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The bHLH protein eHAND is a sensitive marker for cardiovascular precursors in the Xenopus embryo. The earliest site of expression is a broad domain within the lateral plate mesoderm of the tailbudembryo. This domain comprises precursors that contribute to the posterior cardinal veins in later stages. Surprisingly, expression is profoundly asymmetric at this stage and is random with respect to embryo side. XeHAND is also expressed in an anterior domain that encompasses the prospective heart region. Within the myocardium and pericardium, transcripts are also asymmetrically distributed, but in these tissues they are localised in a left-sided manner. Later in development XeHAND transcripts are largely restricted to the ventralaorta, aortic arches and venous inflow tract (sinus venosus) which flank the heart itself, but no expression is detected in neural crest derivatives at any stage. This demonstrates that patterns of XeHAND expression differ markedly amongst vertebrates and that in Xenopus, XeHAND expression identifies all of the earliest formed elements of the cardiovascular system. In animal cap explants, expression of XeHAND (but not other markers of cardiogenic differentiation) is strongly induced by ectopic expression of the TGFbeta family members, BMP-2 and BMP-4, but this can be blocked by coexpression of a dominant negative BMP receptor. This suggests that XeHAND expression in the embryo is regulated by the ventralising signals of bone morphogenetic proteins. High levels of expression are also detected in explants treated with high doses of activin A which induces cardiac muscle differentiation. No such effect is seen with lower doses of activin, indicating that a second pathway may regulate the XeHAND gene during cardiogenesis.
Fig. 3. Whole-mount in situ analysis of XeHAND expression. (A) Lateral view of a Xenopus tailbud embryo (stage 24). Anterior is to the right. Staining in the presumptive heart region is indicated by a white arrow, and staining in the proctodeum is indicated by a black arrow. (B) View of the same embryo from the opposite side. (C) Lateral view of an embryo at stage 27. Anterior is to the right. Staining in the presumptive heart region is indicated by a white arrow. (D) View of the same embryo from the opposite side. (E) Lateral view of a stage 32 embryo. Staining in the vitelline veins is indicated by a black arrow. (F) View of the other side of the same embryo. (G) Lateral view of a stage 33/34 embryo. Anterior is to the left. Staining in the ventral aorta and aortic arches is indicated by a white arrow, and staining in the sinus venosus (venous inflow tract) is indicated by a black arrow. Expression in the presumptive posterior cardinal veins running ventral to the somites can clearly be seen. (H) Ventral view of a stage 33/34 embryo. Overstaining reveals XeHAND expression in the heart tube (white arrow).
Fig. 4. Transverse sections of embryos processed for RNA whole-mount in situ hybridisation using a probe specific for XeHAND. The positions of the sections are indicated in (H). Sections are viewed from an anterior prospect. (A) At early tailbud stages (stage 25), XeHAND is expressed in the lateralmesoderm. (B) At tadpole stages (stage 33/34), XeHAND is expressed in two patches in the head mesenchyme, on either side of the foregut. (C) Moving towards the posterior, the two patches of expression flatten on either side of the ventral aorta lumen (black arrow). (D) At the level of the anterior of the pericardial cavity, XeHAND can be detected throughout the dorsal pericardium (pericardial cavity indicated by a black arrow). (E) Sections through the middle of the heart tube show XeHAND expression throughout the pericardium, including the inner wall (black arrow), as well as in the left side of the myocardial tube (white arrow). (G) A close-up of the section shown in (E). (F) Sections at the level of the flank staining show that XeHAND is expressed in the presumptive posterior cardinal veins, ventral to the somites.
Fig. 5. Double-labelled in situ hybridisation analysis of XeHAND expression. (A) Late tailbudembryo (stage 31), lateral view showing staining for MLC113 (blue) and XeHAND (reddish-brown). Note the distinct gap between the bottom of the somites, and the domain of XeHAND expression (white arrow). (B) Tailbud (stage 28) embryo, ventral view showing staining for XMLC2a (light blue) and XeHAND (purple). Note the thin line of XeHAND expression dividing the two patches of XMLC2a (black arrow). (C) Lateral view of a swimming tadpole (stage 31) showing staining for XMLC2a (light blue; black arrow) and XeHAND (reddish-brown). The arrowhead indicates that portion of the ventral surface that is not covered by mesoderm. (D) Feeding tadpole (stage 40) lateral view showing staining for XMLC2a (light blue) and XeHAND (purple). XMLC2a staining in the heart is indicated by a black arrow, and XeHAND staining in the ventral aorta and first aortic arches are indicated by the white arrow. The embryo was cleared with BB/BA. (E) Lateral view of a tailbudembryo (stage 27) stained for T4 a-globin (purple) and XeHAND (light blue).
Fig. 6. Expression of XeHAND in adult frog tissues. The distribution of XeHAND mRNA in adult frog tissues was analysed by RNase protection assay. Lane 1, undigested probes; lane 2, tRNA control; lanes 3–11, tad- pole embryo (stage 34), intestine, stomach, gall bladder, spleen, skeletal, heart, lung, liver RNA, respectively. Five micrograms of total RNA was used in each assay. Full-length protected fragments for each probe are indicated. As an internal control, a probe for the highly abundant EF1-a was included in the XeHAND assay.
Fig. 7. Induction of XeHAND expression in animal cap explants. Animal cap explants were assayed for the presence of XeHAND, EF1-a, cardiac actin and T4 a-globin by RT-PCR. (A) RNA encoding BMP-2 (lane 2) and BMP-4 (lane 3) both induce high levels of XeHAND and T4 a-globin (a marker for ventral mesoderm differentiation). Both responses are blocked by the truncated BMP receptor (lanes 5 and 6). (B) All doses of soluble FGF-2 strongly induce XeHAND expression (lanes 2â4), but soluble acti- vin A induces little or no XeHAND expression except at exceptionally high doses (lanes 5â7).
Fig. 2. Expression of XeHAND in Xenopus embryos. The distribution of XeHAND transcripts was analysed by RNase protection assay. Lane 1, undigested probes; lane 2, tRNA control; lanes 3–12, unfertilised oocyte, stages 8, 10, 12.5, 14, 17, 20, 23, 34, 42 embryo RNA, respectively. Two embryo equivalents of RNA (10 mg) was used in each case. Full-length protected fragments for each probe are indicated. As an internal control, a probe for XMax2 (which is expressed at constant levels throughout early development (Tonissen and Krieg, 1994)) was included in the XeHAND assay.
