Genome-wide survey of Nodal and Foxh1 targets in early gastrulae. Arrowhead indicates the P- Smad1 band the lower band in the P- Smad1 lanes is low level primary antibody release from the beads.įig. After detection, membranes were re-probed with anti- Smad2 or anti- Smad1 to show efficiency of the immunoprecipitations. Bound proteins were subjected to western immunoblotting using anti-P- Smad2 or anti-P- Smad1, respectively. (F) Early gastrula cleared lysates were immunoprecipitated using either pan anti- Smad2 or anti- Smad1 polyclonal antibodies covalently coupled to beads. The ectodermally enriched markers sox3 and foxh1.2 are included as non- Foxh1 targets for comparison. gsc, chrd, nodal1, nodal3 and mix1 are mesoendodermal markers sox17a is an endodermal marker ventx2.1 is a BMP target gene sia1 is a Wnt target gene. (E) Examination of foxh1 MO effects on different germ-layer markers by RT-qPCR. (D) Early tailbud stage Foxh1 morphants displaying anterior defects and incomplete blastopore closure SB431542-treated embryos lack distinctive A-P or D-V features. (C) Both Foxh1 morphant and SB431542-treated embryos exhibit gastrulation delay (vegetal views). Ctnnb1 protein levels in crude embryo lysates are unaffected by the MO. (B) Embryonic lysates from control or foxh1-MO injected embryos were subjected to immunoprecipitation followed by western blot using anti- Foxh1 antibody. tfap2a, ectodermal marker t/brachyury, mesodermal marker sox17a, endodermal marker rpl11, expressed throughout the embryo. Total RNAs from animal, marginal and vegetal fragments were subjected to RT-qPCR analyses. (A) Distribution of foxh1 transcripts in X. Foxh1 is crucial for mesendoderm formation. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.įig. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1- Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. Nodal/TGFβ signaling regulates diverse biological responses.
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