Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo

The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G-CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiC1 treatment. Furthermore, both the negative spatial control functions and response to LiC1 require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study

Cis-regulatory logic in the endo16 gene: switching from a specification to a differentiation mode of control

The endo16 gene of Strongylocentrotus purpuratus encodes a secreted protein of the embryonic and larval midgut. The overall functional organization of the spatial and temporal control system of this gene are relatively well known from a series of earlier cis-regulatory studies. Our recent computational model for the logic operations of the proximal region of the endo16 control system (Module A) specifies the function of interactions at each transcription factor target site of Module A. Here, we extend sequence level functional analysis to the adjacent cis-regulatory region, Module B. The computational logic model is broadened to include B/A interactions as well as other Module B functions. Module B drives expression later in development and its major activator is responsible for a sharp, gut-specific increase in transcription after gastrulation. As shown earlier, Module B output undergoes a synergistic amplification that requires interactions within Module A. The interactions within Module B that are required to generate and transmit its output to Module A are identified. Logic considerations predicted an internal cis-regulatory switch by which spatial control of endo16 expression is shifted from Module A (early) to Module B (later). This prediction was confirmed experimentally and a distinct set of interactions in Module B that mediate the switch function was demonstrated. The endo16 computational model now provides a detailed explanation of the information processing functions executed by the cis-regulatory system of this gene throughout embryogenesis. Early in development the gene participates in the specification events that define the endomesoderm; later it functions as a gut-specific differentiation gene. The cis-regulatory switch mediates this functional change

Quantitative functional interrelations within the cis-regulatory system of the S. purpuratus Endo16 gene

Embryonic expression of the Endo16 gene of Strongylocentrotus purpuratus is controlled by interactions with at least 13 different DNA-binding factors. These interactions occur within a cis-regulatory domain that extends about 2300 bp upstream from the transcription start site. A recent functional characterization of this domain reveals six different subregions, or cis-regulatory modules, each of which displays a specific regulatory subfunction when linked with the basal promoter and in some cases various other modules (C.-H. Yuh and E. Davidson (1996) Development 122, 1069-1082). In the present work, we analyzed quantitative time-course measurements of the CAT enzyme output of embryos bearing expression constructs controlled by various Endo16 regulatory modules, either singly or in combination. Three of these modules function positively in that, in isolation, each is capable of promoting expression in vegetal plate and adjacent cell lineages, though with different temporal profiles of activity. Models for the mode of interaction of the three positive modules with one another were tested by assuming mathematical relations that would generate, from the measured single module time courses, the experimentally observed profiles of activity obtained when the relevant modules are physically linked in the same construct. The generated and observed time functions were compared, and the differences were minimized by least squares adjustment of a scale parameter. When the modules were tested in context of the endogenous promoter region, one of the positive modules (A) was found to increase the output of the others (B and G), by a constant factor. In contrast, a solution in which the time-course data of modules A and B are multiplied by one another was required for the interrelations of the positive modules when a minimal SV40 promoter was used. One interpretation is that, in this construct, each module independently stimulates the basal transcription complex. We used a similar approach to analyze the repressive activity of the three Endo16 cis-regulatory modules that act negatively in controlling spatial expression. The evidence obtained confirms that the repressive modules act only by affecting the output of module A (C.-H. Yuh and E. Davidson (1996) Development 122, 1069-1082). A new hierarchical model of the cis-regulatory system was formulated in which module A plays a central integrating role, and which also implies specific functions for certain DNA-binding sites within the basal promoter fragment of the gene. Additional kinetic experiments were then carried out, and key aspects of the model were confirmed

Modular cis-regulatory organization of developmentally expressed genes: Two genes transcribed territorially in the sea urchin embryo, and additional examples

The cis-regulatory systems that control developmental expression of two sea urchin genes have been subjected to detailed functional analysis. Both systems are modular in organization: specific, separable fragments of the cis-regulatory DNA each containing multiple transcription factor target sites execute particular regulatory subfunctions when associated with reporter genes and introduced into the embryo. The studies summarized here were carried out on the CyIIIa gene, expressed in the embryonic aboral ectoderm and on the Endo16 gene, expressed in the embryonic vegetal plate, archenteron, and then midgut. The regulatory systems of both genes include modules that control particular aspects of temporal and spatial expression, and in both the territorial boundaries of expression depend on a combination of negative and positive functions. In both genes different regulatory modules control early and late embryonic expression. Modular cis-regulatory organization is widespread in developmentally regulated genes, and we present a tabular summary that includes many examples from mouse and Drosophila. We regard cis-regulatory modules as units of developmental transcription control, and also of evolution, in the assembly of transcription control systems

Complexity of cis-regulatory organization of six3a during forebrain and eye development in zebrafish

<p>Abstract</p> <p>Background</p> <p>Six3a belongs to the SIX family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species. Though the function of Six3a as a crucial regulator of eye and forebrain development has been thoroughly investigated, the transcriptional regulation of <it>six3a </it>is not well understood.</p> <p>Results</p> <p>To elucidate the transcriptional regulation of <it>six3a</it>, we performed an <it>in vivo </it>reporter assay. Alignment of the 21-kb region surrounding the zebrafish <it>six3a </it>gene with the analogous region from different species identified several conserved non-coding modules. Transgenesis in zebrafish identified two enhancer elements and one suppressor. The D module drives the GFP reporter in the forebrain and eyes at an early stage, while the A module is responsible for the later expression. The A module also works as a repressor suppressing ectopic expression from the D module. Mutational analysis further minimized the A module to four highly conserved elements and the D module to three elements. Using electrophoresis mobility shift assays, we also provided evidence for the presence of DNA-binding proteins in embryonic nuclear extracts. The transcription factors that may occupy those highly conserved elements were also predicted.</p> <p>Conclusion</p> <p>This study provides a comprehensive view of <it>six3a </it>transcription regulation during brain and eye development and offers an opportunity to establish the gene regulatory networks underlying neurogenesis in zebrafish.</p

Nestin Is Essential for Zebrafish Brain and Eye Development through Control of Progenitor Cell Apoptosis

BACKGROUND: Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. METHODOLOGY/PRINCIPAL FINDINGS: As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. CONCLUSION/SIGNIFICANCE: These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis

Patchy Interspecific Sequence Similarities Efficiently Identify Positive cis-Regulatory Elements in the Sea Urchin

We demonstrate that interspecific sequence conservation can provide a systematic guide to the identification of functional cis-regulatory elements within a large expanse of genomic DNA. The test was carried out on the otx gene of Strongylocentrotus purpuratus. This gene plays a major role in the gene regulatory network that underlies endomesoderm specification in the embryo. The cis-regulatory organization of the otx gene is expected to be complex, because the gene has three different start sites (X. Li, C.-K. Chuang, C.-A. Mao, L. M. Angerer, and W. H. Klein, 1997, Dev. Biol. 187, 253–266), and it is expressed in many different spatial domains of the embryo. BAC recombinants containing the otx gene were isolated from Strongylocentrotus purpuratus and Lytechinus variegatus libraries, and the ordered sequence of these BACs was obtained and annotated. Sixty kilobases of DNA flanking the gene, and included in the BAC sequence from both species, were scanned computationally for short conserved sequence elements. For this purpose, we used a newly constructed software package assembled in our laboratory, “FamilyRelations.” This tool allows detection of sequence similarities above a chosen criterion within sliding windows set at 20–50 bp. Seventeen partially conserved regions, most a few hundred base pairs long, were amplified from the S. purpuratus BAC DNA by PCR, inserted in an expression vector driving a CAT reporter, and tested for cis-regulatory activity by injection into fertilized S. purpuratus eggs. The regulatory activity of these constructs was assessed by whole-mount in situ hybridization (WMISH) using a probe against CAT mRNA. Of the 17 constructs, 11 constructs displayed spatially restricted regulatory activity, and 6 were inactive in this test. The domains within which the cis-regulatory constructs were expressed are approximately consistent with results from a WMISH study on otx expression in the embryo, in which we used probes specific for the mRNAs generated from each of the three transcription start sites. Four separate cis-regulatory elements that specifically produce endomesodermal expression were identified, as well as ubiquitously active elements, and ectoderm-specific elements. We confirm predictions from other work with respect to target sites for specific transcription factors within the elements that express in the endoderm

Induced pluripotent stem cells and regenerative medicine

AbstractStem cells, a special subset of cells derived from embryo or adult tissues, are known to present the characteristics of self-renewal, multiple lineages of differentiation, high plastic capability, and long-term maintenance. Recent reports have further suggested that neural stem cells (NSCs) derived from the adult hippocampal and subventricular regions possess the utilizing potential to develop the transplantation strategies and to screen the candidate agents for neurogenesis, neuroprotection, and neuroplasticity in neurodegenerative diseases. In this article, we review the roles of NSCs and other stem cells in neuroprotective and neurorestorative therapies for neurological and psychiatric diseases. We show the evidences that NSCs play the key roles involved in the pathogenesis of several neurodegenerative disorders, including depression, stroke, and Parkinson’s disease. Moreover, the potential and possible utilities of induced pluripotent stem cells, reprogramming from adult fibroblasts with ectopic expression of four embryonic genes, are also reviewed and further discussed. An understanding of the biophysiology of stem cells could help us elucidate the pathogenicity and develop new treatments for neurodegenerative disorders. In contrast to cell transplantation therapies, the application of stem cells can further provide a platform for drug discovery and small molecular testing, including Chinese herbal medicines. In addition, the high-throughput stem cell-based systems can be used to elucidate the mechanisms of neuroprotective candidates in translation medical research for neurodegenerative diseases

Network Biology of Tumor Stem-like Cells Identified a Regulatory Role of CBX5 in Lung Cancer

Mounting evidence links cancers possessing stem-like properties with worse prognosis. Network biology with signal processing mechanics was explored here using expression profiles of a panel of tumor stem-like cells (TSLCs). The profiles were compared to their parental tumor cells (PTCs) and the human embryonic stem cells (hESCs), for the identification of gene chromobox homolog 5, CBX5, as a potential target for lung cancer. CBX5 was found to regulate the stem-like properties of lung TSLCs and was predictive of lung cancer prognosis. The investigation was facilitated by finding target genes based on modeling epistatic signaling mechanics via a predictive and scalable network-based survival model. Topologically-weighted measurements of CBX5 were synchronized with those of BIRC5, DNMT1, E2F1, ESR1, MLH1, MSH2, RB1, SMAD1 and TAF5. We validated our findings in another Taiwanese lung cancer cohort, as well as in knockdown experiments using sh-CBX5 RNAi both in vitro and in vivo.National Science Council (China) (NSC grant 100-2325-B-010-010-MY3/98-2314-B-010-024-MY2/97-3111-B075-001-MY3/ 96-2314-075-056-MY3)National Yang-Ming University (Ministry of Education, Aim for the Top University Plan: 96ADD122, 96ADD125, 96ADT191, 97ACD113, 97ACT302, 98ACT302, 98ACD107, 98ACT192 and Brain Research Center-3T-MRI project)))Taipei Veterans General Hospital (98-C1-099/E1-003/ER3-001)Taipei Veterans General Hospital (Joint Projects of VGHUST (98-G6-6/ 98-P1-01/99-P6-39)Chi Mei Medical Center (CMYM9801)Yen-Tjing-Ling Medical Foundation (96/97/98)Taipei City Hospital (96-002-62-092)Technology Development Program for Academia (TDPA; 98-EC-17-A-19-S2-0107)Taiwan. Department of Industrial Technology, Ministry of Economic AffairsNational Science Council (China) (NSC 101-2325-B-010 -009)Taiwan. Department of Health. Cancer Research Center of Excellence (DOH101-TD-C-111-007

Reduced expression of alpha-1,2-mannosidase I extends lifespan in Drosophila melanogaster and Caenorhabditis elegans

Exposure to sub-lethal levels of stress, or hormesis, was a means to induce longevity. By screening for mutations that enhance resistance to multiple stresses, we identified multiple alleles of alpha-1,2-mannosidase I (mas1) which, in addition to promoting stress resistance, also extended longevity. Longevity enhancement is also observed when mas1 expression is reduced via RNA interference in both Drosophila melanogaster and Caenorhabditis elegans. The screen also identified Edem1 (Edm1), a gene downstream of mas1, as a modulator of lifespan. As double mutants for both mas1 and Edm1 showed no additional longevity enhancement, it appeared that both mutations function within a common pathway to extend lifespan. Molecular analysis of these mutants revealed that the expression of BiP, a putative biomarker of dietary restriction (DR), is down-regulated in response to reductions in mas1 expression. These findings suggested that mutations in mas1 may extend longevity by modulating DR