Developmental biology of zebrafish myeloid cells

The zebrafish (Danio rerio) has emerged as an informative vertebrate model for developmental studies, particularly those employing genetic approaches such as mutagenesis and screening. Zebrafish myelopoiesis has recently been characterized, paving the way for the experimental strengths of this model organism to contribute to an improved understanding of the genetic regulation of myeloid development. Zebrafish have a multi-lineage myeloid compartment with two types of granulocyte (heterophil/neutrophil and eosinophil granulocytes), and monocyte/macrophages, each with characteristic morphological features and histochemical staining properties. Molecular markers have been characterised for various myeloid cell types and their precursor cells, for example: stem cells (scl, hhex, lmo2), myeloid lineage precursors (spi1/pu.1, c/ebp1), heterophil granulocytes (mpx/mpo), macrophages (L-plastin, fms). In zebrafish, the sites of early myeloid and erythroid commitment are anatomically separated, being located in the rostral and caudal lateral plate mesoderm respectively. Functional macrophages appear before cells displaying granulocytic markers. By the second day of life, cells expressing granulocyte- and macrophage-specific genes are scattered throughout the embryo, but tend to aggregate in the ventral venous plexus, which may be a site of their production or a preferred site for their residence. Even in early embryos, macrophages are phagocytically active, and granulocytes participate in acute inflammation. Equipped with an understanding of the developmental biology of these various myeloid cells and a set of tools for their identification and functional study, we will now be able to exploit the experimental strengths of this model organism to better understand the genetic regulation of myelopoiesis.     

Flounder (Paralichthys olivaceus) FoxD1 and its regulation on the expression of myogenic regulatory factor, MyoD

It has been reported that FoxD1 plays important roles in formation of several different tissues, such as retina and kidney in vertebrates. The function of FoxD1 in muscle development is, however, unclear although it is expressed in muscle cells in zebrafish. Muscles are the major tissue in fish, which serves as a rich protein source in our diet. To further understand the function of FoxD1 in fish muscle development, here we isolated and characterized the FoxD1 gene from flounder (Paralichthys olivaceus), a valuable sea food and an important fish species in aquaculture in Asia. We analyzed its expression pattern and function in regulating myogenic regulatory factor, MyoD, one of the earliest marker of myogenic commitment. In situ hybridization revealed that FoxD1 was expressed in the tailbud, adaxial cells, posterior intestine, forebrain, midbrain and half of the retina in flounder embryos. Functional studies demonstrated that when flounder FoxD1 was over-expressed in zebrafish by microinjection, MyoD expression was decreased, suggesting that FoxD1 may be involved in myogenesis by regulating the expression of MyoD.     

Haploinsufficiency of TAB2 Causes Congenital Heart Defects in Humans

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-β-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development.     

Jmjd3 activates Mash1 gene in RA‐induced neuronal differentiation of P19 cells

Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri‐methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix‐loop‐helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA‐treated P19 cells. Over‐expression of Jmjd3 in P19 cells also significantly enhances the RA‐induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA‐induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co‐Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA‐induced neuronal differentiation of P19 cells. J. Cell. Biochem. 110: 1457–1463, 2010. © 2010 Wiley‐Liss, Inc.     

Differential Requirement for irf8 in Formation of Embryonic and Adult Macrophages in Zebrafish

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. In particular, genetic tools to analyze the role of Irf8 in zebrafish macrophage development at larval and adult stages are lacking. We generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.