The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish

Abstract

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl ؊/؊ embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNAbinding forms, suggesting tethering to target genes through DNA-binding part- IntroductionLmo2 was first discovered through its homology to the T-cell oncogene lmo1 and has been found to be activated by chromosomal translocations in several T-cell leukemias. 1 Like its partner in erythroid cells, scl, during normal development lmo2 is specifically expressed in hematopoietic progenitors, erythroid cells, and megakaryocytes, and in endothelial cells. 2,3 Targeted gene ablation in mice revealed an essential role for Lmo2 in primitive hematopoiesis, with lmo2 Ϫ/Ϫ embryos dying at embryonic day (E) 9 to 10 due to severe anemia. 2 Lmo2 Ϫ/Ϫ embryonic stem (ES) cells were unable to contribute to any hematopoietic lineage in chimeras, indicating a critical requirement for Lmo2 in definitive hematopoiesis. 4 In addition, although lmo2 Ϫ/Ϫ ES cells were initially able to contribute to normal primary capillary plexus formation, they were unable to contribute to the formation of larger vessels, and chimeras with a large contribution of lmo2 Ϫ/Ϫ ES cells exhibited disrupted vascular organization from E9 onwards, indicating a role for Lmo2 in angiogenesis. 3 The Lmo2 protein comprises 2 LIM zinc finger-like protein interaction domains and has been found to act as a bridging molecule between the Scl/E47 heterodimer and Gata1/2 in the formation of DNA-binding complexes. 5-10 A specific phenylalanine residue in helix 2 of the Scl basic helix-loop-helix (bHLH) domain has been shown to be essential in vitro for hematopoietic activity of Scl. However, rather than being important for heterodimerization with E47, this residue promotes protein interactions with Lmo2. 11 Remarkably, introduction of a phenylalanine residue at the corresponding position in the myogenic bHLH, MyoD, was sufficient to confer hematopoietic activity, rescuing hematopoietic development in scl Ϫ/Ϫ ES cells. The interaction between Scl and Lmo2 is therefore essential for initiation of hematopoiesis in ES cells. However, the in vivo requirement for this interact...

Molecular and developmental biology of the hemangioblast

“…LMO2, an SCLbinding partner, is required for erythroid development and is dispensable for the formation of mesoderm and endothelial cells using gene-targeting in mouse embryos and EBs (Warren et al, 1994). These observations from loss-of-function analyses in mouse embryos are supported by those observed in scl and lmo2 morphant embryos using antisense morpholino knockdown in zebrafish Patterson et al, 2005Patterson et al, , 2007Qian et al, 2007). Mis-expression of either scl, or scl and lmo2 Fig.…”

“…This represents the first single gene mutation that eliminates both endothelial and hematopoietic lineages, probably in the level of hemangioblasts. Epistasis analyses have shown that cloche acts upstream of zbp-89, scl, lmo2, gata1, gata2, runx1, c-myb, fli1, flk1, tie2, hhex, and etsrp in hematopoietic and endothelial cell development in zebrafish (Stainier et al, 1995;Fouquet et al, 1997;Liao et al, 1997Liao et al, , 1998Liao et al, , 2000Brown et al, 2000;Burns et al, 2002;Patterson et al, 2005Patterson et al, , 2007Li et al, 2006;Pham et al, 2006;Sumanas and Lin, 2006). In addition, microarray analyses by comparing cloche mutant and wild-type embryos have revealed that cloche specifically regulates a panel of hematopoietic and endothelial genes (Qian et al, 2005;Sumanas et al, 2005;Weber et al, 2005).…”

“…Zebrafish cloche mutant affects both endothelial and hematopoietic lineages (Stainier et al, 1995). Gain-of-function analysis in zebrafish embryos suggests that scl, lmo2, hhex, and etsrp can specify the hemangioblast from the posterior lateral plate mesoderm (LPM) at the expense of other mesodermal cell fates (Gering et al, 1998;Liao et al, 2000;Gering et al, 2003;Dooley et al, 2005;Pham et al, 2006;Sumanas and Lin, 2006;Patterson et al, 2007). However, it remains to be revealed how these known and other unknown players interact in hemangioblast development and differentiation.…”

Functional characterization of lmo2‐Cre transgenic zebrafish

“…Overexpression of lmo2 and scl, another key regulator in hemangioblasts, can convert non-axial mesoderm into hemangioblasts (Gering et al, 2003). Consistently, knockdown of lmo2 results in the reduced or lack of expression of genes normally expressed in hematopoietic and vascular endothelial cells (Patterson et al, 2007). Recently, a 2.5-kb genomic regulatory sequence upstream of the translational start site of lmo2 gene was cloned and shown to be able to drive the enhanced green fluorescent protein (EGFP) reporter gene expression specifically in the developing hematopoietic progenitor cells and vascular endothelial cells, which mirrors the endogenous lmo2 expression pattern (Zhu et al, 2005).…”

Regulation of Stem Cells in the Zebra Fish Hematopoietic System

“…scl and lmo2 are expressed in hematopoietic progenitors and endothelial cells, possibly acting together to specify the hemangioblast (Patterson et al 2007). scl encodes a basic helix-loop-helix transcription factor, whereas lmo2 is a LIM-domain transcription factor.…”