Expression ofHoxDGenes in Developing and Regenerating Axolotl Limbs

Hoxgenes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior–posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns ofHoxDgenes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary ofHoxd-11expression is conserved with respect to morphological landmarks, but there is no anterior–distal expansion of the posterior domain ofHoxd-11expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5′HoxDgenes are reexpressed during limb regeneration. The change in the expression pattern ofHoxd-11during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. BothHoxd-8andHoxd-10are expressed in non-regenerating wounds, butHoxd-11is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.     

Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb

Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression.     

Alarm reaction and alert state inGambusia Affinis (Pisces,Poeciliidae) in response to chemical stimuli from injured conspecifics

We studied the behavior of the Poeciliid fishGambusia affinis after the introduction of 3 substances into their tank: a homogenization ofGambusia affinis, a homogenization of the Anabantid fishBetta splendens, and a blank made of distilled water. The response of the fish was measured as a change in their spatial distribution in the tank after the introduction of the substance. Two sizes of fish were used, and theGambusia homogenization produced clear alarm reactions in both, the fish fleeing to the bottom of the tank. This is one of a few examples available of recognition of alarm substances in non-ostariophysian fish. In addition, we found that the small fishes that had recently been exposed to the alarm substance stayed in an ‘alert state’, in which they had an increased sensitivity to mechanical and visual fright stimuli.     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.     

Physical Characterization of the Chromosomal Rearrangements That Accompany the Transgene Insertion in thechakragatiMouse Mutant

The circling phenotype of thechakragatimouse is a result of a transgenic insertional mutation. The absence of the phenotype in mice heterozygous for the transgene insertion suggests that this is due to a loss of function of an endogenous gene. Efforts to identify this gene have led to a previous report that sequences flanking the transgene,D16Ros1andD16Ros2,map 10 cM apart in wildtype mice. We present here physical mapping data indicating that the proximity ofD16Ros1andD16Ros2in theckrmouse is explained by a duplication ofD16Ros2and its insertion along with the transgene atD16Ros1.We further demonstrate thatD16Ros1sequences are also duplicated and that this duplication is also part of the insertion at the endogenousD16Ros1locus.     

The mouse CD7 gene: identification of a new element common to the human CD7 and mouse Thy-1 promoters

HumanCD7 (CD7) is a 40000M _r member of the immunoglobulin gene superfamily that is expressed early in natural killer (NK) and T-lymphocyte development.CD7 is involved in lymphocyte activation, as ligation ofCD7 activates NK and TCRγδ T lymphocytes, and ligation ofCD7 on TCRαβ T lymphocytes induces a non-mitogenic calcium flux. We have previously cloned and characterized the gene for humanCD7 (hCDT) and have described its expression in transgenic mice. Recently a mouse cDNA homologous tohCD7 was reported, which we mapped to the corresponding mouse chromosomal location ashCD7. We now report the identification and characterization of a mouseCD7 (mCDT) genomic clone. We demonstrated that themCD7 gene was similar both in size and structural organization tohCD7. Comparison of the 5′ flanking sequences of themCD7 andhCD7 genes revealed two regions of sequence similarity. Electrophoretic mobility shift assay confirmed both of these regions to be sites of tissue-restricted protein binding in vitro. The more 3′ similarity region also shared sequence with a region in the mouseThy-1 gene 5′ flanking region, suggesting that this sequence may be a cis-acting regulatory element common to all three genes. Thus, the promoter regions and exonic organization were similar in the humanCDT, mouseCDT, and mouseThy-1 genes. The nucleotide sequence data reported in thts paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U23462     

利用Tol2转座子介导的增强子诱捕技术获得血管相关转基因斑马鱼系

心血管系统形成于胚胎发育极早期并为其他器官的发育、维持、修复所必需,血管生长异常可造成多种疾病.然而,由于研究对象所限,胚胎血管的发育机制尚未完全阐明,调控血管发育的基因也所知有限.通过Tol2转座子介导的大规模增强子诱捕筛选到26个血管特异表达绿色荧光蛋白(EGFP)报告基因的转基因斑马鱼系,其中有一些品系在胚胎的某些特异血管结构中表达绿色荧光.通过linker-mediated PCR克隆到22个鱼系中Tol2插入位点附近的斑马鱼基因组序列,其中有17个鱼系的Tol2插入可定位到现有的斑马鱼基因组中的单一位点.通过整体胚胎原位杂交对插入位点附近的基因进行表达谱分析,得到8个表达谱与转基因鱼系一致的基因,涵盖了9个鱼系,其中dusp5基因对应于2个不同的鱼系.这8个基因中包括hhex、ets1a和dusp5等3个功能已知的基因,但是大部分(5个)基因在斑马鱼中尚无功能研究,分别为zvsg1、micall2a、arl8b(1of2)、zgc:73355以及hecw2(1of2).hhex和ets1a基因对血管与血细胞前体的发育具有重要作用,所获得的EGFP报告基因受hhex或ets1a基因增强子控制的转基因斑马鱼(mp378b和mp430c-2)为国际首例,为深入研究这两个基因在血管与血液发育中的作用机制提供了新的机遇.筛选到的功能未知基因可以用来进一步研究其在血管发育中的功能;同时,利用所获得的转基因鱼系,可以实现实时、动态观察成血管细胞的起源、分化与基因表达调控,并可用于高通量小分子药物筛选等重要研究.     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Mice transgenic for simian immunodeficiency virusnef are immunologically compromised

An intactnef gene is essential for rapid development of immunodeficiency in human immunodeficiency virus and simian immunodeficiency virus infections. To assess the role ofnef in the immune response, mice transgenic for SIVnef were constructed and the humoral and cellular immune response to herpes simplex virus type-1 (HSV-1), measured. Mice transgenic for SIV-mac239nef exhibited a significantly increased mortality rate when challenged with HSV-1 and also showed unusual antibody kinetics in response to viral challenge. During a 32-week period following exposure to HSV, it was noted that IgG subclass titers continued to rise in thenef+ animals, while titers ofnef– animals decreased. Additionally, following secondary challenge with HSV,nef– mice had a significantly greater rise in HSV-neutralizing antibody titers thannef+ mice. A decreased proliferative response to the T cell mitogen, PHA, was noted in thenef+ animals. These results suggest that the presence ofnef+ is sufficient to induce immune dysfunction.     

Conservation of a functional hierarchy between mammalian and insect Hox/HOM genes.

We have generated several transgenic Drosophila strains containing different mouse Hox genes under heat shock control and studied how their generalized expression affects Drosophila larval patterns. We find that they have spatially restricted effects which correlate with their genetic order and expression pattern in the mouse; as they are expressed more posteriorly in the mouse, they have more extensive effects in Drosophila. The generalized expressions of Hoxd-8 and d-9 modify Drosophila anterior head segment(s), but have no effect in the rest of the body. Hoxd-10 expression affects head and thorax, but not the abdomen. Finally, Hoxd-11 alters head, thorax not the abdomen. Finally, Hoxd-11 alters head, thorax and abdomen. The developmental effect of the Hox genes consists of a homeotic transformation of the affected segment(s), which exhibit a 'ground' pattern similar to that obtained in the absence of homeotic information, suggesting that Hox genes are able to inactivate Drosophila homeotic genes, but do not specify a pattern of their own. A partial exception is Hoxd-11 which, even though it has a general suppressing effect, can also activate the resident Abdominal-B and empty spiracles genes in ectopic positions. Our results strongly suggest a general conservation of the functional hierarchy of homeotic genes that correlates with genetic order and expression patterns.     

Tissue-specific expression of theHLA-DRA gene in transgenic mice

Transgenic mice were produced containing a 33 kilobase (kb) DNA fragment encompassing the five exons and all the known regulatory regions of the class IIHLA-DRA gene. The transgene displayed regulated expression [constitutive and interferon- (IFN)- induced] of the human products in most mouse tissues. The tissue distribution of theDRA transgene products more closely resembled that of their mouse homologues, the endogenous H-2 Ea products, than the wider distribution ofDRA products in humans. This was evident in several tissues (endothelia of small vessels, especially those of glomerular capillaries, Kupffer cells, and epithelial cells lining the gastrointestinal tract), known to differentially express class II molecules in the two species. Thus, the wider human specific pattern of expression requires an exact cis/trans complementation which is incompletely reconstituted in transgenic mice, suggesting that human specific cis-acting elements may have arisen during evolution to direct the expression of class II genes to those anatomical regions which usually lack them in the mouse. The only example of aberrant expression of theDRA gene in the present series of transgenic mice was in the dendritic and/or epithelial cells of the thymic cortex, which displayed greately reducedDRA levels in spite of a normal expression of the endogenous Ea molecules.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Spatial and temporal regulation of alacZ reporter transgene in a binary transgenic mouse system

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrneet al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using theEscherichia coli -galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for -galactosidase activity. Transactivation, as demonstrated by strong -galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern oflacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.