Tbx20-related genes, mid and H15, are required for tinman expression, proper patterning, and normal differentiation of cardioblasts in Drosophila

Tbx20-related T-box genes have been implicated in the regulation of heart development in several vertebrate species. In the present report, we demonstrate that a pair of genes representing Drosophila orthologs of Tbx20, midline (mid) and H15, have important functions during the development of the Drosophila equivalent of the heart, i.e. the dorsal vessel. We show that mid is among the earliest known genes that are specifically expressed in all cardioblasts during early embryogenesis, and H15 expression is subsequently activated in the same cells. Mutant embryos lacking the activity of mid, or both mid and H15, are able to form dorsal vessels with largely normal numbers of cardioblasts and pericardial cells. Furthermore, the mutant cardioblasts express several general cardioblast markers such as Mef2 and Toll at normal levels. However, the expression of tinman (tin), which normally occurs in four out of six cardioblasts in each hemisegment of the dorsal vessel, is almost abolished. Conversely, the expression of the Dorsocross (Doc) T-box genes, which is normally restricted to the two Tin-negative cardioblasts in each hemisegment, is strongly expanded into the majority of cardioblasts in mid mutant and mid+H15-deficient embryos. Altogether, the data from the loss-of-function phenotypes demonstrate that mid, and to a lesser degree H15, have important roles in establishing the metameric patterning of cardioblast identities, but not in specifying cardioblasts as such. Ectopic expression of mid causes ectopic tin expression and, less efficiently, produces extra cardioblasts. We propose that one of the major functions of mid and H15 during cardioblast development is the re-activation of tin expression at a stage when the induction of tin by Dpp in the dorsal mesoderm has ceased. Through this activity, mid and H15 are required for the normal functional diversification of cardioblasts and the expression of tin-dependent terminal differentiation genes within the dorsal vessel.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

Developmental expression of the amphioxus <Emphasis Type=Italic>Tbx1</Emphasis>/<Emphasis Type=Italic>10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18^−/− kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRβ+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18^−/− kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.     

Mesonephric FGF signaling is associated with the development of sexually indifferent gonadal primordium in chick embryos

The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression.     

The epaxial-hypaxial subdivision of the avian somite

In all jaw-bearing vertebrates, three-dimensional mobility relies on segregated, separately innervated epaxial and hypaxial skeletal muscles. In amniotes, these muscles form from the morphologically continuous dermomyotome and myotome, whose epaxial-hypaxial subdivision and hence the formation of distinct epaxial-hypaxial muscles is not understood. Here we show that En1 expression labels a central subdomain of the avian dermomyotome, medially abutting the expression domain of the lead-lateral or hypaxial marker Sim1. En1 expression is maintained when cells from the En1-positive dermomyotome enter the myotome and dermatome, thereby superimposing the En1-Sim1 expression boundary onto the developing musculature and dermis. En1 cells originate from the dorsomedial edge of the somite. Their development is under positive control by notochord and floor plate (Shh), dorsal neural tube (Wnt1) and surface ectoderm (Wnt1-like signalling activity) but negatively regulated by the lateral plate mesoderm (BMP4). This dependence on epaxial signals and suppression by hypaxial signals places En1 into the epaxial somitic programme. Consequently, the En1-Sim1 expression boundary marks the epaxial-hypaxial dermomyotomal or myotomal boundary. In cell aggregation assays, En1- and Sim1-expressing cells sort out, suggesting that the En1-Sim1 expression boundary may represent a true compartment boundary, foreshadowing the epaxial-hypaxial segregation of muscle.