Developmental expression patterns of Tbx1, Tbx2, Tbx5, and Tbx20 in Xenopus tropicalis

Abstract

T-box genes have diverse functions during embryogenesis and are implicated in several human congenital disorders. Here, we report the identification, sequence analysis, and developmental expression patterns of four members of the T-box gene family in the diploid frog Xenopus tropicalis. These four genes-Tbx1, Tbx2, Tbx5, and Tbx20-have been shown to influence cardiac development in a variety of organisms, in addition to their individual roles in regulating other aspects of embryonic development. Our results highlight the high degree of evolutionary conservation between orthologs of these genes in X. tropicalis and other vertebrates, both at the molecular level and in their developmental expression patterns, and also identify novel features of their expression. Thus, X. tropicalis represents a potentially valuable vertebrate model in which to further investigate the functions of these genes through genetic approaches.

Fig. 1

Genomic locus structure of Tbx1, Tbx2, Tbx5, and Tbx20 in Xenopus tropicalis. Tbx1 (a), Tbx2 (b), Tbx5 (c), and Tbx20 (d) cDNAs and their corresponding genomic loci are shown in diagrammatic form (not to scale). Coding regions of each cDNA are shown (boxes) together with their nucleotide positions and the position of the T-box (defined by alignment of the encoded proteins with the T-domain of Xbra) is also indicated. The exons corresponding to the cDNA sequences are shown together with their sizes (in base pairs) plus those of the intervening introns. Note that, as the size of the first exon of each gene is predicted based on the available cDNA sequence, the sizes of these exons may be underestimated here.

Fig. 2

Expression pattern of Tbx1 in Xenopus tropicalis. The results of in situ hybridizations for Tbx1 expression from early neurula to late tail bud stages are shown (embryos uncleared). Except for the anterior views shown in a and d, all embryos are oriented with anterior to the left. a– c: Stage 13 is shown in anterior (a), lateral (b), and dorsal (c) views. Bilateral patches of stronger expression are indicated in a) by arrowheads. d–f: Stage 19 is shown in anterior (d), lateral (e), and dorsal (f) views. Bilateral stripes of stronger expression are indicated in f) by an asterisk. g–n: Tbx1 expression through tail bud stages is shown as follows: stage 25 lateral (g) and ventral (h), stage 26 lateral (i), stage 27 lateral (j) and ventral (k), stage 28 lateral (l) and ventral (m), stage 33 lateral (n). ba1, first branchial arch; ba2, second branchial arch; cg, cement gland; ea, eye anlagen; ha, hyoid arch; ma, mandibular arch; ov, otic vesicle.

Fig. 3

Expression pattern of Tbx2 in X. tropicalis. In situ hybridization results are shown for Tbx2 (embryos uncleared). a– c: Expression at early gastrula (stage 10.5) is shown in lateral (a) and vegetal (b) views of whole-mount embryos and in transverse section (c; ventral to the right). In both a and b, the embryo is oriented with dorsal to the left and the dorsal blastopore lip is indicated by an arrowhead in a. d: A vegetal view of a late gastrula (st12) is shown, ventral side uppermost. e–r: Expression at early neurula (stage 13; e– g) late neurula (stage 19; h,l), and tail bud stages 21/22 (m), 25 (n), 26 (o,p), 29 (q), and 33 (r) are also shown. Expression in the forebrain (telencephalon) at tail bud stages is indicated by an asterisk (o–r). Except for anterior (j) and posterior (d,l) views, all embryos are oriented with anterior to the left. cg, cement gland; crg, cranial ganglia; drg, dorsal root ganglia; fn, frontonasal process; hm, hypaxial muscle; ht, heart tube; lp, lens placode; ov, otic vesicle; pa, placodal area; pd, pronephric duct; pr, proctodeum.

Fig. 4

Expression pattern of Tbx5 in Xenopus tropicalis. The expression pattern of Tbx5 detected by in situ hybridization between late neurula and early tadpole stages is shown (uncleared except for g). a–j; Stages are as follows: stage 19 (a,b), stage 25 (c,d), stage 26 (e,f), stage 31/32 (g; cleared), stage 33 (h), and stage 40 (i,j). Except for the anterior view in b and the ventral view in j, embryos are oriented with anterior to the left. Anterior is to the top in j. ea, eye anlagen, hp, heart primordium, v, ventricle.

Fig. 5

Expression pattern of Tbx20 in Xenopus tropicalis.The expression pattern of Tbx20 detected by in situ hybridization between late neurula and early tadpole stages is shown (embryos uncleared). a– o: Stages are as follows: stage 20 (a– c), stage 25 (d,e), stage 29/30 (f), stage 33 (g–i), stage 36 (j–l), and stage 40 (m– o). Ventral views are shown in i, l, and o. Except for the anterior view in a, embryos are oriented with anterior to the left. a, atrium; cg, cement gland; hb, hindbrain; hp, heart primordium; ht, heart tube; sv, sinus venosus; v, ventricle.

Fig. 6

Cardiac expression of Tbx2, Tbx5, and Tbx20. The in situ hybridization patterns of Tbx2, Tbx5, and Tbx20 in the forming cardiac chambers were examined in transverse sections through the tadpole heart after looping. a: Tbx2 expression was seen in the myocardium of both the atrial (am) and ventricular (vm) regions of the looped heart at stage 36. b: Expression of Tbx5 was restricted primarily to the developing ventricular myocardium at stage 38. c: In contrast to Tbx5, high levels of Tbx20 expression were seen in the atrial region but not in the ventricle (stage 40). Original magnification, ×100.