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Fig. 3. Characterization of the spatial relationship between the Xisl-1 and Nkx2.5 expression domains. Single and double whole mount in situ hybridizations were performed to depict the expression domains of Xisl-1 and Nkx2.5 during early heart development in more detail. Pictures A, F, K, P show detection of Xisl-1 transcripts in light blue. Pictures B, G, L, Q show detection of Nkx2.5 transcripts in red. Pictures C, H, M, R show staining for both transcripts with the overlap of expression exhibiting a darker, rather purple staining. The asterisk behind a letter denotes images of identical embryos. Other embryos were stage matched. Pictures E, J, O depict schematics of the relative expression domains of Xisl-1 (blue dots) and Nkx2.5 (red lines) from embryos shown in pictures D, I, and N. (A–D) At neurula stages, both Xisl-1 and Nkx2.5 demarcate the cardiac crescent, which lies ventrally to the cement gland (arrows in pictures C, D). All embryos are shown from an anterior view. Picture D shows an enlargement of the cardiac crescent region of the embryo depicted in picture C. (F–I) At early tailbud stages, Xisl-1 and Nkx2.5 expression still overlaps largely in the heart region (arrow in picture H). (I) A ventral view of the heart region of an embryo to better demonstrate the overlapping expression of Xisl-1 and Nkx2.5 (arrow) during early tailbud stages. Xisl-1 only (blue) is also strongly expressed in the stomodeal region abutting the cement gland. (K–N) At stage 30 when the linear heart tube begins to form, Xisl-1 transcripts become restricted to a more anterior domain of the heart region (arrowhead) and the overlapping domain with Nkx2.5 decreases (black arrow). The white arrow points to the domain in which only Nkx2.5 is expressed. Picture N is a ventral view of the same embryo shown in picture M; head is to the left. The dotted lines highlight the Xisl-1 (white lines) and Nkx2.5 (black lines) expression domains. The ventral view of a stage matched embryo depicted in picture T shows a single staining for Xisl-1 in dark blue, in which the gap of Xisl-1 expression (asterisk) marks the region that expresses Nkx2.5 as seen in picture N. The arrow points to Xisl-1 expression in the posterior domain of the heart. (P–S) At progressive stages Xisl-1 transcripts are detected in the most anterior part of the heart region and its cardiac expression can only be documented in transverse sections. (U–X) Anterior to posterior series of transverse sections of a st. 28 embryo stained for Xisl-1 (purple) and Nkx2.5 (red). In the most anterior section (U) Xisl-1 transcripts are localized in two circular domains in the mesodermal layer (arrows) and in the pharyngeal endoderm (asterisk). No Nkx2.5 staining is detectable. (V) Along the A–P axis, expression of Xisl-1 becomes crescent-shaped in the mesoderm. (W) The section reveals an extensive overlap of Xisl-1 and Nkx2.5 in the cardiogenic mesoderm (arrows). (X) Further posterior, Xisl-1 becomes restricted to the dorsolateral domain (black arrows). In the ventralmost expression domain only Nkx2.5 transcripts are detected (white arrow). (Y–Z3) Transverse sections through the linear heart tube of st.30 embryos are shown. (Y, Y1) Nkx2.5 expression (red) throughout the heart tube (myocardium, mesocardium and pericardial roof) is depicted. A subsection of the mesocardium and pericardial roof is shown as an enlargement in picture Y1. (Y2, Y3) Nkx2.5 (red) and Xisl-1 (blue) expression is seen in the linear heart tube of a st.30 embryo. Xisl-1 transcripts are absent from the myocardium but are detected in the endocardium and in the foregut endoderm (asterisk). The Xisl-1 and Nkx2.5 expression domains overlap in the mesocardium and pericardial roof (see enlargement Y3). (Z, Z1) TnIc (red) is expressed in differentiated cardiomyocytes of the myocardium. Note the absence of TnIc transcripts in the mesocardium and pericardial roof. The enlargement shows a subsection of these domains (Z1). (Z2, Z3) TnIc (red) and Xisl-1 (blue) expression in the linear heart tube of a st.30 embryo. Note the complementary expression domains of TnIc and Xisl-1 from the myocardium, mesocardium, and pericardial roof, respectively. cg: cement gland; ec: endocardium; mc: myocardium; me: mesocardium; nf: neural fold; np: neural plate; pc: pericardial roof. |