Zhou Hong-Ming, Wang Jian, Rogers Rhonda, Conway Simon J
Cardiovascular Development Group, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Dev Biol. 2008 Mar 15;315(2):369-82. doi: 10.1016/j.ydbio.2007.12.020. Epub 2007 Dec 27.
Pax3 is an essential paired- and homeodomain-containing transcription factor that is necessary for closure of the neural tube, and morphogenesis of the migratory neural crest and myoblast lineages. Homozygous loss-of-function mutation results in mid-gestational lethality with defects in myogenesis, neural tube closure and neural crest-derived lineages including melanocytes, Schwann cells and insufficient mesenchymal cells to septate the cardiac outflow tract. To address the function of Pax3 in later fetal stages and in specific adult tissues, we generated a floxed Pax3 allele (Pax3(flox)). An intermediate allele (Pax3(neo)) was produced via creation of the floxed allele, in which the TK-neo(R) cassette is present between exons 5 and 6. It was deduced to be a hypomorph, as Pax3 protein expression is reduced by 80% and homozygote hypomorphs die postnatally. To assess the consequences of reduced Pax3 levels on the various Pax3-expressing lineages and to determine the underlying cause of lethality, we examined Pax3 spatiotemporal expression and the resultant defects. Defective limb and tongue musculature were observed and lethality was due to an inability to suckle. However, the heart, diaphragm, trunk musculature, as well as the various neural crest-derived lineages and neural tube were all unaffected by reduced Pax3 levels. Significantly, elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic component. The limb and tongue myogenic defects were found to be due to a significant increase in apoptosis within the somites that leads to a paucity of migratory hypaxial myoblasts. These effects were attributed to the hypomorphic effect of the Pax3(neo) allele, as removal of the TK-neo(R) cassette completely relieves the hypomorphic effect, as 100% of the Pax3(flox/flox) mice were normal. These data demonstrate a lineage-specific response to approximately 80% loss of Pax3 protein expression, with myogenesis of limb and tongue being most sensitive to reduced Pax3 levels. Thus, we have established that there are different minimum threshold requirements for Pax3 within different Pax3-expressing lineages.
Pax3是一种重要的含配对结构域和同源结构域的转录因子，对于神经管闭合、迁移性神经嵴和成肌细胞谱系的形态发生至关重要。纯合功能丧失突变导致妊娠中期致死，并伴有肌生成、神经管闭合以及神经嵴衍生谱系（包括黑素细胞、施万细胞和分隔心脏流出道的间充质细胞不足）缺陷。为了研究Pax3在胎儿后期和特定成年组织中的功能，我们构建了一个floxed Pax3等位基因（Pax3(flox)）。通过构建floxed等位基因产生了一个中间等位基因（Pax3(neo)），其中TK-neo(R)盒位于外显子5和6之间。由于Pax3蛋白表达降低了80%且纯合子亚效等位基因小鼠在出生后死亡，因此推断它是一个亚效等位基因。为了评估Pax3水平降低对各种表达Pax3的谱系的影响，并确定致死的潜在原因，我们研究了Pax3的时空表达以及由此产生的缺陷。观察到肢体和舌部肌肉组织存在缺陷，致死原因是无法哺乳。然而，心脏、膈肌、躯干肌肉组织以及各种神经嵴衍生谱系和神经管均未受Pax3水平降低的影响显著的是，在未受影响的神经管和轴旁体成分中，相关的Pax7蛋白水平升高。发现肢体和舌部的肌生成缺陷是由于体节内凋亡显著增加，导致迁移性轴下成肌细胞数量减少。这些影响归因于Pax3(neo)等位基因的亚效作用，因为去除TK-neo(R)盒完全消除了亚效作用，因为100%的Pax3(flox/flox)小鼠是正常的。这些数据表明，对于Pax3蛋白表达约80%的缺失存在谱系特异性反应，肢体和舌部的肌生成对Pax3水平降低最为敏感。因此，我们已经确定在不同的表达Pax3的谱系中，对Pax3有不同的最低阈值要求。
