Control of Ectodermal Development in Vertebrate Embryos

Thomas D. Sargent, PhD, Head, Section on Vertebrate Development
Yoo-Seok Hwang, PhD, Visiting Fellow
Yanhua Xu, PhD, Visiting Fellow
Ting Luo, MD, PhD, Staff Scientist

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. The functions of the genes encoding these proteins are being analyzed 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.

Inca: a novel regulator of cytoskeletal dynamics

Luo, Xu, Hwang, Sargent; in collaboration with Williams

Inca (Induced in Neural Crest by AP2) 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. Inca is also expressed in mesoderm during gastrulation and in additional tissues, such as heart, in tadpole and in later stages of frog development. Homologues of Inca exist in all vertebrates, including mouse, human, and zebrafish, but Inca genes are not found in invertebrates. The Inca protein sequence is novel with no distinguishing features enabling its assignment to existing protein families. The early expression pattern of Inca is conserved in fish and mouse embryos, and we have shown by anti-sense loss-of-function experiments that Inca is required for normal craniofacial development. A collaborative project to target the mouse Inca gene is currently under way in the Williams laboratory. Recently born Inca null mice do not appear to be grossly abnormal, suggesting that other genes in the mouse, in contrast to fish and frogs, might at least partially compensate for Inca function. Longer-term and more detailed analysis of the Inca null mice is in progress.

Using yeast two-hybrid analysis, we identified a p21-activated kinase (PAK4) as an interaction partner for Inca. 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 Inca in NIH 3T3 cells leads to increased levels of active, GTP-bound Rho, resulting in alterations in the actin cytoskeleton, microtubule acetylation, and cell-migratory behavior. PA K4 is known to regulate Rho activation via repression of RhoGE F; we thus hypothesize that Inca affects Rho signaling via the same route. In frog embryos, loss of Inca function inhibits convergent extension (CE) movements in dorsal mesoderm, delaying or preventing the completion of gastrulation. In experiments with overexpression of a deletion mutation of Inca, we removed the conserved 35-residue “inca box” and observed complex effects on both CE and MAPK signaling, apparently through separate mechanisms. In the zebrafish, two Inca genes are equally similar to Xenopus Inca. One is expressed mainly in neural crest, and its knockdown yields a phenotype similar to what we observed in the frog. The second zebrafish Inca, Inca2, is most prominently expressed in the mesoderm, particularly in notochord (where Xenopus Inca is also expressed). Knockdown of zebrafish Inca2 causes dramatic changes in the expression of marker genes for dorsoventral polarity, indicating a role of this Inca in basic embryonic axis formation, including notochord formation. We observed similarities between mesoderm movements during gastrulation and the extension of cranial cartilage following NC migration, suggesting that both processes might require Inca in a fundamentally equivalent manner.

Luo T, Xu Y, Hoffman TL, Zhang T, Schilling T, Sargent TD. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development. Development 2007;134:1279-1289.

Myosin10: a cytoskeletal motor protein required in CNC development

Hwang, Luo, Sargent

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 anti-sense 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.

Collaborator

Trevor Williams, PhD, University of Colorado, Denver, CO

For further information, contact sargentt@mail.nih.gov or visit http://svd.nichd.nih.gov.