Fu Y et al. (1998), Vertebrate tinman homologues XNkx2-3 and XNkx2-...

XB-ART-14114

Development 1998 Nov 01;12522:4439-49. doi: 10.1242/dev.125.22.4439.

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Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in a functionally redundant manner.

Fu Y , Yan W , Mohun TJ , Evans SM .

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Tinman is a Drosophila homeodomain protein that is required for formation of both visceral and cardiac mesoderm, including formation of the dorsal vessel, a heart-like organ. Although several vertebrate tinman homologues have been characterized, their requirement in earliest stages of heart formation has been an open question, perhaps complicated by potential functional redundancy of tinman homologues. We have utilized a novel approach to investigate functional redundancy within a gene family, by coinjecting DNA encoding dominantly acting repressor derivatives specific for each family member into developing Xenopus embryos. Our results provide the first evidence that vertebrate tinman homologues are required for earliest stages of heart formation, and that they are required in a functionally redundant manner. Coinjection of dominant repressor constructs for both XNkx2-3 and XNkx2-5 is synergistic, resulting in a much higher frequency of mutant phenotypes than that obtained with injection of either dominant repressor construct alone. Rescue of mutant phenotypes can be effected by coinjection of either wild-type tinman homologue. The most extreme mutant phenotype is a complete absence of expression of XNkx2-5 in cardiogenic mesoderm, an absence of markers of differentiated myocardium, and absence of morphologically distinguishable heart on the EnNkxHD-injected side of the embryo. This phenotype represents the most severe cardiac phenotype of any vertebrate mutant yet described, and underscores the importance of the tinman family for heart development. These results provide the first in vivo evidence that XNkx2-3 and XNkx2-5 are required as transcriptional activators for the earliest stages of heart formation. Furthermore, our results suggest an intriguing mechanism by which functional redundancy operates within a gene family during development. Our experiments have been performed utilizing a recently developed transgenic strategy, and attest to the efficacy of this strategy for enabling transgene expression in limited cell populations within the developing Xenopus embryo.

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Species referenced: Xenopus laevis
Genes referenced: myl2 nkx2-3 nkx2-5 tnni3

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	Fig. 3. Whole-mount in situ analyses of control and EnHD-injected embryos. Embryos were injected with a total of 200 pg of DNA into one cell at the two cell stage, fixed, and processed for whole-mount in situ analyses utilizing probes for either XMLC2 or XcTnI mRNA. All embryos shown are oriented with anterior to the left, and posterior to the right. The mutant phenotypes shown were observed in embryos injected with either EnHDX2.3 or EnHDX2.5, or with coinjection of both EnHD constructs. However, coinjection resulted in a significantly higher frequency of observed mutant phenotypes (refer to Table 1). The same mutant phenotypes were observed utilizing probes for either XMLC2 or XcTnI mRNA. (A) Lateral view of control En-injected stage 34/37 embryos stained for expression of XMLC2 mRNA. At these stages the normal Xenopus heart expresses high levels of XMLC2 mRNA (Evans et al., 1995). (B) Lateral view of EnHD-injected stage 34/37 embryos stained for expression of XMLC2 mRNA. In contrast to the control embryos (A), the upper embryo exhibits unfused heart primordia, with much lower expression of XMLC2 mRNA on one side (arrow). The lower embryo has a much reduced area expressing XMLC2 mRNA (arrow). The embryos shown were injected with DNA encoding the EnHDX2.5 repressor. (C) Ventrolateral view of control En injected stage 32 embryo stained for expression of XcTnI mRNA, showing that fusion proceeds in an anterior to posterior direction (Balinsky, 1970). (D) Ventrolateral view of EnHDX2.3-injected stage 32 embryo stained for expression of XcTnI mRNA. This embryo exhibits unfused cardiac primordia, with one of the primordia showing greatly reduced staining (arrow). (E) Control and mutant stage 35/36 embryos stained for expression of XcTnI mRNA. The upper EnHD-injected embryo (ventral view) has severely reduced XcTnI staining on one side of the embryo (arrow), and a small asymmetrically located heart tube on the other side. This embryo was coinjected with both EnHDX2.3 and EnHDX2.5. The lower, control En-injected embryo, shown in a ventrolateral view, exhibits a symmetrically located (centered beneath the cement gland) normal-sized heart. (F) Ventral views of control and mutant stage 32 embryos stained for expression of XcTnI mRNA. The upper EnHD-injected embryo exhibits a small heart tube on one side, and no apparent expression of XcTnI mRNA on the opposite side (arrow). This embryo resulted from a coinjection of both dominant repressor Nkx constructs. The lower En controlinjected embryo demonstrates the normal-sized heart primordia. C, cement gland.
	Fig. 4. Transverse paraffin sections of stage 36 control and mutant embryos following whole-mount in situ analyses for expression of either XMLC2 or XcTnI mRNA. Sections were stained for expression of XcTnI, but similar results were observed in sections stained for XMLC2. Dorsal is at the top. (A) En control injected embryo. At this stage, the heart is looped and has partitioned atria and ventricular chambers (Nieuwkoop and Faber, 1956). Note the relatively large size of myocardial tissue in comparison to XcTnI mRNA-positive tissue in the mutant sections shown in B and C. (B) Embryo injected with both EnHDX2.3 and EnHDX2.5 showing the unfused phenotype. Two distinct heart tubes can be seen forming, despite the fact that the primordia have not contacted each other. (C) Embryo injected with both EnHDX2.3 and EnHDX2.5. Whole mount in situ analysis of this embryo revealed a small asymmetrically located heart on one side, with no visible staining for XcTnI on the other. This external phenotype was confirmed by sectioning. Note that there is no morphological evidence of a heart tube on the side which lacks expression of XcTnI. Note general lack of development on this side of the embryo. M, midgut; N, notochord; O, otic vesicle and R, rhombencephalon.
	Fig. 5. Paraffin sections of embryos stained for expression of β- galactosidase, and processed by whole-mount in situ for expression of XcTnI mRNA. Sections shown are from stage 40 embryos, dorsal is at the top. 100 pg of mRNA encoding β-galactosidase was injected as a lineage tracer to indicate the injected side of the embryo. A white line is drawn through the midline of the sections, to emphasize the positioning of the heart. (A) Control embryo, coinjected with En control plasmid and mRNA encoding β-galactosidase. (B). Mutant embryo, coinjected with both dominant repressor Nkx constructs, and lineage tracer mRNA encoding β-galactosidase. Note the asymmetric placement of the XcTnI-positive myocardial the smaller size of the heart relative to that of the control (A). The small heart is formed on the side opposite to that of injection. M, midgut; N, notochord; O, otic vesicle and R, rhombencephalon
	Fig. 6. Whole-mount in situ analyses of embryos stained for expression of XNkx2-5, or XNkx2-5 and XcTnI. Embryos were injected with a total of 200 pg of DNA into one cell at the twocell stage, fixed, and processed for whole-mount in situ analyses staining for XNkx2-5 mRNA alone, or for both XNkx2-5 and XcTnI mRNAs utilizing double-label in situ (see Materials and Methods for details). XNkx2-5 mRNA signal was detected utilizing BCIP/NBT (brownish-purple color), and XcTnI mRNA signal was detected utilizing BCIP alone (light blue color). Overlap of the two signals gives a dark blue signal (E and F). Anterior is to the right. The mutant embryos shown were coinjected with both EnHDX2.3 and EnHDX2.5. (A) Control-injected stage 21 embryo, ventral view, showing symmetrical XNkx2-5 mRNA staining just posterior to the cement gland. (B) EnHD-injected stage 21 embryo, ventral view, with arrow indicating reduction in XNkx2-5 mRNA staining relative to the other side of the embryo. (C) Control injected stage 24 embryo, ventral view, showing XNkx2-5 mRNA in fused cardiac primordia posterior to the cement gland. (D). EnHD-injected stage 24 embryo, ventral view, with arrow indicating absence of XNkx2-5 mRNA staining on one side of the embryo. (E) Control injected stage 32 embryo, ventrolateral view, co-stained for XNkx2-5 and XcTnI mRNAs (dark blue). (F) EnHD-injected stage 33 embryo, ventrolateral view, co-stained for XNkx2-5 and XcTnI mRNAs. In this embryo, the positively staining regions have not yet fused. Although one side stains positively for both XNkx2-5 and XcTnI mRNAs (dark blue staining as indicated by blue arrowhead), the other side shows very faint staining for both mRNAs (black arrow). C, cement gland.
	Fig. 7. Paraffin sections of control and mutant embryos following whole-mount in situ analyses for XNkx2-5 and XcTnI mRNAs. Sections shown were from stage 28 embryos, co-stained for XNkx2-5 mRNA (brownish-purple) and for XcTnI mRNA (blue); dorsal at the top. (A) Control embryo exhibits symmetrical staining of both XcTnI mRNA, in differentiating myocardial cells, and XNkx2-5 mRNA in both differentiating myocardial cells and adjacent mesoderm. (B) Mutant embryo exhibits asymmetrical staining of both XcTnI and XNkx2-5 mRNAs, with one side (arrow) exhibiting greatly reduced signal for both mRNAs. M, midgut; N, notochord; O, otic vesicle and R, rhombencephalon.
	Fig. 8. Enlarged heart phenotype in embryos following injection with wild-type XNkx2-5 DNA. The observation of this phenotype in DNA-injected embryos demonstrates that tinman homologues can affect cardiac cell fate when expressed following the mid-blastula transition. Lateral view of stage 35 embryos, anterior to the right, stained for expression of XcTnI mRNA. The lower embryo was injected with DNA encoding wild-type XNkx2-5. Note the greatly enlarged heart relative to the upper, control-injected embryo. Comparable enlarged hearts were also observed in embryos injected with DNA encoding wild-type XNkx2-3 (data not shown).