Specification of both neural crest cells and Rohon-Beard (RB) sensory neurons involves a complex series of interactions between the neural and non-neural ectoderm. The molecular mechanisms directing this process are not well understood. The zebrafish narrowminded (nrd) mutation is unique, since it is one of two mutations in which defects are observed in both cell populations: it leads to a complete absence of RB neurons and a reduction in neural crest cells and their derivatives. Here, we show that nrd is a mutation in prdm1, a SET/zinc-finger domain transcription factor. A Morpholino-mediated depletion of prdm1 phenocopies the nrd mutation, and conversely overexpression of prdm1 mRNA rescues the nrd RB sensory neuron and neural crest phenotype. prdm1 is expressed at the border of the neural plate within the domain where neural crest cells and RB sensory neurons form. Analysis of prdm1 function by overexpression indicates that prdm1 functions to promote the cell fate specification of both neural crest cells and RB sensory neurons, most likely as a downstream effector of the BMP signaling pathway.
The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocytemacrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These -results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (7) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors.
Physiologic role oferythropoietin in erythroid differentiation Erythropoietin (EPO)' is a 34-kD glycoprotein hormone which is the primary regulator of human erythropoiesis. A cDNA encoding EPO has been isolated (1), and recombinant EPO with oligosaccharide moieties identical to the natural material is available (2). EPO is synthesized and released by the kidney, and it circulates to the bone marrow where it stimulates resident erythroid progenitors via a specific receptor (3). EPO as a pharmacologic agent has clearly demonstrated efficacy in anemic states such as kidney failure (4, 5). In vivo, EPO causes a rapid rise in hematocrit in 7-10 d.Despite the availability of purified recombinant EPO, little is known regarding the interaction of EPO and the erythropoietin receptor (EPO-R) or the physiologic mechanisms by which EPO causes cells to undergo proliferation or differentiation. This is largely due to the lack of adequate quantities of EPO-R for in-depth biochemical study. Only small numbers of surface EPO-R (< 1,000) are present on normal erythroblasts and erythroleukemia cells. EPO provides a proliferative signal to the Burst-forming unit-erythroid (BFU-E), an early EPOresponsive erythroid progenitor, and a differentiative signal to the colony-forming unit-erythroid (CFU-E), a later EPO-responsive erythroid progenitor (3). These two classes of EPOresponsive cells are clearly different populations since they can be separated by unit gravity sedimentation and more recently have been purified to homogeneity (6). The purpose of this review is to describe the recent advances in our understanding of EPO-R the studies described used iodinated recombinant human EPO. Scatchard analysis has been employed to determine the number of binding sites per cell and the affinity constants for the interaction. As shown in Table I (references 6-37), -200 EPO-R are present on the cell surface of purified normal erythroid progenitors. On certain cell lines, that number can increase to about 1,000 per cell. This relatively low number of EPO-R is characteristic of other receptors for hematopoietic cytokines such as G-CSF, GM-CSF, IL-3, and IL-6 (reviewed in references 38-40). Scatchard analysis revealed that certain erythroid cells, such as MEL cells, express only low-affinity receptors whereas other cell lines express high-and low-affinity receptors (Table I). Of note, the lower-affinity receptor has an affinity constant in the 200 pM range and therefore still represents an intimate interaction between receptor and ligand. Although functional differences between the higher-and loweraffinity EPO-R have not been determined, the two affinities may account for different cellular responses to EPO. Friend virus-infected cells respond to EPO with proliferation and differentiation. MEL cells bind EPO only with low affinity and do not appear to respond to the hormone. Affinity cross-linking experiments using radiolabeled EPO (Table I) (Fig. 1). As expected, the EPO-R transcript showed erythroid specific expression (41). Surprisingly, the ...
Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins.
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