Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.     

Lens development depends on a pair of highly conserved Sox21 regulatory elements

Highly conserved non-coding elements (CNEs) linked to genes involved in embryonic development have been hypothesised to correspond to cis-regulatory modules due to their ability to induce tissue-specific expression patterns. However, attempts to prove their requirement for normal development or for the correct expression of the genes they are associated with have yielded conflicting results. Here, we show that CNEs at the vertebrate Sox21 locus are crucial for Sox21 expression in the embryonic lens and that loss of Sox21 function interferes with normal lens development. Using different expression assays in zebrafish we find that two CNEs linked to Sox21 in all vertebrates contain lens enhancers and that their removal from a reporter BAC abolishes lens expression. Furthermore inhibition of Sox21 function after the injection of a sox21b morpholino into zebrafish leads to defects in lens development. These findings identify a direct link between sequence conservation and genomic function of regulatory sequences. In addition to this we provide evidence that putative Sox binding sites in one of the CNEs are essential for induction of lens expression as well as enhancer function in the CNS. Our results show that CNEs identified in pufferfish-mammal whole-genome comparisons are crucial developmental enhancers and hence essential components of gene regulatory networks underlying vertebrate embryogenesis.     

Evolution of regulatory elements producing a conserved gene expression pattern in Caenorhabditis

Natural selection acts at the level of function, not at the logistical level of how organisms achieve a particular function. Consequently, significant DNA sequence and regulatory differences can achieve the same function, such as a particular gene expression pattern. To investigate how regulatory features underlying a conserved function can evolve, we compared the regulation of a conserved gene expression pattern in the related species Caenorhabditis elegans and C. briggsae. We find that both C. elegans and C. briggsae express the ovo-related zinc finger gene lin-48 in the same pattern in hindgut cells. However, the regulation of this gene by the Pax-2/5/8 protein EGL-38 differs in two important ways. First, specific differences in the regulatory sequences of lin-48 result in the presence of two redundant EGL-38 response elements in C. elegans, whereas the redundancy is absent in C. briggsae. Second, there is a single egl-38 gene in C. briggsae. In contrast, the gene is duplicated in C. elegans, with only one copy retaining the ability to regulate lin-48 in vivo. These results illustrate molecular changes that can occur despite maintenance of conserved gene function in different species.     

Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals

Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals, which are not discernible by sequence comparison among mammals.     

Sorting out Sox10 functions in neural crest development

For both vertebrate developmental and evolutionary biologists, and also for clinicians, the neural crest (NC) is a fundamental cell population. An understanding of Sox10 function in NC development is of particular significance since Sox10 mutations underlie several neurocristopathies. Surprisingly, experiments in different model organisms aimed at identifying Sox10's role(s) have suggested at least four distinct functions. Sox10 may be critical for formation of neural crest cells (NCCs), maintaining multipotency of crest cells, specification of derivative cell fates from these cells and their differentiation. Here, I discuss this controversy and argue that these functions are, in part, molecularly interrelated.     

Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis

Wingless (wg)/Wnt family genes encode secreted glycoproteins that function as signalling molecules in the development of vertebrates as well as invertebrates. In a survey of Wnt family genes in the newly sequenced Tribolium genome, we found a total of nine Wnt genes. In addition to wg or Wnt1, Tribolium contains orthologs of the vertebrate Wnt5-7 and Wnt9-11 genes. As in Drosophila, Wnt1, Wnt6 and Wnt10 are clustered in the genome. Comparative genomics indicates that Wnt9 is also a conserved member of this cluster in several insects for which genome sequence is available. One of the Tribolium Wnt genes appears to be a member of the WntA family, members of which have been identified in Anopheles and other invertebrates but not in Drosophila or vertebrates. Careful phylogenetic examination suggests an Apis Wnt gene, previously identified as a Wnt4 homolog, is also a member of the WntA family. The ninth Tribolium Wnt gene is related to the diverged Drosophila WntD gene, both of which phylogenetically group with Wnt8 genes. Some of the Tribolium Wnt genes display multiple overlapping expression patterns, suggesting that they may be functionally redundant in segmentation, brain, appendage and hindgut development. In contrast, the unique expression patterns of Wnt5, Wnt7 and Wnt11 in developing appendages likely indicate novel functions.     

The regulatory mechanisms of early embryogenesis in the mouse

The mechanisms involved in the regulation of preimplantation mammalian development have been considered using the example of mouse embryos. The role of four factors affecting the program of early embryogenesis is discussed: nucleocytoplasmic interactions, "maternal" control of development, cell-to-cell interactions, and genomic imprinting. The current concepts of the spatial and temporal regulation of developmental processes have been reviewed, as well as the perspectives of some trends in the experimental embryology of mammals.     

Sox10-rtTA mouse line for tetracycline-inducible expression of transgenes in neural crest cells and oligodendrocytes

Using gene targeting, we inserted a high-affinity variant of the reverse tetracycline controlled transactivator (rtTA) into the genomic Sox10 locus. This rtTA transgene faithfully recapitulated Sox10 expression in the emerging neural crest, several of its derivatives, and in oligodendrocytes. It was furthermore able to induce expression of a tetracycline inducible transgenic reporter gene in a doxycycline-dependent manner. Induction was fast, with substantial reporter gene expression visible 6 h after the onset of doxycycline treatment. Shut-off, in contrast, exhibited delayed kinetics, which probably correlated with doxycycline clearance rates. This mouse provides a useful tool for generating tetracycline-controlled gene expression in neural crest and oligodendrocytes.     

Tissue-specific expression of the mouse Q10 H-2 class-I gene during embryogenesis

We have studied the pattern of expression of the Q10 gene, a H-2 class-I gene located in the major histocompatibility complex which encodes a soluble class-I molecule, in the mid-gestation mouse embryo, and compared it to those of two other class-I genes, namely Kd and 37, the latter gene located in the thymus leukemia region. We found that the steady-state amount of these different mRNAs gradually increased from day 13 to day 18. By comparison with the level of expression of these genes in adult liver, the increase during gestation was fairly more marked for Q10 mRNA than for the others. Furthermore, we found that the Q10 gene is transiently expressed in the endoderm layer of the visceral yolk sac and in the fetal heart. Expression in the latter tissue decreases abruptly while increasing in the liver. It has been proposed that the Q10 protein is involved in immune tolerance. However, the time course of expression of Q10 mRNA and its tissue distribution during embryogenesis suggest that the Q10 protein could play a role in the differentiation of hematopoietic stem cells.