The beta 3 tubulin gene is a direct target of bagpipe and biniou in the visceral mesoderm of Drosophila

Previous studies have identified the NK homeobox gene bagpipe and the FoxF fork head domain gene biniou as essential regulators of visceral mesoderm development in Drosophila. Here we present additional genetic and molecular information on the functions of these two genes during visceral mesoderm morphogenesis and differentiation. We show that both genes are required for the activation of beta 3Tub60D in the visceral mesoderm, which encodes beta 3 tubulin. We demonstrate that a 254 bp derivative of a previously defined visceral mesoderm-specific enhancer element, vm1, from beta 3Tub60D contains one specific in vitro binding site for Bagpipe and two such sites for Biniou. While the wild-type version of the 254 bp enhancer is able to drive significant levels of reporter gene expression within the entire trunk visceral mesoderm, mutation of either the Bagpipe or the Biniou binding sites within this element results in a severe decrease of enhancer activity. Moreover, mutation of all three binding sites for Bagpipe and Biniou, respectively, results in the complete loss of enhancer activity. Together, these observations suggest that Bagpipe and Biniou serve as direct, partially redundant, and tissue-specific activators of the terminal differentiation gene beta 3Tub60D in the visceral mesoderm.     

Serum response factor, an enriched cardiac mesoderm obligatory factor, is a downstream gene target for Tbx genes

We tested the idea that T-box factors direct serum response factor (SRF) gene activity early in development. Analysis of SRF-LacZ "knock-in" mice showed highly restricted expression in early embryonic cardiac and skeletal muscle mesoderm and neuroectoderm. Examination of the SRF gene for regulatory regions by linking the promoter and 5'-flanking sequences, up to 5.5 kb, failed to target LacZ transgene activity to the heart and the tail pre-somitic mesenchyme. However, linkage of a minimal SRF promoter with the SRF 3'-untranslated region (UTR), inundated with multimeric T-box binding sites (TBEs), restored robust reporter gene activity to embryonic heart and tail. Finer dissection of the 3'-UTR to a small cluster of TBEs also stimulated transgene activity in the cardiac forming region and the tail, however, when the TBEs contained within these DNA sequences were mutated, preventing Tbx binding, transgene activity was lost. Tbx2, Tbx5, and the cardiac-enriched MYST family histone acetyltransferase TIP60, were observed to be mutual interactive cofactors through the TIP60 zinc finger and the T-box of the Tbx factors. In SRF-null ES cells, TIP60, Tbx2, and Tbx5 were sufficient to stimulate co-transfected SRF reporter activity, however this activity required the presence of the SRF 3'-UTR. SRF gene transactivation was blocked by two distinct TIP60 mutants, in which either the histone acetyltransferase domain was inactivated or the Zn finger-protein binding domain was excised. Our study supports the idea that SRF embryonic cardiac gene expression is dependent upon the SRF 3'-UTR enhancer, Tbx2, Tbx5, and TIP60 histone acetyltransferase activity.     

Retinoic acid signaling sequentially controls visceral and heart laterality in zebrafish

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality.     

Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm.

To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression.     

Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors.

Individual somatic muscles and heart progenitors are specified at defined positions within the mesodermal layer of Drosophila. The expression of the homeobox gene even-skipped (eve) identifies one specific subset of cells in the dorsal mesoderm, which give rise to particular pericardial cells and dorsal body wall muscles. Genetic analysis has shown that the induction of eve in these cells involves the combined activities of genes encoding mesoderm-intrinsic factors, such as Tinman (Tin), and spatially restricted signaling activities that are largely derived from the ectoderm, particularly those encoded by wingless (wg) and decapentaplegic (dpp). Here we show that a Dpp-activated Smad protein, phosphorylated Mad, is colocalized in eve-expressing cells during an extended developmental period. We demonstrate further that a mesodermally active enhancer of eve contains several Smad and Tin binding sites that are essential for enhancer activity in vivo. This enhancer also contains a number of binding sites for the Wg-effector Pangolin (Pan/Lef-1), which are required for full levels of enhancer activity. However, we find that their main function is to prevent ectopic enhancer activity in the dorsal mesoderm. This suggests that, in the absence of Wg signaling, Pan binding serves to abrogate the synergistic activities of Smads and Tin in eve activation while, in cells that receive Wg signals, Pan is converted into a coactivator that promotes eve induction. Together, these data show that the eve enhancer integrates several regulatory pathways via the combinatorial binding of the mesoderm-intrinsic regulator Tin and the effectors of the Dpp and Wg signals.     

Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.

In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.