Mammalian Developmental Genetics And Animal Models Of Hu

哺乳动物分子遗传学(SMMG)部分的重点是胚胎发育的分子遗传学。转录调控是发育调控的关键因素，涉及一系列复杂的顺式调控靶基因序列，以及多种调控基因表达的因素的协同作用和物理作用。核心元件存在于许多细胞环境中，被认为与细胞或组织特异性因子形成复合体，以建立对基因表达的正向和负向控制，并在发育中的胚胎中产生细胞规格和组织特性。我们研究了这一过程的分子遗传学。我们的大部分工作涉及在小鼠发育过程中对LIM类同源框基因(称为LHX基因)成员的功能评估。在这些实验过程中，我们鉴定了两类新的蛋白质，分别由LDB和SSDP基因家族编码。这些都是辅助因子，它们与LHX编码的LIM同源结构域因子以及其他转录调节因子的相互作用对胚胎发育至关重要。我们研究的其他主题是Wnt抑制剂DKK家族的成员，该家族形成了lhx基因的作用和发育中的中枢神经系统模式形成之间的联系。 密切相关的Lhx1和Lhx5基因是我们正在进行的工作的主题。这两个基因在发育中的中枢神经系统的许多区域都有显著的表达，包括脊髓和小脑。早期的工作表明，Lhx1缺失胚胎缺乏前头部结构，Lhx5缺失突变体在海马区发育中受到损害。Lhx1缺失的胚胎在发育的早期就会死亡，这排除了对可能的大脑缺陷的彻底分析。我们还担心Lhx1和Lhx5之间可能存在功能冗余。因此，我们产生了条件性Lhx1突变体和Lhx1/5双突变体，以便能够更密切地研究这两个基因在脑发育中的单独或联合作用。分析的重点是这些突变体中发育中的小脑。缺乏Lhx1或Lhx5的胚胎的小脑表型并不显著。然而，对Lhx1/5双突变体的检测显示，原始小脑的浦肯野细胞大部分缺失，其定义是缺少Calbindin标记。我们的实验表明，Lhx1和Lhx5对于Calbindin阳性浦肯野细胞的产生是必不可少的，并且在这一功能方面存在冗余。 我们目前工作的另一个方面涉及LHX2在脑下垂体发育过程中的功能。我们之前的研究已经确定了该基因在大脑和眼睛发育以及造血中的作用。空胚胎是无眼症，因为在视杯形成之前，眼球血管发生了发育停滞。此外，前脑细胞增殖不足导致新皮质发育不良和海马神经原发育不全。LHX2缺陷突变体在子宫中死亡，可能是因为最终的红细胞生成的细胞非自主缺陷导致了严重的贫血。这里我们描述一个额外的表型。LHX2基因缺失会损害脑下垂体后叶的形成。我们观察到该组织中的前体细胞过度增殖和缺乏适当的分化。LHX2功能的丧失也会导致垂体腺的前叶和中间叶的紊乱，可能继发于后叶的形成缺陷。我们早期的工作已经确定Lhx3和Lhx4是垂体发育的重要调节因子。目前的工作表明，LHX2在这一过程中也起着重要的作用。 Wnt抑制剂DKK家族的成员在发育中的胚胎中广泛表达。我们之前曾报道过Dkk1，它是头部诱导过程中lhx和其他转录活动的中介。严重的吻部缺陷是Dkk1功能性消融的结果。我们最近的工作涉及Dkk2基因敲除小鼠的表型。这些突变体的特征是角膜细胞周转严重缺陷，表明Wnt通路调节对于维持眼表面完整性是必不可少的。角膜是一种非常有序的透明结构，其上皮每隔三到四周就会被位于角膜缘的干细胞衍生物取代，角膜缘是角膜和毗邻的结膜上皮之间的过渡区。年轻成年Dkk2基因缺失小鼠的角膜是不透明的，含有通常在皮肤和结膜中发现的细胞，包括毛囊、皮脂腺和杯状细胞。WNT信号在突变的角膜区上调。因此，Dkk2被认为是一种Wnt抑制因子，可以调节角膜上皮细胞的更新。

The Section on Mammalian Molecular Genetics (SMMG) is focused on the molecular genetics of embryonic development. Transcriptional control is a key element of developmental regulation, involving an elaborate repertoire of cis-regulatory target gene sequences as well as the cooperation and physical interaction of multiple factors that regulate gene expression. Core elements, present in many cell contexts, are thought to form complexes with cell- or tissue-specific factors to establish positive and negative control of gene expression and to bring about cell specification and tissue identity in the developing embryo. We study the molecular genetics of this process. Much of our work deals with the functional evaluation of members of the LIM class of homeobox genes (termed Lhx genes) during mouse development. During the course of these experiments, we identified two novel classes of proteins, encoded by the Ldb and Ssdp gene families, respectively. These are co-factors whose interaction with the Lhx-encoded LIM-homeodomain factors and with other transcriptional regulators is essential for embryonic development. Additional topics of our studies are members of the Dkk family of Wnt inhibitors that form a link between the action of Lhx genes and pattern formation in the developing central nervous system. The closely related Lhx1 and Lhx5 genes are topics of our ongoing work. The two genes are prominently expressed in many regions of the developing central nervous system, including the spinal cord and the cerebellum. Earlier work had shown that Lhx1 null embryos lack anterior head structures and that Lhx5 null mutants are impaired in hippocampal development. Lhx1 null embryos die at an early stage of development, precluding a thorough analysis of possible brain defects. We were also concerned that functional redundancies may exist between Lhx1 and Lhx5. We therefore generated conditional Lhx1 mutants as well as Lhx1/5 double mutants to be able to examine more closely the individual or combined roles of both genes in brain development. The analysis is focused on the developing cerebellum in these mutants. The cerebellar phenotype of embryos that lack either Lhx1 or Lhx5 is not remarkable. However, the examination of Lhx1/5 double mutants revealed that the Purkinje cells of the primordial cerebellum are largely missing, as defined by the absence of the Calbindin marker. Our experiment shows that both Lhx1 and Lhx5 are essential for the generation of Calbindin-positive Purkinje cells and that there is redundancy in this functional aspect. Another aspect of our current work concerns functions of Lhx2 in the context of the development of the pituitary gland. Our previous studies had established a role for this gene in brain and eye development and in hematopoiesis. Null embryos were anophthalmic because of a developmental arrest of the eye anlagen prior to the formation of the optic cup. In addition, deficient cell proliferation in the forebrain resulted in hypoplasia of the neocortex and aplasia of the hippocampal anlagen. The Lhx2-deficient mutants died in utero, possibly because of a cell non-autonomous defect of definitive erythropoiesis that caused severe anemia.Here we describe an additional phenotype. Deletion of Lhx2 impairs formation of the posterior lobe of the pituitary gland. We observe over-proliferation and a lack of proper differentiation of precursor cells in this tissue. Loss of Lhx2 function also leads to a disorganization of the anterior and intermediate lobes of the pituitary gland, possibly secondary to defects in the formation of the posterior pituitary. Our earlier work had identified Lhx3 and Lhx4 as essential regulators of pituitary development. The present work shows that Lhx2 plays an important role in the process as well. Members of the Dkk family of Wnt inhibitors are widely expressed in the developing embryo. We previously reported on Dkk1, a mediator of Lhx and other transcriptional activity during head induction. Severe rostral defects are the result of Dkk1 functional ablation. Our recent work has dealt with the phenotype of Dkk2 knockout mice. These mutants are characterized by a profound defect in cornea cell turnover, indicating that Wnt pathway regulation is essential for the maintenance of ocular surface integrity. The cornea is a very ordered, transparent structure whose epithelium is replaced every three or four weeks by derivatives of stem cells that reside in the limbus, a transitional zone between the corneal and the abutting conjunctival epithelium. The cornea of young adult Dkk2 null mice is opaque and contains cells normally found in skin and conjunctiva, including hair follicles, sebaceous glands and goblet cells. Wnt signals are upregulated in the mutant limbus region. Dkk2 has therefore been identified as a Wnt inhibitor that regulates the turnover of corneal epithelia.