Circuit dissection of the role of somatostatin in itch and pain

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide

Conditional gene expression and lineage tracing of tuba1a expressing cells during zebrafish development and retina regeneration

The tuba1a gene encodes a neural-specific Α-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in MÜller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a -expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreER T2 and Β- actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to label tuba1a -expressing cells conditionally and permanently via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in MÜller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a -expressing progenitors contribute to multiple cell lineages during development and that tuba1a -expressing MÜller glia are retinal progenitors in the adult. J. Comp. Neurol. 518:4196–4212, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77972/1/22448_ftp.pd

Prospective Identification and Isolation of Enteric Nervous System Progenitors Using Sox2

The capacity to identify and isolate lineage-specific progenitor cells from developing and mature tissues would enable the development of cell replacement therapies for disease treatment. The enteric nervous system (ENS) regulates important gut functions, including controlling peristaltic muscular contractions, and consists of interconnected ganglia containing neurons and glial cells. Hirschsprung's disease (HSCR), one of the most common and best understood diseases affecting the ENS, is characterized by absence of enteric ganglia from the distal gut due to defects in gut colonization by neural crest progenitor cells and is an excellent candidate for future cell replacement therapies. Our previous microarray experiments identified the neural progenitor and stem cell marker SRY-related homoebox transcription factor 2 (Sox2) as expressed in the embryonic ENS. We now show that Sox2 is expressed in the ENS from embryonic to adult stages and constitutes a novel marker of ENS progenitor cells and their glial cell derivatives. We also show that Sox2 expression overlaps significantly with SOX10, a well-established marker of ENS progenitors and enteric glial cells. We have developed a strategy to select cells expressing Sox2, by using G418 selection on cultured gut cells derived from Sox2βgeo/+ mouse embryos, thus allowing substantial enrichment and expansion of neomycin-resistant Sox2-expressing cells. Sox2βgeo cell cultures are enriched for ENS progenitors. Following transplantation into embryonic mouse gut, Sox2βgeo cells migrate, differentiate, and colocalize with the endogenous ENS plexus. Our studies will facilitate development of cell replacement strategies in animal models, critical to develop human cell replacement therapies for HSCR. Stem Cells 2011;29:128–14

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases

The regulation of gene expression by estrogen receptor-α (ERα) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERα as bait in a yeast two-hybrid screen, we have identified a novel ERα-interacting protein, ZNF366, which is a potent corepressor of ERα activity. The interaction between ZNF366 and ERα has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERα corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen

Dbx1-Expressing Cells Are Necessary for the Survival of the Mammalian Anterior Neural and Craniofacial Structures

Development of the vertebrate forebrain and craniofacial structures are intimately linked processes, the coordinated growth of these tissues being required to ensure normal head formation. In this study, we identify five small subsets of progenitors expressing the transcription factor dbx1 in the cephalic region of developing mouse embryos at E8.5. Using genetic tracing we show that dbx1-expressing cells and their progeny have a modest contribution to the forebrain and face tissues. However, their genetic ablation triggers extensive and non cell-autonomous apoptosis as well as a decrease in proliferation in surrounding tissues, resulting in the progressive loss of most of the forebrain and frontonasal structures. Targeted ablation of the different subsets reveals that the very first dbx1-expressing progenitors are critically required for the survival of anterior neural tissues, the production and/or migration of cephalic neural crest cells and, ultimately, forebrain formation. In addition, we find that the other subsets, generated at slightly later stages, each play a specific function during head development and that their coordinated activity is required for accurate craniofacial morphogenesis. Our results demonstrate that dbx1-expressing cells have a unique function during head development, notably by controlling cell survival in a non cell-autonomous manner

Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreERTm and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1PB-CreERTm;R26RlacZ mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs

Comprehensive overview of the structure and regulation of the glucocorticoid receptor

Glucocorticoids are among the most prescribed drugs worldwide for the treatment of numerous immune and inflammatory disorders. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. There are several GR isoforms resulting from alternative RNA splicing and translation initiation of the GR transcript. Additionally, these isoforms are all subject to several transcriptional, post-transcriptional, and post-translational modifications, all of which affect the protein's stability and/or function. In this review, we summarize recent knowledge on the distinct GR isoforms and the processes that generate them. We also review the importance of all known transcriptional, post-transcriptional, and post-translational modifications, including the regulation of GR by microRNAs. Moreover, we discuss the crucial role of the putative GR-bound DNA sequence as an allosteric ligand influencing GR structure and activity. Finally, we describe how the differential composition and distinct regulation at multiple levels of different GR species could account for the wide and diverse effects of glucocorticoids