Postimplantation Mga expression and embryonic lethality of two gene-trap alleles.

BACKGROUND: The dual-specificity T-box/basic helix-loop-helix leucine zipper transcription factor MGA is part of the MAX-interacting network of proteins. In the mouse, MGA is necessary for the survival of the pluripotent epiblast cells of the peri-implantation embryo and a null, gene-trap allele MgaGt results in embryonic lethality shortly after implantation. We have used this allele to document expression of Mga in postimplantation embryos and also investigated a second, hypomorphic gene-trap allele, MgaInv. RESULTS: Compound heterozygotes, MgaGt/MgaInv, die prior to midgestation. The extraembryonic portion of the embryos appears to develop relatively normally while the embryonic portion, including the pluripotent cells of the epiblast, is severely retarded by E7.5. Mga expression is initially limited to the pluripotent inner cell mass of the blastocyst and epiblast, but during organogenesis it is widely expressed, notably in the central nervous system and sensory organs, reproductive and excretory systems, heart, somites and limbs. CONCLUSIONS: Widespread yet specific areas of expression of Mga during organogenesis raise the possibility that the transcription factor may play roles in controlling proliferation and potency in the progenitor cell populations of different organ systems. Documentation of these patterns sets the stage for the investigation of specific progenitor cell types.

Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos.

Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos.

Mga is essential for the survival of pluripotent cells during peri-implantation development.

The maintenance and control of pluripotency is of great interest in stem cell biology. The dual specificity T-box/basic-helix-loop-helix-zipper transcription factor Mga is expressed in the pluripotent cells of the inner cell mass (ICM) and epiblast of the peri-implantation mouse embryo, but its function has not been investigated previously. Here, we use a loss-of-function allele and RNA knockdown to demonstrate that Mga depletion leads to the death of proliferating pluripotent ICM cells in vivo and in vitro, and the death of embryonic stem cells (ESCs) in vitro. Additionally, quiescent pluripotent cells lacking Mga are lost during embryonic diapause. Expression of Odc1, the rate-limiting enzyme in the conversion of ornithine into putrescine in the synthesis of polyamines, is reduced in Mga mutant cells, and the survival of mutant ICM cells as well as ESCs is rescued in culture by the addition of exogenous putrescine. These results suggest a mechanism whereby Mga influences pluripotent cell survival through regulation of the polyamine pool in pluripotent cells of the embryo, whether they are in a proliferative or quiescent state.

Diverse functional networks of Tbx3 in development and disease.

The T-box transcription factor Tbx3 plays multiple roles in normal development and disease. In order to function in different tissues and on different target genes, Tbx3 binds transcription factors or other cofactors specific to temporal or spatial locations. Examining the development of the mammary gland, limbs, and heart as well as the biology of stem cells and cancer provides insights into the diverse and common functions that Tbx3 can perform. By either repressing or activating transcription of target genes in a context-dependent manner, Tbx3 is able to modulate differentiation of immature progenitor cells, control the rate of cell proliferation, and mediate cellular signaling pathways. Because the direct regulators of these cellular processes are highly context-dependent, it is essential that Tbx3 has the flexibility to regulate transcription of a large group of targets, but only become a active on a small cohort of them at any given time or place. Moreover, Tbx3 must be responsive to the variety of different upstream factors that are present in different tissues. Only by understanding the network of genes, proteins, and molecules with which Tbx3 interacts can we hope to understand the role that Tbx3 plays in normal development and how its aberrant expression can lead to disease. Because of its myriad functions in disparate developmental and disease contexts, Tbx3 is an ideal candidate for a systems-based approach to genetic function and interaction.

Expression of Slit and Robo genes in the developing mouse heart.

Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.