呼吸中枢发育的基因调控研究进展

脑干呼吸中枢组成一个复杂的网络系统,产生和调控节律性呼吸。近年来,人们利用分子生物学技术研究发现,小鼠某些基因突变可影响呼吸中枢特定神经元的发育,并由此导致特殊的呼吸表型。最近,在人类某些中枢性呼吸疾病中,也发现了相应的基因突变。因此,基因水平的研究为深入认识节律性呼吸活动的产生和调控机制提供了新的研究途径。本文就目前研究较多、相对较深入的一些呼吸中枢发育调节基因的研究进展作一综述。     

Transferring genes into cultured mammalian embryos by electroporation

Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.     

Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes.     

Impaired adult myeloid progenitor CMP and GMP cell function in conditional c-myb-knockout mice

The differentiation of myeloid progenitors to mature, terminally differentiated cells is a highly regulated process. Here, we showed that conditional disruption of the c-myb proto-oncogene in adult mice resulted in dramatic reductions in CMP, GMP and MEP myeloid progenitors, leading to a reduction of neutrophils, basophils, monocytes and platelets in peripheral blood. In addition, c-myb plays a critical role at multiple stages of myeloid development, from multipotent CMP and bipotent GMP to unipotent CFU-G and CFU-M progenitor cells. c-myb controls the differentiation of these cells and is required for the proper commitment, maturation and normal differentiation of CMPs and GMPs. Specifically, c-myb regulates the precise commitment to the megakaryocytic and granulo-monocytic pathways and governs the granulocytic-monocytic lineage choice. c-myb is also required for the commitment along the granulocytic pathway for early myeloid progenitor cells and for the maturation of committed precursor cells along this pathway. On the other hand, disruption of the c-myb gene favors the commitment to the monocytic lineage, although monocytic development was abnormal with cells appearing more mature with atypical CD41 surface markers. These results demonstrate that c-myb plays a pivotal role in the regulation of multiple stages in adult myelogenesis.     

Expression of various genes is controlled by DNA methylation during mammalian development

Despite thousands of articles about 5-methylcytosine (m(5)C) residues in vertebrate DNA, there is still controversy concerning the role of genomic m(5)C in normal vertebrate development. Inverse correlations between expression and methylation are seen for many gene regulatory regions [Heard et al., 1997; Attwood et al., 2002; Plass and Soloway, 2002] although much vertebrate DNA methylation is in repeated sequences [Ehrlich et al., 1982]. At the heart of this debate is whether vertebrate DNA methylation has mainly a protective role in limiting expression of foreign DNA elements and endogenous transposons [Walsh and Bestor, 1999] or also is important in the regulation of the expression of diverse vertebrate genes involved in differentiation [Attwood et al., 2002]. Enough thorough studies have now been reported to show that many tissue- or development-specific changes in methylation at vertebrate promoters, enhancers, or insulators regulate expression and are not simply inconsequential byproducts of expression differences. One line of evidence comes from mutants with inherited alterations in genes encoding DNA methyltransferases and from rodents or humans with somatically acquired changes in DNA methylation that illustrate the disease-producing effects of abnormal methylation. Another type of evidence derives from studies of in vivo correlations between tissue-specific changes in DNA methylation and gene expression coupled with experiments demonstrating cause-and-effect associations between DNA hyper- or hypomethylation and gene expression. In this review, I summarize some of the strong evidence from both types of studies. Taken together, these studies demonstrate that DNA methylation in mammals modulates expression of many genes during development, causing major changes in or important fine-tuning of expression. Also, I discuss previously established and newly hypothesized mechanisms for this epigenetic control.     

Expression levels of heat shock factors are not functionally coupled to the rate of expression of heat shock genes

The expression patterns of two mammalian heat shock factors (HSFs) were analysed in cell systems known to reflect an altered heat shock response. For being able to discriminate between the two closely related factors HSF 1 and HSF 2, specific cDNA sequences were cloned and used to generate antisense RNAs as hybridization probes. In general, in various cell lines expression of the two heat shock factors was clearly different. These expression patterns of the HSF genes were not influenced by retinoic acid-induced differentiation of human NT2 and mouse F9 teratocarcinoma cells. Generally, HSF 2 expression was extremely low, whereas the significantly higher expression of HSF 1 revealed cell specific differences. The highest expression rates of both HSFs were observed in 293 cells. To examine whether these high levels are involved in the constitutive expression of heat shock genes in these cells, we analysed the binding pattern of 293 cell proteins to the heat shock elements (HSEs). As with other cells, HSE-binding activity in 293 cells was only observed after heat shock treatment. This points to an HSE-independent way for high level expression of heat shock genes in these cells.