Thomas D. Sargent, PhD, Head, Section on Vertebrate Development
Ting Luo, MD, PhD, Staff Scientist
Janaki Rangarajan, BS, Graduate Student
Yeo-Sook Hwang, PhD, Visiting Fellow
Yanhua Xu, PhD, Visiting Fellow
Using the frog Xenopus laevis as an experimental model organism, we work to identify factors and mechanisms that are responsible for control of early vertebrate development, focusing on the neural crest (NC) and other ectodermal derivatives. Our previous research revealed a central role in ectodermal development for the transcription activator TFAP2. We have continued our efforts by identifying downstream regulatory targets of TFAP2 and are now concentrating on three such genes that are essential for proper NC development. The encoded proteins are Inca, a novel protein associated with p21-activated kinase; PCNS, a novel protocadherin; and MyosinX, a non-muscle myosin expressed in NC, sensory placodes, and other tissues. We are analyzing the functions of these genes by inhibiting expression with gene-specific antisense oligonucleotides and by overexpression strategies in embryos and cultured cells. We are also using yeast two-hybrid screening to identify protein-protein interactions.
Inca, a novel regulator of cytoskeletal dynamics and apoptosis
Luo, Xu, Sargent; in collaboration with Schilling, Williams
Inca (Induced in Neural Crest by AP2) is intensely expressed in the neural crest, beginning after gastrulation and continuing throughout development. Such 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 heart, as development proceeds. Homologues of Inca exist in all vertebrates, including mouse, human, and zebrafish, but not in invertebrates such as Drosophila or other phyla. The Inca protein sequence is entirely 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, reinforcing the hypothesis that Inca has an important developmental function. Thomas Schilling's laboratory confirmed Inca's developmental function in loss-of-function experiments using antisense morpholino oligonucleotides, which resulted in severe defects in neural crest-derived craniofacial bone and cartilage in both Xenopus and zebrafish. A collaborative mouse knockout project is currently under way in Trevor Williams's laboratory.
Using yeast two-hybrid screening of a mouse embryo cDNA library with mouse Inca, we identified a p21-activated kinase (PAK4) as a candidate interaction partner with Inca and proved that the interaction does indeed occur in unmanipulated cells. As a group, PAK proteins transduce cell-cell signals mediated by the Rho-class GTPases Rac and Cdc42, with the proteins implicated in the regulation of cytoskeletal dynamics and apoptosis. PAK4 exhibits the same functions and is upregulated in many tumor lines. In this context, it is interesting that overexpression of Inca modifies the cytoskeleton and cell-cell adhesion in the early embryo. Furthermore, loss of Inca causes an increase in apoptosis in frog and fish embryos while overexpression has the opposite effect in frogs and cultured cells. The regulation of PAK4/5 has been relatively poorly understood, and we expect Inca to be an important new tool for solving this and other biological problems.
PCNS, a novel protocadherin required for somite and neural crest development
Rangarajan, Luo, Sargent
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 have named it PCNS (Protocadherin in Neural crest and Somites). This gene is transiently expressed in somites in an anterior-posterior wave correlating with the condensation of somites from paraxial mesoderm and is strongly expressed also in premigratory and migratory neural crest. Late in development, PCNS mRNA vanishes in derivatives of both these embryonic tissues but appears in the heart and ear vesicle. Loss of PCNS function, achieved via antisense oligonucleotides as well as by a dominant negative approach, leads to striking phenotypes in neural crest and somites. The neural crest cells lacking PCNS are induced normally but fail to migrate. The defect appears fairly early and is likely to occur in the epithelial/mesenchymal transition. In the somite, loss of PCNS prevents the orchestrated rotation of somite cells into an orderly periodic array. PCNS does not appear to be a particularly strong adhesive molecule, and we hypothesize that it functions in one or more signaling pathways to the control of cytoskeleton or cell polarity. Preliminary evidence suggests that the protein kinase JNK may be regulated in part via PCNS-dependent interactions.
Myosin10, a cytoskeletal motor protein expressed and required in cranial NC 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 (Weber et al., Nature 2004;431:325). The gene was strongly induced by TFAP2a in our microarray screen and is expressed in NC, placodes, and paraxial mesoderm. Myo10 is encoded by a maternal mRNA in Xenopus, and suppression of this mRNA 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. Preliminary results indicate that the protein is required for craniofacial cartilage development, with a loss-of-function phenotype similar in some aspects to that obtained for Inca and PCNS. In future experiments, we will look for microtubule and microfilament interactions with Myo10 in NC cells and attempt to determine what specific cellular function Myo10 protein might perform in the developing cranial NC.
COLLABORATORS
Thomas F. Schilling, PhD, University of California, Irvine, Irvine, CA
Trevor Williams, PhD, University of Colorado, Denver, CO
For further information, contact tsargent@nih.gov.