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Fig. 1. Sequence alignment of Zcchc24 family members. Comparison of the predicted amino acid sequence of X. laevis Zcchc24 with its chick, human, mouse and rat homologs. X. laevis Zcchc24 (Genbank accession no. KF438010) shares 90.8% of identity with chick ZCCHC24 (XP_421599), 86.3% with human ZCCHC24 (NP_699198), 84.6% with mouse ZCCHC24 (NP_001094903), and 84.2% with ZCCHC24 rat (NP_001101864) homologs. Conserved residues are shaded in orange while identical amino acids among some of the sequences are shaded in light blue. The absence of residues at the corresponding region is indicated by dashes. Both zf-CCHC and zf-3CxxC zinc finger domains are displayed in green.
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Fig. 2. zcchc24 expression from blastula to gastrula stages. Whole mount in situ hybridization using DIG labelled zcchc24 (A-C’, E,E’) or fluorescein labelled myoD (D,F) was performed on embryos from blastula to gastrula stages. (A,A’) At blastula stages, zcchc24 is expressed as a ring around the marginal zone in both dorsal and ventral sides. The embryos display the animal hemisphere to the top. (B,B’) In the beginning of gastrulation (st 10.5), zcchc24 expression is restricted to the dorsal mesoderm in both involuting and non-involuting marginal zone. (C,C’, E,E’) At late gastrula (st 11 and 12), zcchc24 transcripts are detected in two lateral mesoderm stripes around the blastopore that culminate in the dorsal mesoderm, the somitogenic mesoderm, but is excluded from the midline. (D,F) myoD is expressed in the somitogenic mesoderm. (A’ , B’) Hemisections of (A,B), respectively. (C‘, E’) Transverse sections of st 11 and st 12 embryos, respectively, with dorsal side displayed to the top. (B,C) Vegetal views. (D,E,F) Dorsal views. Dashed lines delimitate the blastopore. An, animal; Vg, vegetal; D, dorsal; V, ventral; dbl, dorsal blastopore lip; sm, somitogenic mesoderm.
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Fig. 3. zcchc24 expression during neurula stages. Whole mount in situ hybridization using DIG labelled zcchc24 (A-B’’, E) or fluorescein labelled nkx2.5 (D) and double whole mount in situ hybridization with DIG labelled zcchc24 and fluorescein labelled nkx2.5 (C) were performed on embryos at neurula stages. (A) At late gastrula/early neurula (st 13), zcchc24 is expressed as two stripes in the dorsal side of the embryo along the antero-posterior axis but is excluded from the notochord. (B-B’’, E) During neurula stages, zcchc24 is expressed in anterior and somitogenic mesoderm, decreasing the expression from the posterior to the anterior region. Comparison between the expression of zcchc24 and nkx2.5 (B,C,D) shows that the most anterior expression of zcchc24 is adjacent to the cardiac mesoderm. (A,B, C-E) Embryos in a dorsal view. (B’, B’’) Transversal sections of st 15 embryos with the dorsal region displayed to the top. Red arrows indicate the heart precursor region. A, anterior; P, posterior; n, notochord; sm, somitogenic mesoderm.
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Fig. 4. Expression pattern of zcchc24 during tailbud stages. Whole mount in situ hybridization using DIG labelled zcchc24 (A, B, F-G’), fluorescein labelled nkx2.5 (C,H) or fluorescein labelled twi (D,J), and double whole mount in situ hybridization using DIG labelled zcchc24 and fluorescein labelled twi (E,K) or DIG labelled zcchc24 and fluorescein labelled nkx2.5 (I) were performed on embryos at tailbud stages. (A-E) At early tailbud stages, zcchc24 is strongly expressed in the unsegmented somitogenic mesoderm, somites, and migrating neural crest but is absent in the region of differentiating cardiac muscle. (F) Later, at st 28, zcchc24 transcripts start to be detected in the forming heart, and in the entire head except the region between the branchial arches. (G-K) This expression is maintained throughout tailbud development, being zcchc24 expressed in the somites, branchial arches, head and heart. All the embryos are displayed in a lateral view with the anterior region to the left. (G’) Transversal section of the heart region of a st 32 embryo. The orange arrows indicate the neural crest/branchial arches, and the red arrows indicate heart-forming region/heart. A, anterior; P, posterior; ec, endocardium; pc, pericardium; mc, myocardium; bam, branchial arch mesenchyme; cg, cement gland.
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Fig. 6. Schematic representation of the localization of heart precursors and zcchc24 expression during X. laevis embryonic development. Red areas represent the heart precursors and the gray areas represent the expression of zcchc24. (A) Vegetal view, (B) anterior view, (C,D) lateral views with the anterior region to the left. A, anterior; P, posterior; D, dorsal; V, ventral; R, right; L, left.
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zcchc24 (zinc finger, CCHC domain containing 24) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
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zcchc24 (zinc finger, CCHC domain containing 24) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
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zcchc24 (zinc finger, CCHC domain containing 24) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, dorsal up, anterior left.
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zcchc24 (zinc finger, CCHC domain containing 24) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, dorsal up, anterior left.
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Fig. 5. In chicken embryos, ZCCHC24 is expressed in the heart and somites. Whole mount in situ hybridization using DIG labelled ZCCHC24 in chick embryos at HH10. (A, Aâ) At this stage, ZCCHC24 is expressed in the heart (red arrow), somites (yellow arrow) and in head mesenchyme. (Aâ) Magnification of (A).
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