Burgess Shawn, Reim Gerlinde, Chen Wenbiao, Hopkins Nancy, Brand Michael
Massachusetts Institute of Technology, Center for Cancer Research, E17-340, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Development. 2002 Feb;129(4):905-16. doi: 10.1242/dev.129.4.905.
In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.
在胚胎发育早期,沿前后轴大脑被分为三个主要区域:前脑、中脑和后脑。通过斑马鱼中的逆转录病毒插入诱变和化学诱变实验,我们分离出了导致后脑组织异常和中脑 - 后脑边界(MHB)无法形成的突变,MHB区域是相邻脑区的组织者。这些突变不能互补spiel-ohne-grenzen(spg)突变,spg突变会导致类似的表型,但受影响的基因未知。我们通过遗传定位、原病毒插入位点克隆和等位基因测序表明,spg突变破坏了pou2,pou2是一个编码Pou2转录因子的基因。基于染色体同源性、系统发育序列比较以及表达和功能数据,我们认为pou2是小鼠Oct3/Oct4和人类POU5F1在斑马鱼中的直系同源基因。对于哺乳动物基因,迄今为止尚未描述其在脑发育中的功能。在缺乏功能性pou2的情况下,中脑、MHB和后脑原基的标志物(pax2.1、wnt1、krox20)的表达严重降低,这与pou2的神经外胚层特异性表达阶段相关。向spg突变胚胎中注射pou2 mRNA可恢复这些缺陷,但不会异位激活这些标志物,这表明pou2具有许可作用。低浓度注射pou2 - 吗啉代寡核苷酸可模拟spg表型,进一步证明spg编码pou2。两项观察结果表明pou2还有一个更早的功能:更高浓度的pou2 - 吗啉代寡核苷酸会特异性导致原肠胚形成前细胞分裂和形态发生的停滞,并且在此阶段spg纯合胚胎中pou2 mRNA自身的表达会降低。这些实验表明pou2有两个作用。最初,Pou2在卵裂球的早期增殖和形态发生过程中发挥作用,类似于哺乳动物在形成内细胞团期间的Oct3/4。在斑马鱼脑形成过程中,Pou2随后第二次发挥作用,激活中脑和后脑原基中的基因表达,这在后期表现为spg胚胎中MHB的特异性缺失以及中脑和后脑的相关缺陷。