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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2009)

The initiation of tissue specific genetic programs requires that a signaling pathway must activate a subset of its complete repertoire of targets at the appropriate time and in the correct location. This is an extreme challenge for a pathway that operates in multiple tissues during the same stage of development. To achieve this level of specificity, tissue specific transcription of target genes relies on the activity of local factors that define a zone of activation competence.

The Drosophila Hedgehog (Hh) signaling pathway functions in a variety of tissues, effecting gene activation by modulating the activity of its downstream transcription factor, Cubitus interruptus (Ci). Cells receiving the Hh signal produce a full-length Ci transcriptional activator. Cells that do not receive Hh signal convert full length Ci into a transcriptional repressor. Little concerted effort has been put forth to identify the direct downstream targets of Ci that mediate the effect of Hh signaling. In this work, I use a combination of genomic approaches to identify regions in the genome where Ci binds (DAMID) and to determine the genes that respond to Hh signaling (expression array analysis) in the Drosophila embryo.

I find that DAMID signals for repressor (DamCiRep) and activator (DamCiAct) forms of Ci overlap hundreds of times indicating that repressor and activator forms of Ci recognize the same sequences in vivo. Transcriptional profiling of Hh pathway mutants uncovers genes that respond to all genetic backgrounds as well as sub-clusters of genes that change expression levels in specific mutant backgrounds. High confidence putative Ci targets are found to be largely tissue specific in their expression and function. A validation of putative Ci targets via in situ hybridization reveals a tissue specific response to Ci in three developmental systems: the embryonic visual system, dorsal ectoderm, and the developing tracheal system. Sequence specific binding sites for tissue specific regulators are present in Ci enhancers leading to a model by which Ci functions in synergy with local factors for optimal gene activation.

    Cover page: Targets of Cubitus interruptus regulation in the Drosophila embryo
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2002)

    Background

    In the Drosophila larva, imaginal discs are programmed to produce adult structures at metamorphosis. Although their fate is precisely determined, these organs remain largely undifferentiated in the larva. To identify genes that establish and express the different states of determination in discs and larval tissues, we used DNA microarrays to analyze mRNAs isolated from single imaginal discs.

    Results

    Linear amplification protocols were used to generate hybridization probes for microarray analysis from poly(A)+ RNA from single imaginal discs containing between 10,000 and 60,000 cells. Probe reproducibility and degree of representation were tested using microarrays with approximately 6,000 different cDNAs. Hybridizations with probes that had been prepared separately from the same starting RNA pool had a correlation coefficient of 0.97. Expression-profile comparisons of the left and right wing imaginal discs from the same larva correlated with a coefficient of 0.99, indicating a high degree of reproducibility of independent amplifications. Using this method, we identified genes with preferential expression in the different imaginal discs using pairwise comparisons of discs and larval organs. Whereas disc-to-disc comparisons revealed only moderate differences, profiles differed substantially between imaginal discs and larval tissues, such as larval endodermal midgut and mesodermal fat body.

    Conclusions

    The combination of linear RNA amplification and DNA microarray hybridization allowed us to determine the expression profiles of individual imaginal discs and larval tissues and to identify genes expressed in tissue-specific patterns. These methods should be widely applicable to comparisons of expression profiles for tissues or parts of tissues that are available only in small amounts.
      Cover page: Expression profiling of Drosophilaimaginal discs
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2012)
      Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b(-/-) and Sprouty mutant mice.
        Cover page: BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2012)
        Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases.
          Cover page: Expression and Characterization of Drosophila Signal Peptide Peptidase-Like (sppL), a Gene That Encodes an Intramembrane Protease
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2015)
          Proper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology.
            Cover page: Opposing Activities of Notch and Wnt Signaling Regulate Intestinal Stem Cells and Gut Homeostasis
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2013)
            Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.
              Cover page: Characterization of Dental Epithelial Stem Cells from the Mouse Incisor with Two-Dimensional and Three-Dimensional Platforms
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