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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. |
