Paul E. Mead

Bipotential Primitive-Definitive Hematopoietic Progenitors in the Vertebrate Embryo

Two regions of the vertebrate embryo, the blood islands and the dorsal lateral plate (DLP), participate in early hematopoietic development. In Xenopus, primitive erythrocytes are derived solely from the ventral blood islands (VBI), while definitive hematopoietic cells such as lymphocytes are derived from both VBI and DLP. We have utilized a transplantation technique to demonstrate in vivo that all hematopoietic cells (embryonic, fetal, or adult) originate from ventral mesoderm. Reciprocal grafts between VBI and DLP demonstrated that both regions are bipotential with respect to primitive and definitive hematopoiesis. Commitment of the VBI to primitive erythropoiesis and restriction of the DLP to definitive hematopoiesis occurs during neurula stages. Thus, hematopoietic development involves the induction of the blood program on the ventral axis of the embryo followed by environmentally regulated specification to the primitive or definitive lineages.

Small pool expression screening: Identification of genes involved in cell cycle control, apoptosis, and early development

Publisher Summary This chapter discusses the identification of genes involved in cell cycle control, apoptosis, and early development. Traditional genetic and biochemical methods have been quite successful in identifying genes that are essential for cell cycle progression and early embryonic development, among other diverse biological processes. Nevertheless, only a small fraction of the genes in the vertebrate genome has been functionally characterized. This chapter describes a systematic and broadly applicable approach to cloning genes based solely on the biological activities or biochemical properties of the gene products. It describes several potential applications of this expression cloning approach, and also discusses its use in related types of screening procedures. It describes general methods used to prepare library pools of cDNA, RNA, and protein, and the sib selection techniques used to subdivide a pool once it is found to contain a candidate activity.

BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation.

Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. We demonstrate that a member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells. Moreover, XGrhl1 is regulated by the BMP4 signaling cascade. Disruption of XGrhl1 activity in vivo results in a severe defect in terminal epidermal differentiation, with inhibition of XK81A1 epidermal keratin gene expression, a key target of BMP4 signaling. Furthermore, transcription of the XK81A1 gene is modulated directly by binding of XGRHL1 to a promoter-localized binding motif that is essential for high-level expression. These results establish a novel developmental role for XGrhl1 as a crucial tissue-specific regulator of vertebrate epidermal differentiation.

Neptune, a Kruppel-like transcription factor that participates in primitive erythropoiesis in xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta-globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptunes DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Molecular insights into early hematopoiesis

&NA; During embryogenesis, a pool of pluripotential, self‐renewing hematopoietic stem cells is specified from undifferentiated ventral mesoderm. These cells proliferate and differentiate to yield all the peripheral blood lineages. Although the mechanisms involved in the commitment and differentiation of multipotential progenitors have been well documented, little focus has been directed to the molecular events underlying the formation of hematopoietic stem cells. In this review we discuss the recent advances made in defining a bone morphogenetic protein‐responsive signal cascade that regulates the earliest phase of blood formation during embryogenesis.

Embryonic Expression and Function of the Xenopus Ink4d Cyclin D-Dependent Kinase Inhibitor.

Here we report the cloning and functional characterization of the cyclin D-dependent kinase 4 and 6 (Cdk4/6) inhibitory protein Cdkn2d/p19Ink4d of Xenopuslaevis (Xl-Ink4d). Xl-Ink4d is the only Ink4 family gene highly expressed during Xenopus development and its transcripts were detected maternally and during neurulation. The Xl-Ink4d protein has 63% identity to mouse and human Cdkn2d/p19Ink4d and its function as a negative regulator of cell cycle traverse is evolutionary conserved. Indeed, Xl-lnk4d can functionally substitute for mouse Cdkn2d in binding to mouse Cdk4 and inhibiting cyclin-D1-dependent CDK4 kinase activity. Further, enforced expression of Xl-lnk4d arrests mouse fibroblasts in the G1 phase of the cell cycle. These findings indicate that CDKN2d/p19Ink4d is conserved through vertebrate evolution and suggest Xl-lnk4d may contribute to the development of Xenopuslaevis.