Id Genes Are Direct Targets of Bone Morphogenetic Protein Induction in Embryonic Stem Cells

TLDR

Bone morphogenetic proteins (BMPs) are essential morphogenetic signaling molecules that drive embryonic patterning. The study sought to identify new genes directly activated by BMP signaling and proposes that Msx and Id genes are direct BMP4 targets in vivo. The authors used CDM‑cultured mouse embryonic stem cells stimulated with BMP2/4, activin A, and bFGF, and applied differential display cDNA cloning to identify BMP‑direct target genes. BMP2/4 stimulation of CDM‑cultured ES cells rapidly induced Msx‑1, Msx‑2, and JunB, and differential display identified six direct BMP targets—including Id3, Id1, Id2, Cyr61, DEK, eIF4AII, and a GC‑binding protein—while in vivo ectopic Id3 and Msx‑2 expression in Ft/+ embryos supported their direct BMP4 regulation.

Abstract

Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. <i>In vitro</i> cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes <i>Msx-1</i> and<i>Msx-2</i> and the proto-oncogene <i>JunB</i>, whereas c-<i>jun</i> transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include <i>Id3</i>, which showed strong mRNA induction, and the moderately induced<i>Cyr61</i>, <i>DEK</i>, and <i>eIF4AII</i> genes, as well as a gene encoding a GC-binding protein. Besides <i>Id3</i>, also the <i>Id1</i> and <i>Id2</i> genes were activated by BMP4 in both ES cells and a range of different cell lines.<i>Id</i> genes encode negative regulators of basic helix-loop-helix transcription factors. <i>In vivo</i> we observed local ectopic expression <i>of Id3</i> and <i>Msx-2</i>mRNAs in <i>Ft/+</i> embryos at overlapping regions of ectopic<i>Bmp4</i> misexpression. We therefore propose that the<i>Msx</i> and <i>Id</i> genes are direct target genes of embryonic BMP4 signaling <i>in vivo</i>.