Rax is a selector gene for mediobasal hypothalamic cell types.

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

Pilus-mediated epithelial cell death in response to infection with Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen that can cause serious infections in cystic fibrosis (CF) patients. The ET12 lineage appears particularly virulent in CF; however, its pathogenesis is poorly understood and may be associated with host response. To help characterize this response, the ability of B. cenocepacia to induce cytotoxicity and apoptosis in an epithelial cell model was examined. Upon infection with B. cenocepacia strain K56-2, A549 human lung epithelial cells underwent significant cell death; propidium iodine staining and DNA fragmentation assays suggested apoptosis. Initiation of cell death was independent of the type III secretion system, biofilm formation, and secreted bacterial cytotoxins. However, the frequency of cell death was lower in cells infected with a non-piliated mutant, K56-2 cblA::Tp. Furthermore, purified cbl pili were found to directly induce cytotoxicity in A549 cells and activate caspase-9, -8, -7, and -3, the major cysteine proteinases involved in apoptosis. It appears that B. cenocepacia cbl pili, which are a distinctive feature of the ET12 lineage, act as an initiator of cytotoxicity and apoptosis. Understanding the role of cbl pili in the pathogenesis of B. cenocepacia infections offers the potential for decreasing the virulence of these potentially life-threatening organisms in CF patients.

[Y chromosome variations and hyplogroups from 15 biallelic markers in six Chinese populations].

By using 15 biallelic markers, 342 male individuals from six populations in China were genotyped with ASPCR (allele specific PCR). The 15 biallelic markers included M1 (YAP), M15 (9 bp insertion), M89 (C-->T), M9 (C-->G), M119 (A-->C), M50 (T-->C), M110 (T-->C), M103 (C-->T), M95 (C-->T), M88 (A-->G), M111 (2-bp deletion), M45 (G-->A), M122 (T-->C), M7 (C-->G) and M134 (1 bp deletion). The distribution of variation frequencies of 15 biallelic markers in six populations showed that with the extremely high frequencies of M9G (96.20% & 96.43%) and Han nationality displayed higher diversity than the four minority populations. It's noteworthy that M95T (82.14%) in Sichuan Han and M45A (18.57%) in Hui gave prominace to the two populations. The six populations displayed 34 (Fujian Han), 21 (Sichuan Han), 14 (Mongol), 26 (Hui), 10 (Xibo) and 8 (Hezhe) haplogroups respectively with 2, 1, 2, 1, 2 and 2 prominent haplogroups among them. Furthermore, the haplogroup analysis revealed that one predominant haplogroup was shared in the four minority populations and even two predominant haplogroups were shared in Mongol, Hezhe and Xibo. Unlike Han populations, the minority populations showed strikingly different haplogroups which were close to the ancestral pattern. However, the two Han populations exhibited divergence between them with the distinct frequencies of M89T and M95T. With the comparison of the number of people sharing the common haplogroups between any two of the four minority populations, relative genetic distance among them was deduced.

[Establishment and preservation of immortal lymphoblastoid cell lines of the 10 ethnic groups in China].

The immortal lymphoblastoid cell lines were established by EBV transformation of B cells and addition of cyclosporin A to inhibit the activity of T cells. In the present study,549 immortal cell lines of different ethnic groups of Hazak, Manchu, Korea, Hozhe, Mongolia, Sibe, Hui, Puyi, Han in Fujian and Han in Sichuan were established. Through our research,we found it is harmful for B lymphocytes to transform if excessful leucocytes are inoculated.And it is crisis that cyclosporine A shoud be added the last. Our work is an important part of the research of human genome diversity for the exploration of the origin and evolution of different ethnic groups,and it also provides enough research materials for further studies.Moreover,we have sent 50 cell lines of Hozhe, Mongolia, Sibe, Daur, Oroqen to CEPH. Thus it is possible for us to utilize the genetic resources of CEPH freely.

Efficient gene delivery into multiple CNS territories using in utero electroporation.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos(1,2) that we have successfully adapted(3,4). It is largely based on the success of in ovo electroporation technologies that are commonly used in chick(5). Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described(6). Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells. To date, in utero electroporation has been most widely used for the analysis of neocortical development(1,2,3,4). More recent studies have targeted the embryonic retina(7,8,9) and thalamus(10,11,12). Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3(rd) ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

A new haplogroup pattern displayed in Fujian Han in China.

Human Y-chromosomal binary polymorphisms have been considered to preserve the paternal genetic legacy and provide evidence on human evolution and the genetic relationships among and demographic history of different populations. To reveal the genetic origin and immigration of the Fujian Han, 13 binary markers on the Y chromosome were used to screen Fujian Han by allele-specific polymerase chain reaction. The results indicated that the M9G marker was highly prevalent (96.20%), suggesting a significant genetic drift. In addition, M122C frequency was only 22.78%, and M45A and M103T were default. The distinctive haplogroup frequencies (H1, H5, and H6/7/8) imply that the haplogroup pattern is a relatively ancestral and interim type.