Numb protein isoforms differentially regulate Notch1, Notch2 and Notch3

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ABSTRACTS / Developmental Biology 306 (2007) 339–344

epithelial cell differentiation and increased cell apoptosis, as shown by changed cell morphology, reduced expression of lung epithelial markers (CCSP, SP-C, AQP5), and lack of surfactant secretion. Therefore, these data indicate that Alk3-mediated BMP signaling, particularly during birth, is essential for neonatal respiratory function by promoting peripheral lung epithelial cell terminal differentiation and linage maintenance. doi:10.1016/j.ydbio.2007.03.190

Program/Abstract # 132 Genetic analysis of BMP-mediated cell survival in the limb bud mesenchyme Sangeeta Underwood 1, Yuji Mishina 2, Uta Grieshammer 3, Gail Martin 3, Trevor Williams 4, Mark Lewamdoski 1 1 Cancer and Developmental Biology Laboratory, NCIFrederick, NIH, Frederick, MD, USA 2 Laboratory of Reproductive and Developmental Toxicology, NIEHS, NIH, Research Triangle Park, NC, USA 3 Department of Anatomy and Program in Developmental Biology, UCSF, San Francisco, CA, USA 4 Cell and Developmental Biology, University of Colorado Health Science Center, Auorora CO, USA To study BMP signaling in the developing limb bud mesenchyme, we conditionally inactivated the Bmp receptor gene, Bmpr1a, in a wildtype or a Bmpr1b−/− background using one of two Cre lines, Ap2-Cre (active at E9.5) or Osr-Cre (active interdigitally). Skeletal elements are lost distally in Ap2-Cre; Bmpr1a mutants and such loss occurs more proximally as BMP signaling is further attenuated by also removing Bmpr1b alleles. We attribute these phenotypes to aberrantly high cell death starting at E10.5, resulting in a smaller progenitor population for formation of skeletal elements, indicating that BMPs can act as cell survival factors for the early limb bud mesenchyme. Alternatively, during interdigital development at E12.5, loss of BMP signaling causes a decrease in cell death and hence syndactyly after birth. In recent published work we have shown that BMP signals indirectly control interdigital cell death by controlling FGF signaling from the bud apical ectodermal ridge (Development, in press). However, AER-FGF signaling is unaffected in Osr-Cre limb buds, indicating that BMPs can also directly control interdigital cell death. Together these data indicate that BMP signaling in different subsets of the developing limb bud can have different effects on cell survival. doi:10.1016/j.ydbio.2007.03.191

Program/Abstract # 133 The role of Sprouty in regulating cell proliferation during ocular lens development Lixing W. Reneker, Leike Xie Department of Ophthalmology, University of Missouri, Columbia, MO, USA

Lens cell proliferation, differentiation and survival are tightly regulated to achieve the normal developmental pattern and structure of the ocular lens. Growth factors are the key regulators of these cellular events, but very little is known about their signaling pathways in the lens. In the past, we demonstrated that small GTP-binding protein Ras is required for cell proliferation but not for the initiation of fiber cell differentiation during normal lens development. In this study, we used transgenic mouse technique to assess the role of Sprouty (Spry) in receptor tyrosine kinase (RTK)-activated Ras-ERK pathway in mouse lens. Expression of Spry 1, 2 and 3 were detected in mouse lens by reverse transcriptase-polymerase chain reaction (RT-PCR) during normal development. By expressing Spry2 or dominant negative (dn) Spry2 in the transgenic mouse lens, we found that Spry2 functions as a negative regulator to control lens epithelial cell proliferation but not differentiation of the secondary fiber cells during lens development. Western blot analysis indicated that ERK (but not Akt) activity in the transgenic mouse lenses were affected. Genetic crosses between the Spry2 mice and transgenic mice overexpressing PDGF-A or FGF-3 in the lens indicated that Spry2 is capable of blocking cell proliferation induced by these growth factors. Our data are consistent with the hypothesis that Spry2 functions as a negative regulator of cell proliferation, potentially contributing to the establishment and maintenance of the monolayer structure of the lens epithelium. doi:10.1016/j.ydbio.2007.03.192

Program/Abstract # 134 Numb protein isoforms differentially regulate Notch1, Notch2 and Notch3 Brian J. Beres 1, Jane McGlade 2, Alan Rawls 1, Jeanne Wilson-Rawls 1 1 School of Life Sciences, Arizona State University, Tempe, AZ, USA 2 The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Ca 3 Basic Medical Sciences University of Arizona College of Medicine-Phoenix, Phoenix AZ, USA Signaling through Notch1, Notch2 and Notch3 regulate diverse cellular decisions during development. Numb is a cytoplasmic protein that inhibits Notch. There are four proteins isoforms of mammalian Numb that arise through alternative splicing. It has been reported that Numb mediates the ubiquitination of Notch and blocks activation of the bHLH Orange repressors. In the somite, as in other developing tissues and organs, Notch1–3 and Numb overlap in expression. In order to understand this complex signaling milieu, the inhibitory effect of the different Numb isoforms was determined for each Notch receptor. Experiments were done to examine whether all three receptors were targets of all four Numb isoforms using a 4XCSL-Luciferase reporter. These studies demonstrated that Notch1 is targeted by all Numb isoforms, Notch2 is variably

ABSTRACTS / Developmental Biology 306 (2007) 339–344

repressed and Notch3 is not a target of any Numb protein. The ability of Numb isoforms to relieve Notch mediated repression of MyoD-induced myogenic conversion of C3H10T1/2 cells recapitulated the data from the transcription assays. The ability of Numb proteins to induce ubiquitination of the Notch receptors was examined. These data indicate a varying ability of Numb isoforms to effectively modulate the function of specific Notch receptors and their downstream signaling. doi:10.1016/j.ydbio.2007.03.193

Program/Abstract # 135 Possible role of Sorcin during mouse development Itzel Ricaño, Erika P. Zarate-Díaz, Alfredo Varela-Echavarría, Anaid Antaramian Instituto de Neurobiología, UNAM, Querétaro, México Sorcin is a 21.6-kDa Ca2+ binding protein with 5 EF hands motifs. It has been implicated in the regulation of intracellular Ca2+ cycling and in cardiac excitation–contraction coupling known as the Ca 2+ induce Ca2+ -release (CICR) by the inhibition of the type 2 Ryanodine receptor (RyR2) activity. We have demonstrated that Sorcin inhibits the activity of the type 2 Ryanodine receptor both in mouse cardiomyocytes in vitro and in lipid bilayer assays. Three types Ryanodine receptors exist, type 1 is expressed in the skeletal muscle, type 2 is expressed in cardiac muscle, whereas the type 3 is expressed in brain tissue. Since Sorcin seems to have and important role in calcium homeostasis and it could be a calcium buffer in the heart and other developing organs, we analyzed its expression during embryonic development in mouse embryos. Using fluorescent inmunostaining, we studied the expression of Sorcin in mouse embryos from 9 to 12 days of gestational age (E9 to E112). In addition to the expected expression in the heart, in E9 to E11 embryos we observed prominent expression in neuronal groups of the central and peripheral nervous system. Our results suggest two possibilities. First, since the cardiovascular system is one of earliest to develop and the heartbeat begins at E8, we think it is likely that Sorcin regulates the activity of the cardiac type Ryanodine receptor (RyR2) during early development. Second, our results also suggest that Sorcin is involved in the development of specific structures of the central and peripheral nervous systems. We are grateful for the technical assistance of Martín García Servín. doi:10.1016/j.ydbio.2007.03.194

Program/Abstract # 136 Glucose transporters on atrial natriuretic peptide-induced glucose uptake by adult and neonatal hypoxic cardiomyocytes Roxana Carbo, Veronica Sosa, Veronica Guarner Dept. Physiology, Instituto Nacional de Cardiologia, Mexico D.F., Mexico

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Natriuretic peptides, beside their endocrine actions, have paracrine functions including glucose uptake and metabolism. Atrial natriuretic peptide (ANP) actions are mediated by cGMP implicated in the metabolic adaptation of glucose metabolism to oxygen deprivation in the heart. Although it has been reported that ANP increases glucose uptake, cGMP decreases it. In this paper we evaluate the role of the glucose transporters 1 and 4 (GLUTS), in glucose uptake produced by ANP in adult and neonatal cardiomyocytes under oxygenation and hypoxia. We also explored if the calcium–calmodulin complex participates in ANP-induced increase in glucose uptake. Neonatal cells had a higher glucose uptake than adult cells and GLUT 1 participated in basal uptake in both cell types. Hypoxia increased glucose uptake in adult cardiomyocytes but not in neonatal cells and this increase in glucose uptake was mediated by GLUT4. ANP increased glucose uptake in both adult and neonatal myocytes, under oxygenation and hypoxia, and GLUT4 favored this increase. Neonatal cells were less sensitive to ANP. Trifluoperazine, a calcium–calmodulin blocker, inhibited the ANP-induced increase in glucose uptake. This suggests that ANP promotes GLUT 4 calcium-mediated recruitment to the cell membrane. In conclusion, glucose uptake regulation is one of the paracrine metabolic effects of ANP in adult and neonatal cardiomyocytes under oxygenation and hypoxia. This effect of this peptide could explain the beneficial effects found in the internal medicine and surgical fields. doi:10.1016/j.ydbio.2007.03.195

Program/Abstract # 137 Yolk sac vascular remodeling mediated by PDGF Wendy J. French, Michelle D. Tallquist UT Southwestern, Dallas, TX Platelet derived growth factor receptors (PDGFR)α and β are receptor tyrosine kinases involved in diverse cellular functions essential for embryonic development. While analysis of the null alleles has demonstrated independent roles for PDGFRα and PDGFRβ, the cooperative roles for the receptors have yet to be determined. We found embryos lacking both PDGFR resulted in embryonic lethality at E9.5 with disrupted yolk sac vascular development. To investigate which cell population was responsible for the aberrant vasculature, we deleted the receptors in either the mesothelium or the vascular smooth muscle cells (VSMC). We found that only deletion of the receptors in the mesothelium lead to embryonic lethality at E10.5 while loss of the receptors in VSMC resulted in normal vasculature. The mutant yolk sacs formed a vascular plexus similar to wild type; however, vascular remodeling failed to occur. Proliferation and cell survival were normal in the mutant yolk sac. Using markers for specific cell populations of the yolk sac we determined there was a decrease in the VSMC populations suggesting a role for the PDGFR in mesothelium differentiation. Further analysis of mouse mutants deficient in downstream signaling pathways of the receptors demonstrated that PI3 kinase signaling was not essential for vascular