Real time monitoring of BMP Smads transcriptional activity during mouse development.

SUMMARY: Bone morphogenetic protein (BMP) signaling is a key pathway in the patterning and development of organisms as diverse as fruit fly and humans. However, the determination of net BMP signaling, paramount to understanding organogenesis, is limited to the analysis of fixed material. We generated a transgenic mouse that reports the transcriptional response of BMP Smad activation by coupling a well established BMP response element (BRE), isolated from the Id1 promoter, to green fluorescent protein (BRE:gfp). We monitored BMP Smad transcriptional activity in fresh and fixed BRE:gfp embryos. GFP expression was observed where expected on the basis of known signaling sites, but also in specific cell populations in which BMP signaling had been implicated but not directly demonstrated. Deletion of Smad5 reduced GFP in vivo as expected. The BRE:gfp transgenic mice are a novel tool, which will facilitate the identification of specific BMP Smad responsive cell types and allow BMP Smad signaling to be monitored in real time, supporting studies in development and disease.

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.

Isl1Cre reveals a common Bmp pathway in heart and limb development.

A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 (Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.

Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins.

Signaling by bone morphogenetic proteins is essential for a wide variety of developmental processes. Receptor-regulated Smad proteins, Smads 1 and 5, are intracellular mediators of bone morphogenetic protein signaling. Together with Smad4, these proteins translocate to the nucleus and modulate transcription by binding to specific sequences on the promoters of target genes. We sought to map transcriptional Smad1/5 activity in development by generating embryonic stem cell lines carrying a Smad1/5-specific response element derived from the Id1 promoter coupled to beta-galactosidase or luciferase as reporters. Three independent lines (BRE-lac1, BRE-lac2 and BRE-luc) have shown the existence of an autocrine bone morphogenetic protein signaling pathway in mouse embryonic stem cells. Reporter activity was detected in chimeric embryos, suggesting sensitivity to physiological concentrations of bone morphogenetic protein. Reporter activity in embryos from transgenic mouse lines was detected in tissues where an essential role for active bone morphogenetic protein signaling via Smads 1 or 5 had been previously established. We have thus generated, for the first time, an in vivo readout for studying the role of Smad1/5-mediated transcriptional activity in development.

BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo.

Deletion of various bone morphogenetic proteins (BMPs) and their downstream Smads in mice have clearly shown that BMP signaling is essential for the formation of primordial germ cells (PGCs). However, the molecular mechanism through which this takes place is still unclear. Here, we demonstrate that BMP4 produced in the extraembryonic ectoderm signals through ALK2, a type I BMP receptor, in the visceral endoderm (VE) to induce formation of PGCs from the epiblast. Firstly, embryonic day 5.5-6.0 (E5.5-E6.0) embryos cultured on fibronectin formed PGCs in the presence of VE, but not in its absence. Secondly, Alk2-deficient embryos completely lacked PGCs and the heterozygotes had reduced numbers, resembling Bmp4-deficient phenotypes. Thirdly, expression of constitutively active ALK2 in the VE, but not in the epiblast, was sufficient to rescue the PGC phenotype in Bmp4-deficient embryos. In addition, we show that the requirement for the VE at E5.5-E6.0 can be replaced by culturing embryos stripped of VE on STO cells, indicating that STO cells provide or transduce signals necessary for PGC formation that are normally transmitted by the VE. We propose a model in which direct signaling to proximal epiblast is supplemented by an obligatory indirect BMP-dependent signal via the VE.