Congress Abstracts - Society for Developmental Biology

Program/Abstract # 195
Coordination of mitosis and morphogenesis: Role of a prolonged G2 phase during chordate neural tube closure
Ogura, Yosuke (University of Tsukuba, Japan); Sakaue-Sawano, Asako (Brain Science Institute, RIKEN, Japan); Nakagawa, Masashi
(University of Hyogo, Japan); Satoh, Nori (Okinawa Institute of Science and Technology Promotion Corporation, Japan); Sasakura,
Yasunori (University of Tsukuba, Japan)
Chordates undergo a characteristic morphogenetic process during neurulation to form a dorsal hollow neural tube. Neurulation begins
with the formation of the neural plate and ends when the left epidermis and right epidermis overlying the neural tube fuse to close the
neural fold. During these processes, mitosis and the various morphogenetic movements need to be coordinated. In this study, we
investigated the epidermal cell cycle in Ciona intestinalis embryos in vivo using a fluorescent ubiquitination-based cell cycle indicator
(Fucci). Epidermal cells of Ciona undergo 11 divisions as the embryos progress from fertilization to the tadpole larval stage. We
detected a long G2 phase between the tenth and eleventh cell divisions, during which fusion of the left and right epidermis occurred.
Characteristic cell shape change and actin filament regulation were observed during the G2 phase. CDC25 is probably a key regulator
of the cell cycle progression of epidermal cells. Artificially shortening this G2 phase by overexpressing CDC25 caused precocious cell
division before or during neural tube closure, thereby disrupting the characteristic morphogenetic movement. Delaying the precocious
cell division by prolonging the S phase with aphidicolin ameliorated the effects of CDC25. These results suggest that the long
interphase during the eleventh epidermal cell cycle is required for neurulation.
Program/Abstract # 196
A Proteomics Approach to Investigate Developmental Disturbances in Forebrain Formation of LRP2 Deficient Mice Using
Mass Spectrometry
Paul, Fabian; Popp, Oliver; Dittmar, Gunnar; Hammes, Annette (Max Delbrueck Center for Molecular Medicine, Germany)
Recently, our lab revealed that the LDL receptor-related protein 2 (LRP2) is an essential component of the sonic hedgehog (SHH)
machinery to orchestrate forebrain development (Christ et al., 2012). Mice deficient for LRP2 exhibit a broad range of severe
forebrain malformations including forms of holoprosencephaly. However, little is known about the detailed dynamics and the role of
LRP2 within the major signaling pathways. To this end, we want to further dissect potential modulatory effects of LRP2 on a
proteomic level. Using state of the art mass spectrometry (MS) we will quantify proteins involved in formation of the central nervous
system (CNS) expressed at crucial developmental stages (E8.5 to E9.5) in mouse embryos. This will enable us to detect differences in
the CNS proteome of LRP2 null mice versus wild type controls. In collaboration with our MS core facility we tested chemical labeling
(dimethyl label) and fathomed the protein amount that is sufficient to robustly quantify most of the CNS proteome at specific
embryonic stages. The first MS runs revealed that for embryonic stage E8.5 the tissue (neural folds) of six embryos per group is
adequate to achieve robust protein identification and quantification results. For E9.5, tissue from two embryos is sufficient. Overall,
we were able to quantify over 3000 proteins at E8.5 and over 5000 proteins for embryonic stage E9.5 respectively. The first MS
dataset with the LRP2 mutant embryos was already obtained and the analysis is currently ongoing. This will help us to further dissect
the role of LRP2 in CNS formation. With the MS approach we also want to establish a workflow, which could serve as a blueprint for
large-scale mutant analysis on the proteome level.
Program/Abstract # 197
SCO-spondin from embryonic cerebrospinal fluid is required for neurogenesis during early brain development
Vera, America; Stanic, Karen; Montecinos, Hernán; Caprile, Teresa (Universidad de Concepcion, Chile)
The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF)
and surrounded by neuroepithelial cells. Several lines of evidence suggest that the eCSF contains diffusible factors regulating the
survival, proliferation and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One
possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof
plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo
function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain
development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that
SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular
differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. Additionally, at the
level of the posterior commissure, SCO-spondin is required for axon attraction and fasciculation. We further corroborated the longrange
function of this protein in vitro, showing that the addition of inhibitory antibodies against SCO-spondin causes a reduction of
neurodifferentiation and an increase of mitosis in mesencephalic explants cultured in eCSF. Our results suggest that SCO-spondin is a
crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.
Program/Abstract # 198
Ift88 has an extraciliary role during neural convergent extension
McFarland, Rebecca J.; Brewster, Rachel (University of Maryland-Baltimore, USA)
Neural convergent extension (NCE), an early stage of neurulation, narrows and elongates the neural ectoderm prior to the formation of
the neural tube. In zebrafish, NCE is driven by polarized cell migration directed towards the midline. Several studies have implicated
ciliary genes in NCE, as they appear to interact with members of the planar cell polarity (PCP) signaling pathway, a key player in this
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