Evans SM et al. (1995), tinman, a Drosophila homeobox gene required for...

XB-ART-19065

Development 1995 Nov 01;12111:3889-99. doi: 10.1242/dev.121.11.3889.

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tinman, a Drosophila homeobox gene required for heart and visceral mesoderm specification, may be represented by a family of genes in vertebrates: XNkx-2.3, a second vertebrate homologue of tinman.

Evans SM , Yan W , Murillo MP , Ponce J , Papalopulu N .

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tinman is a Drosophila Nk-homeobox gene required for heart and visceral mesoderm specification. Mutations in tinman result in lack of formation of the Drosophila heart, the dorsal vessel. We have isolated an Nk-homeobox gene from Xenopus laevis, XNkx-2.3, which appears by sequence homology and expression pattern to be a homologue of tinman. The expression pattern of XNKx-2.3 both during development and in adult tissues partially overlaps with that of another tinman homologue, Csx/NKx-2.5/XNkx-2.5. We have found that embryonic expression of both XNkx-2.3 and XNkx-2.5 is induced at a time when cardiac specification is occurring. XNkx-2.3 is expressed in early cardiac primordia before the expression of a marker of cardiac differentiation. XMLC2, as well as in pharyngeal endoderm. In adult tissues, XNkx-2.3 is expressed in the heart and several visceral organs. As the helix-loop-helix factor Twist is thought to regulate tinman expression in Drosophila, we have compared the expression of XNkx-2.3 and Xtwist during embryonic development in Xenopus. There appears to be no overlap in expression patterns of the two RNAs from the neurulae stages onward, the first time at which the RNAs can be visualized by in situ hybridization. The overlapping expression patterns of XNkx-2.3 and mNkx-2.5/XNkx-2.5 in conjunction with evidence presented here that other Nk-homeodomains are expressed in adult mouse and Xenopus heart suggests that tinman may be represented by a family of genes in vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: mt-tr myl2 nkx2-3 nkx2-5 nkx3-1 tac1 tbx2 trna twist1

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	Fig. 1. A Comparison of NK-homeodomains to the homeodomain of XNkx-2.3. The top line shows the consensus sequences for homeodomains (Guazzi et al., 1990). To obtain PCR clones from Xenopus and mouse adult heart, degenerate oligonucleotide primers were designed to the underlined amino acid regions (for details, see Materials and Methods). The PCR clones obtained in this manner corresponded to each homeodomain shown here, with the exception of Bagpipe and Tinman, which are shown for purposes of comparison only. XPCR-3 and mPCR-13 are two clones that were obtained and which appear to have novel NK-homeodomains. References for all the listed homeodomains are in the text. The amino acid sequence is shown as single-letter code, and dashes indicate identity with XNkx-2.3.
	Fig. 2. DNA sequence comparison of the three XNkx-2.3 cDNA clones. (A) DNA sequences of XNkx-2.3b1, XNkx-2.3b2, and XNkx-2.3a were aligned with the Gap Program, University of Wisconson. The ATG start and TAG stop codons are indicated in capital letters and underlined to demarcate the coding sequences. Dashed lines indicate nucleotide identity with XNkx-2.3b1, or, in the 3¢ untranslated regions where XNkx- 2.3b1 is truncated, identity to XNkx- 2.3b2. Dots represent gaps. GenBank accession numbers for XNkx-2.3a, XNkx- 2.3b1 and XNkx- 2.3b2 are L38674, L38675 and L38676, respectively. (B) Percent identities for pairwise comparisons between the three cDNAs are shown for the 5¢ untranslated (5¢ UT), coding, and 3¢ untranslated (3¢UT) regions.
	Fig. 3. Predicted amino acid sequences of XNkx-2.3a, XNkx-2.3b1 and XNkx-2.3b2, aligned to that of XNkx-2.5. The homeobox domain is boxed. A decapeptide conserved in mNkx-2.5/XNkx-2.5, mNkx-2.1 and Tinman (Lints et al., 1993; Tonissen et al., 1994) is found just downstream of the predicted amino terminus and is underlined. The NK2 domain downstream of the homeodomain (Price et al., 1992) is doubly underlined. Amino acid numbering is indicated on the right.
	Fig. 4. Northern blot analysis of XNkx-2.3 expression in adult Xenopus tissues. 2 mg of poly(A)+ RNA were fractionated on formaldehyde agarose gels and probed for XNkx-2.3 mRNA (see Materials and Methods). In tissues positive for XNkx-2.3 mRNA expression, a major RNA species of 2.4 kb was observed. Secondary bands of approximately 3.0 kb were also observed in all positive tissues. A smaller RNA band of approximately 2.0 kb was also observed in the intestine. The autoradiogram shown was exposed for 4 days at -70°C. The origin of the A+ RNAs: H, heart; I, intestine; K, kidney; Li, liver; Lu, lung; Sk, skeletal muscle; Sp, spleen; St, stomach; T, tongue.
	Fig. 5. RNAse protection analyses of XNkx-2.3a and XNkx-2.3b expression in adult Xenopus tissues: (A,B) Radiolabelled riboprobes specific for each XNkx-2.3 allele were hybridized to 20 mg of total RNA from various adult Xenopus tissues. XNkx-2.3 sequences contained in each probe are diagrammed above each autoradiogram (for details, see Materials and Methods). The probe for XNkx-2.3b2 also recognizes XNkx-2.3b1. (C) Analytical formaldehyde agarose gel of RNA samples. RNA samples were monitored for integrity and quantity on analytical formaldehyde agarose gels before using. 10 mg of each RNA were loaded per lane. P, radiolabelled probe alone. These probes include the XNkx-2.3 sequences as diagrammed, plus vector sequences, resulting in a radiolabelled probe of greater length than the fully protected XNkx-2.3 sequences; t, tRNA control lane; H, heart RNA; I, intestine RNA; K, kidney RNA; Li, liver RNA; Lu, lung RNA; Pa, pancreas RNA; Sk, Skeletal muscle RNA; Sp, spleen RNA; St, stomach RNA.
	Fig. 6. RNAse protection analyses of XNkx-2.3 and XNkx-2.5 expression during Xenopus embryonic development. Radiolabelled probes for each XNkx-2.3 allele (A,B) and for XNkx-2.5 (C) were hybridized to 20 mg of total RNA extracted from staged Xenopus embryos (Nieuwkoop and Faber, 1967). RNA samples were monitored for integrity and quantity on analytical formaldehyde agarose gels before using. The protected RNA species as indicated by arrowheads in each figure correspond to full protection of probe sequences complementary to each cDNA. For probe details, see Materials and Methods. P, probe alone, including vector sequences and regions complementary to each cDNA; t, tRNA control; stage 9- 38, RNA from stages 9 through 38 embryos.
	Fig. 7. Whole-mount in situ analyses of XNkx-2.3, Xtwist and XMLC2 RNA expression in Xenopus embryos. For details on whole-mount procedure and probes used, see Materials and Methods. Arrowheads indicate relevant stained areas. In B, C, the boundaries of the anterior neural plate are outlined with a dotted line. cg, cement gland, h, heart, np, neural plate, ph, pharyngeal region. (A) Anterior view of stage 13.5 embryo hybridized to probe for XNkx-2.3 RNA. Dorsal is at the top. Bluish staining band above purple-brown hybridisation signal (arrowhead) is a result of artefactual trapping of probe in the archenteron (Harland, 1991). (B) Anterior view of stage 16 embryo stained for XNkx-2.3 RNA expression. Dorsal is at the top. Staining is observed ventral to the neural folds, immediately posterior to the cement gland. (C) Anterior view of stage 16 embryo hybridized to probe for Xtwist. twist expression can be seen in the forming cephalic neural crest (Hopwood et al., 1989). (D) Ventral view of stage 19 embryo, hybridized to XNkx-2.3 probe. Anterior is to the right. Staining is observed in an anteroventral position. (E) Ventral view of stage 23 embryo, showing two areas of staining for XNkx-2.3 just posterior to the cement gland. The posterior staining is clearly bilateral and corresponds to the partially fused cardiac primordia. Anterior is to the right. (F) Lateral view of same embryo as in E. Anterior is to the right. Note two streaks of staining just caudal to the cement gland. (G) Ventral view of stage 27 embryo hybridized to probe for XNkx-2.3. Anterior is at the top right. Arrowheads mark the bilateral staining that corresponds to the prospective heart region (see H). The staining also extends further rostral, abutting the cement gland. (H) Ventral view of stage 27 embryo, hybridized to a probe for XMLC2, a marker for differentiated cardiac mesoderm. Anterior is to the right. Note that the bilateral staining here corresponding to the cardiac mesoderm does not extend rostrally to the cement gland, as did the staining shown in G for XNkx-2.3. (I) Lateral view of stage 26 embryo stained for XNkx-2.3 RNA expression. Anterior is to the right, dorsal is at the top. Arrowheads indicate two regions of staining, in the heart tube (see J) and in the pharyngeal region more rostrally. (J) Lateral view of stage 26 embryo stained for XMLC2 expression. Staining is confined in the heart tube. (K) Lateral view of stage 26 embryo stained for Xtwist expression. Note the heavy staining in the pharyngeal region, whereas there is no staining in the cardiac region. (L) Lateral view of stage 34 embryo, hybridized to probe for XNkx-2.3. Anterior is to the right, dorsal at the top. Staining is seen in the looped heart tube and more rostrally in the pharyngeal region. (M) Lateral view of stage 34 embryo, hybridized to probe for XMLC2. Staining is evident in the looped heart tube. (N) Lateral view of stage 34 embryo, hybridized to probe for Xtwist. Some staining remains in the pharyngeal region, and is absent from the heart. Embryos shown in I, K-N were cleared in benzyl:benzoate (Harland, 1991) before photographing.
	Fig. 8, Transverse sections from embryos stained by wholemount in situ for XNkx-2.3 and Xtwist RNA expression. For experimental details, refer to Materials and Methods. ec, ectoderm; en, endoderm; ey, eye anlage; h, heart region; m, mesoderm; my, myocardium of early heart tube; nc, branchial arch neural crest; ng, neural groove; ph, pharynx. Embryos were viewed and photographed using Nomarski optics. In C-E, the outline of each section and the pharynx are shown with dotted lines to facilitate orientiation. Dorsal is at the top. (A) Anterior transverse section from a stage 16 embryo stained for XNkx-2.3 RNA expression. Note staining in mesoderm, and faint staining in the endoderm. (B) Transverse section from same embryo as in A, but further posterior. Note staining in mesoderm and stronger staining of endoderm than in A. (C) Transverse section at the level of the eye anlage of stage 30 embryo stained for XNkx-2.3 expression. Staining is observed in the pharyngeal endoderm. (D) Transverse section just posterior to that shown in C, indicating staining for XNkx-2.3 in pharyngeal endoderm and in myocardium. (E) Transverse section of stage 30 embryo stained for Xtwist RNA expression. The neural crest invading the pharyngeal mesoderm is stained for twist. This staining pattern was never observed for embryos stained for XNkx-2.3 expression. The endoderm and the heart do not stain but their position is shown to facilitate comparison with D.