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Vertebrate homologues of the Drosophila tinman transcription factor have been implicated in the processes of specification and differentiation of cardiac mesoderm. In Xenopus three members of this family have been isolated to date. Here we show that the XNkx2-3, Xnkx2-5, and XNkx2-10 genes are expressed in increasingly distinctive patterns in endodermal and mesodermal germ layers through early development, suggesting that their protein products (either individually or in different combinations) perform distinct functions. Using amphibian transgenesis, we find that the expression pattern of one of these genes, XNkx2-5, can be reproduced using transgenes containing only 4.3 kb of promoter sequence. Sequence analysis reveals remarkable conservation between the distalmost 300 bp of the Xenopus promoter and a portion of the AR2 element upstream of the mouse and human Nkx2-5 genes. Interestingly, only the 3' half of this evolutionarily conserved sequence element is required for correct transgene expression in frog embryos. Mutation of conserved GATA sites or a motif resembling the dpp-response element in the Drosophila tinman tinD enhancer dramatically reduces the levels of transgene expression. Finally we show that, despite its activity in Xenopus embryos, in transgenic mice the Xenopus Nkx2-5 promoter is able to drive reporter gene expression only in a limited subset of cells expressing the endogenous gene. This intriguing result suggests that despite evolutionary conservation of some cis-regulatory sequences, the regulatory controls on Nkx2-5 expression have diverged between mammals and amphibians.
FIG. 1. Comparison of expression of Xenopus tinman homologues. Tail bud embryos were stained in whole mount for the expression of
the three Xenopus tinman homologues (stage 22, A; stage 24, D; stage 27/29, G) along with tadpoles at the linear heart tube stage
(stage 30/32, J) and at the onset of chamber formation (stage 38/40, L, M). XNkx2-5 is shown in A, D, G, J, and M; XNkx2-3 in B, E, H,
K and N; and XNkx2-10 in C, F, I, and L. In all cases lateral views are shown with anterior to the left. In (D) a black arrow indicates where
the XNkx2-5 expression in the endoderm begins to be partially occluded by neural crest.
FIG. 2. Comparison of expression of Xenopus tinman homologues. Ventral views of the same embryos depicted in Fig. 1. In all cases
anterior is to the left.
FIG. 3. The Xenopus tinman homologues are expressed in distinctive patterns in the different germ layers. Embryos were stained by whole-mount in situ hybridisation for XNkx2-5 (A), XNkx2-3
G, H), and XNkx2-10 (I) expression. Transverse sections are shown in A and I and a parasaggital section in H. Expression is shown
in dark blue and nuclei are counter-stained in red with Fuelgen.
Embryos were at early tail bud (stage 26, A), heart trough (stage 27/28, B, G), linear heart tube (stage 32, C, H, I), or chamber formation stages (stage 40, D). TO, outflow tract; V, ventricle; A,
atrium; SV, sinus venosus; PE, pharyngeal endoderm; M, myocardium; DM, dorsal mesocardium; CG, cement gland; Me, mesoderm.
FIG. 5. Expression of XNkx2-5/GFP transgene in Xenopus embryos. Normal and transgenic embryos were stained by wholemount in situ hybridisation for XNkx2-5 (A, E, G) or GFP (B, D, H) expression, respectively. Embryos were at early tail bud (stage 22, A, B), late tail bud (stage 27/28, E, F), and heart chamber formation
stages (stage 40, G, H). Transverse sections of embryos shown in A and B are shown in C and D, respectively, with expression shown
in dark blue and nuclei counterstained in red with Fuelgen. Black arrows indicate expression in the branchial arches. PE, pharyngeal endoderm; M, mesoderm; H, heart.
FIG. 6. The XNkx2-5 proximal promoter is not required for transgene expression. Normal and transgenic embryos at lateneurula or early tail bud stages were stained by whole-mount in situ hybridisation for XNkx2-5 (A, B) or GFP (C) expression,
respectively. Each pair of images shows lateral and ventral views of the same embryo. (A, B) Endogenous XNkx2-5 expression. (C, D) DNk2 transgene. (E, F) D659tK transgene. In all cases anterior is to
the left.
