Integrative biology and the developing limb bud

The identification or selective construction of mutations within genes has allowed researchers to explore the downstream effects of gene disruption. Although these approaches have been successful, a limitation in our assessment of the consequences of conditional changes, and thereby our understanding of roles or function of genes, limits the degree to which we examine the effects of our manipulations. It is also clear that linear associations are incorrect models for describing development, and newer methods now give us an opportunity to practice an integrative biology. In our attempts to explore the consequences of Hoxa13 disruption in mice and humans, it has become clear that a better understanding of the consequences of gene alteration may be achievable by taking a broader approach with a long-term view. Fundamental questions regarding Hox gene function in vertebrates, including those related to the number of target genes; the degree of overlap of target gene regulation among paralogs; the magnitude of modulation exerted; and the identity of genes that are activated versus repressed need to be explored if a more thorough mechanistic understanding is to be achieved. To begin to address these questions, we undertook a comprehensive analysis of the expression of genes within developing limb buds of mice, and here we present some of our preliminary results. Our efforts will further (1) the exploration of the broader genetic relationships of expressed genes, (2) the determination of parallels or variations in target usage for a given gene in different tissues and between different organisms, (3) the evaluation of limb patterning mechanisms in other animal model systems, and (4) the exploration of gene expression hierarchies regulated by HOX proteins in developmental systems.     

Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5′-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.