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Dev Biol
2009 Mar 15;3272:376-85. doi: 10.1016/j.ydbio.2008.12.028.
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Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5.
Espinoza-Lewis RA
,
Yu L
,
He F
,
Liu H
,
Tang R
,
Shi J
,
Sun X
,
Martin JF
,
Wang D
,
Yang J
,
Chen Y
.
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The pacemaker is composed of specialized cardiomyocytes located within the sinoatrial node (SAN), and is responsible for originating and regulating the heart beat. Recent advances towards understanding the SAN development have been made on the genetic control and gene interaction within this structure. Here we report that the Shox2 homeodomain transcription factor is restrictedly expressed in the sinus venosus region including the SAN and the sinus valves during embryonic heart development. Shox2 null mutation results in embryonic lethality due to cardiovascular defects, including an abnormal low heart beat rate (bradycardia) and severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation. Genetically, the lack of Tbx3 and Hcn4 expression, along with ectopic activation of Nppa, Cx40, and Nkx2-5 in the Shox2(-/-) SAN region, indicates a failure in SAN differentiation. Furthermore, Shox2 overexpression in Xenopus embryos results in extensive repression of Nkx2-5 in the developing heart, leading to a reduced cardiac field and aberrant heart formation. Reporter gene expression assays provide additional evidence for the repression of Nkx2-5 promoter activity by Shox2. Taken together our results demonstrate that Shox2 plays an essential role in the SAN and pacemaker development by controlling a genetic cascade through the repression of Nkx2-5.
Fig. 1. Shox2 expression in the murine developing heart. (A) Shox2 expression is initially detected in the sinus venosus region (arrowhead) at E8.5 embryo. At E10.5 (B) and E11.5 (C), Shox2 expression gradually becomes restricted to the right side of the heart to the dorsal wall of the right atrium, the SAN region (arrow, C) and sinus valves (arrowheads, B, C). At E12.5 (D), restricted Shox2 expression in the SAN and sinus valves (arrowheads) is clearly observed. (E) Shox2 expression at E16.5 follows the same pattern as previous stages. (F) Whole mount in situ hybridization showing the expression of Shox2a isoform in the sinus venosus region (yellow arrowhead). A, atrium; V, ventricle; D, dorsal side.
Fig. 3. Altered gene expression in the Shox2−/− heart. Expression patterns of Hcn4 (A–D), Tbx3 (EH), Nkx2-5 (I–L), Nppa (M–P), Cx40 (Q–T) and cTnt (U–X) at E10.5 and at E11.5 in the wild type and Shox2−/− hearts are shown. Hcn4 is expressed in the dorsal right wall of the right atrium in the wild type (A, C) and is reduced or absent in the Shox2 mutant (B, D). Tbx3 is expressed in the dorsal right wall of the right atrium and in the sinus valves (E, G), however is absent in the Shox2 mutant (F, H) at both stages. Nppa, Cx40, and Nkx2-5 are ectopically expressed in the dorsal right wall of the Shox2–/– right atrium at both stages (J, N, R, L, P, T), as compared to the wild type controls (I, M, Q, K, O, S). cTnt shows comparable expression level in the Shox2–/– heart (V, X) and the wild type controls (U, W). In all panels, the arrows point to the SAN region, while the arrowheads point to the sinus valves. A, atrium. Scale bars represent 50 μm.
Fig. 4. Shox2 overexpression downregulates Nkx2-5 expression. XNkx2-5 and XcTnI expression was analyzed by in situ hybridization in control embryos at stage 26 (A, E) and at stage 41 (C, G) and in GR-Shox2 mRNA injected induced embryos at same stages (B, F, D, H). XNkx2-5 expression is clearly reduced at the midline of injected induced embryos at stage 26 (B), as compared to control embryos (A). XcTnI expression is maintained in injected induced embryos laterally, but is absent at the midline at stage 26 (F), as compared to the controls (E). At stage 41, XNkx2-5 expression is absent in injected induced embryos (D) compared to control embryos (C). XcTnI expression in injected induced embryos (D) is comparable to that of control embryos (H). Altered gene expression is accompanied by the abnormal heart morphology when comparing control and injected induced embryos (C, D, G, H). Histological transverse sections of Xenopus embryos showing loss of mesodermal cardiac progenitor cells in the ventral midline in injected induced embryos (B’, D’, arrowheads), as evidenced by the loss of XNkx2-5 expression in the corresponding region when compared to control embryos (A’, C’, arrowheads). Scale bars represent 50 μm.
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