Elucidating the framework for specification and determination of the embryonic retina.

How cell determination is regulated remains a major unsolved problem in developmental biology. The early embryonic rudiments of many tissues and organs are difficult or impossible to identify, isolate and study at the time when determination occurs. We have examined the commitment process leading to retina formation in Xenopus laevis, where presumptive eye tissue can be identified and studied to assay its biological properties during the events leading up to determination. We find that for the retina, specification, the point at which a tissue placed in neutral culture medium can first properly differentiate, occurs during mid-gastrulation. By late gastrulation, determination, the final, irreversible step in commitment, has occurred. At this stage, the presumptive retina will differentiate and cannot be reprogrammed even if exposed to other active inducers, e.g. when challenged by transplantation to ectopic sites in the embryo. Key eye regulatory genes are initially expressed in the retinal field during specification and/or determination (e.g. rax, pax6, lhx2, and fzd5) potentially linking them, or genes that regulate them, to these processes. This study provides essential groundwork for defining the mechanisms for how these important developmental transitions occur.

Comparative genomics-based identification and analysis of cis-regulatory elements.

Identification of cis-regulatory elements, such as enhancers and promoters, is very important not only for analysis of gene regulatory networks but also as a tool for targeted gene expression experiments. In this chapter, we introduce an easy but reliable approach to predict enhancers of a gene of interest by comparing mammalian and Xenopus genome sequences, and to examine their activity using a co-transgenesis technique in Xenopus embryos. Since the bioinformatics analysis utilizes publically available web tools, bench biologists can easily perform it without any need for special computing capability. The co-transgenesis assay, which directly uses polymerase chain reaction products, quickly screens for the activity of the candidate elements in a cloning-free manner.

Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.

BACKGROUND: Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY: We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS: These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation.

Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements.

In addition to the canonical Wnt/beta-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila [1] and the Wnt/calcium pathway in vertebrate embryos [2]. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation [3-7]. To further test this hypothesis, we have identified two zebrafish homologs of the Drosophila PCP gene prickle (pk) [8], both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/beta-catenin signaling. Like noncanonical Wnts [9-10], overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. Our results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways.