Comparative Genetic Studies of Limb Development in Bats and Mice

[0220458 . behringer]该项目的主要目标是了解哺乳动物物种之间形态差异背后的遗传差异。Behringer博士假设，这些遗传差异中有许多存在于调节发育控制基因转录的DNA序列中。他选择专注于脊椎动物肢体发育的经典发育生物学范式。他建议对老鼠和蝙蝠肢体形态发生的功能进行比较，因为与老鼠相比，蝙蝠的肢体有很大的变化。他提议研究的发育控制基因Prx1编码一对同源结构域转录因子，这是小鼠四肢和颅面发育所必需的。他定位了一个600 bp的DNA片段，编码一个转录增强子，该转录增强子在小鼠发育过程中以prx1特异性模式指导肢体表达。他已经从我们生成的蝙蝠（Carollia perspicillata）文库中分离出Prx1同源物。他正在通过全载和RNA原位杂交来表征该基因在蝙蝠胚胎中的表达模式。他利用蝙蝠Prx1基因保守的DNA序列和相对于小鼠和人类同源基因的基因组位置，确定了假定的肢体增强子。他将在转基因小鼠中使用lacZ报告试验来测试这种假定的肢体增强子的活性。然后，他将通过在小鼠胚胎干（ES）细胞中进行基因靶向，将小鼠Prx1基因的内源性肢体增强子替换为蝙蝠Prx1肢体增强子。因此，在蝙蝠Prx1肢体增强子的控制下，小鼠Prx1基因的表达将产生小鼠。他将评估这种调节元件在肢体中改变的形态学后果。他提出，这种高度集中的体内测试将为形态学差异的遗传基础提供重要的见解。此外，他的研究将进一步加深我们对调控多样性如何调节器官发生的理解。

0220458BehringerThe primary goal of this project is to understand the genetic differences that underlie morphological variation between mammalian species. Dr. Behringer hypothesizes that many of these genetic differences reside in DNA sequences that regulate the transcription of developmental control genes. He has chosen to focus upon the classic developmental biology paradigm of vertebrate limb development. He proposes to make functional comparisons between mouse and bat limb morphogenesis because compared to mouse, bats have highly modified limbs. The developmental control gene that he proposes to study, Prx1, encodes a paired homeodomain transcription factor that is required in mice for limb and craniofacial development. He has localized a discreet 600 bp DNA segment encoding a transcriptional enhancer that directs limb expression in a Prx1-specific pattern during mouse development. He has isolated the Prx1 orthologue from bat (Carollia perspicillata) libraries that we have generated. He is characterizing the expression pattern of this gene in bat embryos by whole-mount and section RNA in situ hybridization. He has identified the putative limb enhancer of the bat Prx1 gene by virtue of its conserved DNA sequence and genomic position relative to mouse and human orthologues. He will test the activity of this putative limb enhancer using a lacZ reporter assay in transgenic mice. He will then replace the endogenous limb enhancer of the mouse Prx1 gene with the bat Prx1 limb enhancer by gene targeting in mouse embryonic stem (ES) cells. Thus, mice will be generated where the mouse Prx1 gene is expressed under the control of the bat Prx1 limb enhancer. He will assess the morphological consequences of this regulatory element alteration in the limbs. He proposes that this type of highly focused in vivo test will provide important insights into the genetic basis of morphological divergence. In addition, his studies should further our understanding of how regulatory diversity can modulate organogenesis.