Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+E-cadherin(ECD)+PDGFRα(αR)+population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ECD+αR- and Gsc+ECD-αR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages,respectively. The presence of mesendodermal cells in nascent Gsc+ECD+αR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.

Previous studies on mice bearing various mutations within the c‐kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c‐kit gene and a respectable understanding of the molecular nature of c‐kit, however, it is not clear at what stage of gestation c‐kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti‐c‐kit antibody, ACK2, as an antagonistic blocker of c‐kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid‐gestation, c‐kit is functionally required during a restricted period around day 14.5 post‐coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c‐kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.

The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.Supplemental material is available at http://www.genesdev.org.