Pem homeobox gene regulatory sequences that direct androgen-dependent developmentally regulated gene expression in different subregions of the epididymis

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.     

Primary structure and embryonic expression pattern of the mouse Hox-4.3 homeobox gene

We report the cloning, genomic localization, primary structure and developmental expression pattern of the novel mouse Hox-4.3 gene. This gene is located within the HOX-4(5) complex, at a position which classifies it as a member of the Hox-3.1 and -2.4 subfamily, the DNA and predicted protein sequences further confirmed this classification. Hox-4.3 has a primary structure characteristic of a Hox gene but, in addition, contains several monotonic stretches of amino acids, one of the 'paired'-like type. As expected from its presence and position within the complex. Hox-4.3 is developmentally expressed in structures of either mesodermal or neurectodermal origin located or derived from below a precise craniocaudal level. However, a very important offset between anteroposterior boundaries within neuroectoderm versus mesoderm derivatives is observed. Like other genes of the HOX-4(5) complex, Hox-4.3 is expressed in developing limbs and gonads, suggesting that 'cluster specificity' could be a feature of the HOX network.     

Identification and expression of zebrafish Iroquois homeobox gene irx1

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.     

Stage-specific expression of a developmentally regulated gene in Dirofilaria immitis.

A previously reported cDNA clone encoding 34 kDa antigenic polypeptide of Dirofilaria immitis (lambda cD34) was studied to elucidate the mechanism of stage-specific gene expression. The 34 kDa polypeptide was a larva-specific antigen and the mRNA was detectable in microfilariae but not in adult worms and eggs. The lambda cD34 gene was not sex linked and was contained in the genome of D. immitis at each stage. The stage-specific expression of the developmentally regulated gene in D. immitis may be controlled primarily at the mRNA level.     

Sequence and expression pattern of ziro7, a novel, divergent zebrafish iroquois homeobox gene.

We have isolated the zebrafish ziro7 gene, a novel, divergent member of the Iroquois family. ziro7 is expressed at early epiboly stages in the dorsal half of the zebrafish embryo, with a higher level in the dorso-lateral margin. From mid-gastrulation stages onward, ziro7 is expressed in a large transversal stripe in the future neural plate, which subsequently divides into thinner stripes located in the diencephalon, midbrain and hindbrain.     

Primary phloem-specific expression of a Zinnia elegans homeobox gene.

Some plant homeobox genes are expressed specifically in vascular cells and are assumed to function in the differentiation of specific types of vascular cells. However, homeobox genes exhibiting primary phloem-specific expression have not been reported. To elucidate the molecular mechanisms of vascular development, we undertook to isolate from Zinnia elegans primary phloem-specific homeobox genes that may function in phloem development. An HD-Zip type homeobox gene, ZeHB3, was isolated. This gene encodes a class I HD-Zip protein, and constitutes a gene subfamily with the Daucus carota gene CHB6, and Arabidopsis thaliana genes Athb-5, Athb-6, and Athb-16. In situ hybridization of 1-, 14- and 50-day-old plants demonstrated that ZeHB3 mRNA accumulation is restricted to a few cells destined to differentiate into phloem cells and to the immature phloem cells surrounding the sieve elements and companion cells. ZeHB3 protein was also localized to immature phloem cells. These findings clearly indicate that ZeHB3 is a novel homeobox gene that marks, and may function in, the early stages of phloem differentiation.     

Cloning and developmental expression of a zebrafish meis2 homeobox gene

We show here that a zebrafish meis2 gene homolog has a dynamic expression pattern in the developing mesoderm and central nervous system. Meis family homeodomain proteins are known to act as cofactors with other homeodomain proteins. We find expression of meis2.1 in the developing zebrafish hindbrain and somites, correlating with reported sites of zebrafish hox gene expression, as well as in presumptive cerebellum, midbrain, retina and ventral forebrain. The expression pattern shares some, but not all, features with that of murine Meis2.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

[This corrects the article on p. 1503 in vol. 5.].     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

Acetylcholine is a major excitatory neurotransmitter in the central nervous system of insects. Using DNA probes of the Torpedo nicotinic acetylcholine receptor (AChR) we have isolated two overlapping cDNA clones encoding a putative neuronal AChR protein from the fruitfly, Drosophila melanogaster. The predicted mature protein consists of 497 amino acids, has a calculated mol. wt of 57 340 and shows extensive homology to known AChR subunits from different species along its entire amino acid sequence. Northern analysis revealed a hybridizing mRNA of 3.2 kb in late embryo and in pupae. Expression of the corresponding AChR gene thus characterizes periods of neuronal differentiation in Drosophila.     

Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.