CLONAL ANALYSIS OF CARDIAC MORPHOGENESIS IN THE CHICKEN-EMBRYO USING A REPLICATION-DEFECTIVE RETROVIRUS .1. FORMATION OF THE VENTRICULAR MYOCARDIUM
CLONAL ANALYSIS OF CARDIAC MORPHOGENESIS IN THE CHICKEN-EMBRYO USING A REPLICATION-DEFECTIVE RETROVIRUS .1. FORMATION OF THE VENTRICULAR MYOCARDIUM
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DOI：
10.1002/aja.1001930104
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发表时间：
1992-01-01
影响因子：
2.5
通讯作者：
FISCHMAN, DA
中科院分区：
文献类型：
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作者：
MIKAWA, T;BORISOV, A;FISCHMAN, DA
心前中胚层细胞（4 - 6期）和早期心脏的分裂心肌细胞（10 - 15期）用一种无复制能力的逆转录病毒（CXL）（Mikawa等人，1991b）进行标记，该病毒编码细菌β - 半乳糖苷酶（β - gal）。使用了两种方案来感染生心细胞。
（1）从原肠胚期胚胎（4 - 6期）解剖出前外侧中胚层的小块组织（约50μm²），并在含有逆转录病毒的液体培养基中孵育。去除CXL后，将组织分散成单细胞悬液，然后加压注射到受体胚胎（4 - 6期）的心前区域。这些胚胎随后在37℃下体外孵育2天（New，1968），有心跳的胚胎用X - gal组织化学法固定并进行石蜡连续切片。
（2）在卵内（胚胎4 - 15期）将CXL加压注射到生心组织中，然后将卵放回孵化器。在发育的选定阶段，将胚胎或整个心脏固定，用X - gal染色，并在石蜡包埋后进行连续切片。
第一种方法表明：（1）心前中胚层细胞可被逆转录病毒感染；（2）移植的细胞会分化为有跳动的心肌细胞；（3）β - gal的表达足够高，可通过组织化学方法检测到。通过第二种方法我们可以表明：（1）β - gal标记的细胞在心肌中形成集落；（2）标记的细胞完全是心肌细胞；（3）每个集落的细胞数量随着胚胎发育年龄的增加而增加；（4）左心室的集落比右心室的大；（5）许多集落是透壁的，即它们从心肌的心外膜层延伸到心内膜层，并且通常呈圆锥体或漏斗形，圆锥体的底部最靠近心外膜；（6）每个集落内的心肌细胞在心肌的不同层的方向不同；（7）圆锥体中同时包含β - gal⁺和β - gal⁻心肌细胞。用[³H] - 胸腺嘧啶核苷进行的DNA标记研究表明，在发育的第一周，生心细胞每16 - 18小时分裂一次，并且CXL标记的细胞与未标记的心肌细胞分裂情况无法区分。基于这些观察结果，提出了一个心肌生长的模型。
Cells of the precardiac mesoderm (stages 4-6) and dividing myocytes of early hearts (stages 10-15) were tagged with a replication-incompetent retrovirus (CXL) (Mikawa et al., 1991b) encoding bacterial beta-galactosidase (beta-gal). Two protocols were used to infect the cardiogenic cells. (1) Small blocks (approximately 50-mu-m2) of anterolateral mesoderm were dissected from gastrula-stage embryos (stages 4-6) and incubated in liquid medium containing the retrovirus. After removal of CXL, the tissues were dispersed into single-cell suspensions and pressure injected into the precardiac areas of recipient embryos (stages 4-6). Such embryos were then incubated in vitro at 37-degrees-C for 2 days (New, 1968), and those embryos with beating hearts were fixed for X-gal histochemistry and paraffin serial sectioning. (2) CXL was pressure injected in ovo (embryonic stages 4-15) into cardiogenic tissues and the eggs subsequently returned to an incubator. At selected stages of development embryos or whole hearts were fixed, stained with X-gal, and serially sectioned after paraffin embedding. The first method showed that (1) cells of the precardiac mesoderm could be infected with the retrovirus, (2) the transplanted cells would differentiate into beating myocytes, and (3) beta-gal expression was sufficiently high to be detected histochemically. With the second procedure we could show that (1) beta-gal-tagged cells formed colonies in the myocardium, (2) the labeled cells were exclusively myocytes, (3) the number of cells per colony increased with increasing age of embryonic development, (4) the size of colonies was larger in the left than the right ventricle, (5) many of the colonies were transmural, i.e., they extended from epicardial to endocardial layers of the myocardium and generally exhibited a cone or funnel-shape with the base of the cone nearest the epicardium, (6) the orientation of myocytes within each colony changed at different layers of the myocardium, and (7) the cones contained both beta-gal+ and beta-gal- myocytes. DNA labeling studies with [H-3]thymidine indicated that cardiogenic cells divided every 16-18 hr during the first week of development and that the CXL-labeled cells divided indistinguishably from unlabeled myocytes. Based on these observations a model for the growth of the myocardium is presented.