CONTROL OF ECTODERMAL DEVELOPMENT IN XENOPUS
, Head, Section on Vertebrate Development
Ting Luo, MD, PhD, Staff Scientist
Yoo-Seok Hwang, PhD, Visiting Fellow
Yanhua Xu, PhD, Visiting Fellow
Janaki Rangarajan, BS, Graduate Student
The aim of our work is to identify factors and mechanisms that are responsible for the control of early vertebrate development, focusing on the neural crest (NC) and other ectodermal derivatives. We use the frog Xenopus laevis as an experimental model organism. Our previous research revealed a central role for the transcription activator TFAP2 in ectodermal development. We have continued to advance this project by identifying downstream regulatory targets of TFAP2 and are now concentrating on three genes that are essential for proper NC development: Inca, which encodes a novel protein associated with p21-activated kinase; PCNS, which encodes a novel protocadherin; and MyosinX, which encodes a non-muscle myosin expressed in the NC, sensory placodes, and other tissues. We are analyzing the functions of the genes by inhibiting expression with gene-specific antisense oligonucleotides and by overexpression strategies in embryos and cultured cells.
Inca: a novel regulator of cytoskeletal dynamics
Inca (Induced in Neural Crest by AP2) is intensely expressed in the neural crest, beginning after gastrulation and continuing throughout development. Expression is dependent on TFAP2 activity in frog and zebrafish. Inca is also expressed in mesoderm during gastrulation as well as in additional tissues, such as the heart, and in tadpole and later stages. 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, exhibiting no distinguishing features to enable its unambiguous assignment to existing protein families. Inca’s early expression pattern is conserved in fish and mouse embryos, and we have shown by antisense loss-of-function experiments that Inca is required for normal craniofacial development. A collaborative project to target the mouse Inca gene is currently in progress in Trevor Williams’s laboratory.
Using yeast two-hybrid analysis, we identified a p21-activated kinase (PAK4) as an interaction partner for Inca. PAKs transduce cell-cell signals mediated by the Rho class of GTPases Rac and Cdc42 and have been implicated in the regulation of cytoskeletal dynamics and apoptosis. We find that overexpression of Inca in NIH 3T3 cells leads to increased levels of active, GTP-bound Rho. PAK4 is known to regulate Rho activation via repression of RhoGEF; thus, it is likely that Inca affects Rho signaling via the same mechanism, resulting in alterations in the actin cytoskeleton, microtubule acetylation, and cell migratory behavior. In frog embryos, both gain and loss of Inca function inhibit convergent extension movements in dorsal mesoderm, delaying or preventing the completion of gastrulation. We have preliminary evidence suggesting that this effect may be attributable to modulation of the function of cadherin adhesion molecules by Inca. This effect could also result from alterations in Rho-type signaling but could also involve changes in the MAP kinase cascade. Given some similarities between mesoderm movements during gastrulation and the extension of cranial cartilage following neural crest migration, 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;134:1279-89.
PCNS, a novel protocadherin required for somite and neural crest development
Protocadherins are a large subfamily (about 70 genes in mammals) of the cadherin superfamily of calcium-dependent cell adhesion molecules. We discovered a novel protocadherin that is strongly upregulated by TFAP2a and named it PCNS (Protocadherin in Neural crest and Somites). The gene is expressed throughout mesoderm during gastrulation and is transiently expressed in somites in an anterior-posterior wave correlating with the condensation of somites from paraxial mesoderm; it is also strongly expressed in premigratory and migratory neural crest. Late in development, PCNS mRNA vanishes in mesoderm and neural crest but appears in the heart and ear vesicle. Loss of PCNS function in neural crest results in failure to migrate. In the somites, loss of PCNS prevents the orchestrated rotation of somite cells into an orderly periodic array. In early mesoderm, loss of PCNS function inhibits convergent extension while overexpression enhances and modifies these movements. Related protocadherins are thought to function in part by modulating cell-cell adhesion, primarily by affecting cadherin function. The role of PCNS in cell adhesion is subtle, and detection depends on the assay used, but we hypothesize that these protocadherins may also function by modulating adhesion and are testing the idea with more sophisticated adhesion assays.
Rangarajan J, Luo T, Sargent TD. PCNS: a novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus. Dev Biol 2006;295:206-18.
Myosin10: a cytoskeletal motor protein required in cranial NC development
Myosin10 (Myo10), a member of the large superfamily of non-muscle myosins, has been recently shown to interact with both microtubules and F-actin filaments (Weber et al., Nature 2004;431:325). Our microarray screen showed that the gene is strongly induced by TFAP2a and is expressed in NC, placodes, and paraxial mesoderm. Myo10 is encoded by a maternal mRNA in Xenopus, and suppression of its translation causes several defects early in development. To assess Myo10 function later in development, we designed two antisense morpholino oligonucleotides that significantly block processing of Myo10 precursor RNA. Loss of function of Myo10 results in abnormal spreading of neural crest cells on fibronectin in vitro and inhibition of migration in vivo. The Myo10 protein has four major domains in the C-terminal interaction region. We have prepared deletion mutants lacking each interaction domain and plan to test the ability of RNAs encoding the altered Myo10 proteins to rescue the loss-of-function morpholino phenotype. The results should help point to candidate cytoskeletal and/or membrane targets for Myo10 function in the neural crest. As another strategy for identifying Myo10-interacting factors in the frog embryo, we have prepared bait constructs for use in yeast two-hybrid screening.
Publication Related to Other Work
Sargent TD. Transcriptional regulation at the neural plate border. Adv Exp Med Biol 2006;589:32-44.
COLLABORATORS
Trevor Williams, PhD, University of Colorado, Denver, CO
For further information, contact sargentt@mail.nih.gov.
