TY - JOUR TI - Heterotaxin: A novel TGF-beta signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos AU - Nascone-Yoder, Nanette M. AU - Dush, Michael AU - McIver, Andrew AU - Parr, Meredith AU - Young, Douglas AU - Fisher, Julie AU - Hauck, Marlene AU - Deiters, Alexander T2 - Developmental Biology DA - 2010/8// PY - 2010/8// DO - 10.1016/j.ydbio.2010.05.483 VL - 344 IS - 1 SP - 526 J2 - Developmental Biology LA - en OP - SN - 0012-1606 UR - http://dx.doi.org/10.1016/j.ydbio.2010.05.483 DB - Crossref ER - TY - JOUR TI - An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain AU - Jacquet, Benoit V. AU - Ruckart, Philip AU - Ghashghaei, H. Troy T2 - Journal of Visualized Experiments AB - Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds. DA - 2010/12/11/ PY - 2010/12/11/ DO - 10.3791/2486 IS - 46 J2 - JoVE LA - en OP - SN - 1940-087X UR - http://dx.doi.org/10.3791/2486 DB - Crossref ER - TY - JOUR TI - Development of a Model of Sacrocaudal Spinal Cord Injury in Cloned Yucatan MiniPigs for Cellular Transplantation Research AU - Lim, Ji-Hey AU - Piedrahita, Jorge A. AU - Jackson, Lauren AU - Ghashghaei, Troy AU - Olby, Natasha J. T2 - CELLULAR REPROGRAMMING AB - Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery. DA - 2010/12// PY - 2010/12// DO - 10.1089/cell.2010.0039 VL - 12 IS - 6 SP - 689-697 SN - 2152-4998 ER - TY - JOUR TI - Photocaged Morpholino Oligomers for the Light-Regulation of Gene Function in Zebrafish and Xenopus Embryos AU - Deiters, Alexander AU - Garner, R. Aaron AU - Lusic, Hrvoje AU - Govan, Jeane M. AU - Dush, Mike AU - Nascone-Yoder, Nanette M. AU - Yoder, Jeffrey A. T2 - JOURNAL OF THE AMERICAN CHEMICAL SOCIETY AB - Morpholino oligonucleotides, or morpholinos, have emerged as powerful antisense reagents for evaluating gene function in both in vitro and in vivo contexts. However, the constitutive activity of these reagents limits their utility for applications that require spatiotemporal control, such as tissue-specific gene disruptions in embryos. Here we report a novel and efficient synthetic route for incorporating photocaged monomeric building blocks directly into morpholino oligomers and demonstrate the utility of these caged morpholinos in the light-activated control of gene function in both cell culture and living embryos. We demonstrate that a caged morpholino that targets enhanced green fluorescent protein (EGFP) disrupts EGFP production only after exposure to UV light in both transfected cells and living zebrafish (Danio rerio) and Xenopus frog embryos. Finally, we show that a caged morpholino targeting chordin, a zebrafish gene that yields a distinct phenotype when functionally disrupted by conventional morpholinos, elicits a chordin phenotype in a UV-dependent manner. Our results suggest that photocaged morpholinos are readily synthesized and highly efficacious tools for light-activated spatiotemporal control of gene expression in multiple contexts. DA - 2010/11/10/ PY - 2010/11/10/ DO - 10.1021/ja1053863 VL - 132 IS - 44 SP - 15644-15650 SN - 1520-5126 ER - TY - JOUR TI - Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut AU - Chung, Mei-I AU - Nascone-Yoder, Nanette M. AU - Grover, Stephanie A. AU - Drysdale, Thomas A. AU - Wallingford, John B. T2 - DEVELOPMENT AB - Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates. DA - 2010/4/15/ PY - 2010/4/15/ DO - 10.1242/dev.044610 VL - 137 IS - 8 SP - 1339-1349 SN - 1477-9129 KW - Pitx1 KW - Pitx2 KW - Shroom KW - Apical constriction KW - Epithelium KW - Gut KW - Xenopus ER -