Dynamic in vivo binding of transcription factors to cis-regulatory modules ofcerandgscin the stepwise formation of the Spemann–Mangold organizer

Abstract

SUMMARYHow multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann-Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs

“…This amino acid region is outside the homeodomain (15). In addition, anti-Mix1 is specific for Mix1 and it does not cross-react with the paralogous protein Mixer (15). This epitope region is conserved in Xenopus tropicalis (75% amino acid identity).…”

“…We used rabbit polyclonal antibodies (abs) against Mix1 and VegT from X. laevis (15) to detect these proteins in embryos of four additional frog species. The Mix1 polyclonal ab was raised against the C-terminal region of X. laevis Mix1 (amino acids 156−377; accession no.…”

“…The Mix1 polyclonal ab was raised against the C-terminal region of X. laevis Mix1 (amino acids 156−377; accession no. NP_001081294) (15). This amino acid region is outside the homeodomain (15).…”

“…NP_001081294) (15). This amino acid region is outside the homeodomain (15). In addition, anti-Mix1 is specific for Mix1 and it does not cross-react with the paralogous protein Mixer (15).…”

“…mix1 encodes a paired-like homeodomain transcription factor expressed ubiquitously in the endoderm and mesoderm (14). Its expression is detected in the mesendoderm shortly after the mid-blastula transition and expression peaks in the presumptive endoderm and mesoderm of the X. laevis gastrula (14,15). mix1 has not been sequenced in frogs other than Xenopus/Silurana.…”