, even though Twi activity is required for muscles to achieve their final pattern and morphology. Taken together, we present evidence that the repressive activity of the Twi/Da dimer is dependent on the Da REP domain and that the activity of the REP domain is sensitive to tissue context and developmental timing.

Discrete levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis.

Twist (Twi), a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT), and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. In this study, we derive a twi allelic series by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, we find that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, we show that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, we can reconstitute gastrulation and produce viable adult flies. Our results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur.

Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite.

Cell migration and morphogenesis are key events in tissue development and organogenesis. In Caenorhabditis elegans, the migratory path of the distal tip cells determines the morphology of the hermaphroditic gonad. The distal tip cells undergo a series of migratory phases interspersed with turns to form the gonad. A wide variety of genes have been identified as crucial to this process, from genes that encode components and modifiers of the extracellular matrix to signaling proteins and transcriptional regulators. The connections between extracellular and transmembrane protein functions and intracellular pathways are essential for distal tip cell migration, and the integration of this information governs gonad morphogenesis and determines gonad size and shape.

Analysis of cell migration using Caenorhabditis elegans as a model system.

The nematode Caenorhabditis elegans is an excellent model system in which to study long-distance cell migration in vivo. This chapter describes methods used to study a subset of migratory cells in the hermaphrodite nematode, the distal tip cells. These methods take advantage of the organism's transparent body and the expression of green fluorescent protein to observe cell migration and behavior. Additionally, the availability of nematode mutants and gene knockdown techniques that affect cell migration allow the analysis and comparison of wild-type and aberrant migratory paths. Methods for nematode growth and maintenance, strain acquisition, observation and live imaging, gene knockdown, and analysis of cell migration defects are covered.