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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 zebrafish as the primary experimental model organism, but also work with the frog Xenopus laevis. 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, concentrated on a novel gene, Inka, that we have found to be essential for proper craniofacial development, at least in the fish and frog embryo. Inka encodes a p21-activated kinase (PAK)–associated protein that has at least two functional roles in early development—one in cytoskeletal regulation and the other in modulation of FGF signaling during axis formation. The connection between these early functions and the role of Inka in neural crest development is under investigation in both frog and fish embryos. We are also investigating the functions of PAK proteins in zebrafish embryos and have found that some aspects are related to Inka and others are independent.
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 in anti-sense loss-of-function experiments that Inka is required for normal craniofacial development. A collaborative project with the laboratory of Trevor Williams to target the mouse Inka gene 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 Inka, which could fulfill such a function. We will pursue this question initially in zebrafish and possibly in Inka.
Using yeast two-hybrid analysis, we identified 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 cells resulted in alterations in the actin cytoskeleton, greatly reduced microtubule acetylation, and enhanced cell-migratory behavior. We are investigating the function of PAK4 in zebrafish by morpholino knockdown and by dominant negative approaches. Loss of PAK4 in mouse results in numerous abnormalities and in embryonic lethality. Our results indicate that, in zebrafish, PAK4 may play a different role, with specific functions in blood and somite development. Zebrafish PAK4 also appears to be important in axis formation, possibly in conjunction with Inka.
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.
Publications
- Reid BS, Sargent TD, Williams T. Generation and characterization of a novel neural crest marker allele, Inka1-LacZ, reveals a role for Inka1 in mouse neural tube closure. Dev Dyn. 2010;239:1188-1196.
Collaborators
- Trevor Williams, PhD, University of Colorado, Denver, CO