Martinsen BJ et al. (2004), Cardiac neural crest ablation alters Id2 gene e...

XB-ART-3308

Dev Biol 2004 Aug 01;2721:176-90. doi: 10.1016/j.ydbio.2004.04.030.

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Cardiac neural crest ablation alters Id2 gene expression in the developing heart.

Martinsen BJ , Frasier AJ , Baker CV , Lohr JL .

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Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.

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Species referenced: Xenopus laevis
Genes referenced: id2 myh1 snai2 tbx2
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	Fig. 3. Loss of chick Id2 expression in a day 5.5 heart following early cardiac neural crest ablation (3–8-somite stage). Selected sections showing chick Id2 expression in a control day 5.5 heart and a day 5.5 heart after early cardiac neural crest ablation. (A, C, E, G) Expression of Id2 from proximal to distal within the developing outflow tract and heart of a control day 5.5 heart. (B, D, F, H) Loss of Id2 expression from proximal to distal within a day 5.5 outflow tract and heart following early cardiac neural crest ablation. (H) Early cardiac neural crest ablation resulted in a single outflow vessel (So) with no septation or cardiac neural crest derived condensed mesenchyme (cm). Red p and arrowhead, prong of cardiac neural crest (CNC) with Id2 expression. Blue p and arrowhead, prong of CNC with no Id2 expression. Green p and arrowhead, loss of CNC prong and Id2 expression after early CNC ablation. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; Ao, aorta; P, pulmonary trunk; PR, proximal right endocardial cushion; PL, proximal left endocardial cushion; SI, septum intermedium; DD, distal dorsal endocardial cushion; AIC, aorta intercalated endocardial cushion; PIC, pulmonary intercalated endocardial cushion; DRV, distal right ventral endocardial cushion; DLV, distal left ventral endocardial cushion; AVS, atrioventricular sulcus; So, single outflow vessel; SoR, single outflow vessel right endocardial cushion; SoL, single outflow vessel left endocardial cushion. OFT labeling based on that of Qayyum et al. (2001).
	Fig. 5. xId2 expression in staged Xenopus embryos. A, C, E, and G are dorsal views with the anterior of the embryo to the right. B, D, F, and H are right lateral views of the embryos. (A–F) Whole-mount in situ hybridization for xId2 shows expression in the neural folds (NF) and along the length of the closing neural tube (NT). (G, H) shows xId2 expression in the somites (S) and branchial arches (BA).
	Fig. 6. Results of Xenopus neural crest ablation and xId2 expression patterns. (A, B) xSlug expression in the neural crest was used as a guide (A) for stage 15 neural crest ablations (B, abl). (C, D) Whole-mount xId2 in situ hybridization (ISH) was carried out on stage 39 control and neural crest ablated embryos. (C) There is xId2 expression in both the inflow tract (IFT) (green arrow) and outflow tract (OFT) (red arrow) of the developing heart, as well as the otic vesicle (O), eye (E), and branchial arches (BA) in the control embryo. (D) In the ablated stage 39 embryo, there is lack of expression in the IFT (green arrowhead), OFT (red arrowhead), and reduced expression in the BA. (E–G) Control embryos underwent whole-mount in situ hybridization (ISH) for xId2 and were fixed at stage 37/38 for whole-mount analysis and at stage 39 for sectional analysis. (E) xId2 expression is located in the branchial arches (BA), eye (E), otic vesicle (O), and heart (H). (F, G) xId2 expression is located in the neural tube (NT), eye (E), outflow tract (OFT), pharynx (P), nerve ganglion (green arrows), and splanchnic mesoderm (SM, red arrows).
	Fig. 7. Analysis of the heart morphology and xId2 expression patterns of stage 33 Xenopus embryos, following neural crest ablation and in situ hybridization (ISH) for xId2. (A, B) Transverse sections of a stage 33 control embryonic heart immunostained with the myocardial marker MF20. White arrows indicate (MF20 negative) undifferentiated splanchnic mesoderm cells continuous with the newly formed cardiac myocardial cells expressing myosin heavy chain (MF20 positive). (C, D) Sections through a stage 33 control embryo following ISH for xId2. In the control embryos, there is a visible myocardium (m), endocardium (e), and pharynx (P). [See Mohun et al. (2000) for reference to Xenopus heart morphology.] There is xId2 expression in the splachnic mesoderm (red arrows) as well as the outflow track myocardium (green arrow). (E, F) Sections through a stage 33 embryo following stage 15 neural crest ablation and ISH for xId2. There is a lack of xId2 expression in the splachnic mesoderm (red arrowheads) and the outflow tract myocardium (green arrowhead). There are also morphological differences in heart development following neural crest ablation. Note the enlarged myocardium (m), the thickened pericardial layer (Pc), and the pericardial edema (PcE). Morphology is variable among neural crest ablated embryos; (E) sometimes there is visible formation of a myocardium (m) and endocardium (e), (F) whereas in other embryos there is only an undistinguishable mass of heart cells (h).
	Fig. 8. Analysis of morphology and xId2 expression patterns of stage 37 Xenopus embryos following neural crest ablation and in situ hybridization (ISH) for xId2. (A, B) Transverse sections of a stage 37 control embryonic heart immunostained with the myocardial marker MF20. White arrows indicate (MF20 negative) undifferentiated splanchnic mesoderm cells continuous with the newly formed cardiac myocardial cells expressing myosin heavy chain (MF20 positive). (C, D) Sections through a stage 37 control embryo following ISH for xId2. (C) There is Id2 expression in the branchial arches (BA), and in a more caudal section (D) there is expression in the splachnic mesoderm (red arrows), myocardium of the outflow tract (green arrow), and the branchial arches (white arrow). Note that the expression in the splachnic mesoderm is continuous with the distal outflow tract myocardium. There is a visible pharynx (P), pericardium (Pc), myocardium (m), endocardium (e), aortic sac (as, black arrow), and truncus arteriosus (ta). [See Mohun et al. (2000) for reference to Xenopus heart morphology.] (E, F) Sections through a stage 37 embryo following stage 15 neural crest ablation and ISH for xId2. There is a lack of xId2 expression in both the splachnic mesoderm (red arrowheads) and myocardium of the outflow tract (green arrowheads). Morphologically, there is pericardial edema (PcE), an enlarged myocardium (m), and an irregular pericardial layer (pc). (E) The heart that formed in this ablated embryo is an unlooped linear heart tube.