RESUMO
Despite extensive study, the earliest steps of vertebrate axis formation are only beginning to be elucidated. We previously showed that asymmetric localization of maternal transcripts of the conserved zebrafish TGFß factor Squint (Sqt) in 4-cell stage embryos predicts dorsal, preceding nuclear accumulation of ß-catenin. Cell ablations and antisense oligonucleotides that deplete Sqt lead to dorsal deficiencies, suggesting that localized maternal sqt functions in dorsal specification. However, based upon analysis of sqt and Nodal signaling mutants, the function and mechanism of maternal sqt was debated. Here, we show that sqt RNA may function independently of Sqt protein in dorsal specification. sqt insertion mutants express localized maternal sqt RNA. Overexpression of mutant/non-coding sqt RNA and, particularly, the sqt 3'UTR, leads to ectopic nuclear ß-catenin accumulation and expands dorsal gene expression. Dorsal activity of sqt RNA requires Wnt/ß-catenin but not Oep-dependent Nodal signaling. Unexpectedly, sqt ATG morpholinos block both sqt RNA localization and translation and abolish nuclear ß-catenin, providing a mechanism for the loss of dorsal identity in sqt morphants and placing maternal sqt RNA upstream of ß-catenin. The loss of early dorsal gene expression can be rescued by the sqt 3'UTR. Our findings identify new non-coding functions for the Nodal genes and support a model wherein sqt RNA acts as a scaffold to bind and deliver/sequester maternal factors to future embryonic dorsal.
Assuntos
Regiões 3' não Traduzidas , Padronização Corporal/genética , Padronização Corporal/fisiologia , Ligantes da Sinalização Nodal/genética , Ligantes da Sinalização Nodal/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligantes da Sinalização Nodal/antagonistas & inibidores , Oligonucleotídeos Antissenso/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
BACKGROUND: The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type beta family such as Vg1, activin B, the nodal-related proteins and derrière. METHODOLOGY AND PRINCIPLE FINDINGS: Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17alpha/beta, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. CONCLUSIONS/SIGNIFICANCE: These observations reveal distinct functions for these two classes of the TGF-beta family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.
