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Control of Ectodermal Development in Vertebrate Embryos
- Thomas D. Sargent, PhD, Head, Section on Vertebrate Development *
- Yoo-Seok Hwang, PhD, Visiting Fellow
- Hiu Wan Law, Visiting Fellow
- Ting Luo, MD, PhD, Staff Scientist
- Yanhua Xu, PhD, Visiting Fellow
We identify factors and mechanisms that are responsible for controlling early vertebrate development, focusing on the cranial neural crest (CNC). We use the frog Xenopus laevis and zebrafish as the primary experimental model organisms. Our earlier research revealed a central role in epidermal and neural crest (NC) development for the transcription activator TFAP 2. We have built on that research by identifying downstream regulatory targets of TFAP2 and, in the past year, have concentrated on two genes shown to be essential for proper craniofacial development. The genes encode Inca, a novel p21-activated kinase–associated protein involved in cytoskeletal regulation, and MyosinX, a non-muscle motor protein expressed in NC, sensory placodes, and other tissues. We are analyzing the functions of the genes encoding these proteins by inhibiting expression with gene-specific anti-sense oligonucleotides and by overexpression strategies in embryos and cultured cells. We have discovered that, in addition to its function in CNC, Inca plays an important role in controlling cell movements during gastrulation. We are pursuing the molecular basis of this role in both frog and fish embryos.
Inka: a novel regulator of cytoskeletal dynamics
Inka is expressed strongly in the neural crest, beginning after gastrulation and continuing throughout development. As shown by gene knockdown experiments and standard genetics, maintenance of this expression is dependent on TFAP2 activity in the frog and zebrafish. Inka is also expressed in mesoderm during gastrulation and in additional tissues, such as heart, in tadpole and in later stages of frog development. Homologs of Inka exist in all vertebrates, including mouse, human, and zebrafish, but Inka genes are not found in invertebrates. The Inka protein sequence is novel, with no distinguishing features enabling its assignment to existing protein families. The early expression pattern of Inka is conserved in fish and mouse embryos, and we have shown by anti-sense loss-of-function experiments that Inka is required for normal craniofacial development. A collaborative project to target the mouse Inka gene in the laboratory of Trevor Williams has been completed. In contrast to the loss-of-function findings in lower vertebrates, Inka null mice appear to be normal and fertile, suggesting that other genes in the mouse might compensate for Inka function. We have identified an Inka-related gene, which we called Inka2, which could fulfill such a function. We will pursue this question initially in zebrafish and possibly in Xenopus.
Using yeast two-hybrid analysis, we identified a p21-activated kinase (PAK4) as an interaction partner for Inka. PAKs function in the transduction of cell-cell signals mediated by the Rho class GTPases Rac and Cdc42 and have been implicated in the regulation of cytoskeletal dynamics as well as in other cell processes. Interestingly, PAK4 is upregulated in many tumors. We found that overexpression of Inka in NIH 3T3 resulted in alterations in the actin cytoskeleton, greatly reduced microtubule acetylation, and enhanced cell-migratory behavior. In the zebrafish, two Inka genes are equally similar to Xenopus Inka. One, inka1a, is expressed mainly in neural crest, and its knockdown yields a phenotype similar to what we observed in the frog. The second, inka1b, is most prominently expressed in the mesoderm, particularly in notochord (where Xenopus Inka is also expressed). Knockdown of zebrafish inka1b disrupts basic embryonic axis formation. We are currently trying to determine the molecular link among the various phenotypic effects. We are testing the hypothesis that cytoskeletal alterations resulting from changes in inka expression affect protein transport, which could in turn modulate cell-cell signaling. Another possibility under investigation stems from our observation that inka expression can also affect acetylation of histones, which might lead to chromatin remodeling and concomitant transcriptional reprogramming.
Myosin10: a cytoskeletal motor protein required in CNC development
Myosin10 (Myo10) is a member of the large superfamily of non-muscle myosins. It has been recently shown to interact with both microtubules and F-actin filaments. The gene encoding Myo10 was strongly induced by TFAP 2a in our microarray screen and is expressed in NC, placodes, and paraxial mesoderm. Myo10 is encoded by a maternal mRNA in Xenopus; suppression of this mRNA translation causes mitotic defects and arrest during early cleavage stages. To assess Myo10 function later in development, we designed two antisense morpholino oligonucleotides that inhibit processing of Myo10 precursor RNA. Loss of function of Myo10 results in inhibition of migration in vivo. NC cells migrate most efficiently on fibronectin, using integrin alpha5beta1 as the receptor. In vitro migration assays on fibronectin using explanted CNC cells showed significant inhibition of cell attachment as well as spreading and migration resulting from MyoX knockdown. Rhodamin-phalloidin staining revealed malformation of membrane processes, including filopodia. It has been reported that the FERM-domain of MyoX physically interacts with a conserved NPXY motif present in the cytoplasmic domain of most beta integrins. To test for such an interaction in the NC, we co-expressed beta3 integrin labeled with mCherry and MyoX labeled with GFP in frog embryos. We then excised NC tissue and used live confocal imaging after the NC cells had spread on a fibronectin substrate. We observed co-localization, particularly at the tips of filopodia, which was disrupted by MyoX knockdown, supporting our hypothesis that MyoX is required for integrin transport and/or activation in migrating NC. Our work is the first demonstration of such a function for MyoX in embryonic cells.
Publications
- Hwang YS, Luo T, Xu Y, Sargent TD. MyosinX is required for cranial neural crest cell migration in Xenopus laevis. Dev Dyn 2009 238:2522-2529.
Collaborators
- Trevor Williams, PhD, University of Colorado, Denver, CO