Congress Abstracts - Society for Developmental Biology

volume, and cardiac output. We characterized the typical variation observed over the course of a heartbeat at 55 hours postfertilization,
and plan to extend this analysis over developmental time. Slowing the heart rate approximately 25% by decreasing
temperature nevertheless left heart function (as measured by the hallmark parameters) relatively intact. However, slowing the heart
rate to a similar extent by other methods (drugs) drastically altered flow patterns across the inlet region and avj, indicating heart
function was differentially affected. These protocols therefore represent an opportunity to define the downstream morphological or
genetic responses that occur in the embryonic heart when flow is quantitatively altered in different areas of the heart, or in different
portions of the cardiac cycle.
Program/Abstract # 459
Regulation of genome architecture during heart development
Gómez Velázquez, Melisa; Badía Careaga, Claudio (Ctr Nac de Investigaciones Cardiovasculares, Spain); Galjart, Niels (Erasmus
Med Coll, Netherlands); Gómez Skarmeta, José Luis (Ctr Andaluz de Biol del Desarrollo, Spain); Manzanare, Miguel (Ctr Nac de
Investigaciones Cardiovasculares, Spain)
The central question in developmental biology is to understand how a single cell becomes a complex multicellular organism. For this
to occur, gene expression must be highly and tightly controlled in time and space. The information regarding the genes that need to be
expressed at a certain time and place is coded in regulatory elements that sit throughout the genome. The distribution and location of
these elements are going to define on which genes they can act. This is one of the reasons why the genome needs to be exquisitely
organized in three dimensions. CTCF, an 11 zinc finger protein, has been recently associated at multiple levels in this process, as it
can act as an insulator factor, a looping factor and an enhancer-promoting factor. We are interested in understanding how genome
architecture is involved in early development. Here, we focus our attention in heart development and in order to decipher the role that
CTCF plays here, we are specifically deleting the gene in cardiac tissue by using a conditional Ctcf allele and tissue-specific Cre
drivers. When doing so, the embryos die at stage E13. As a first approach to understand the underlying defects we are analyzing by in
situ hybridization at E9.5 and E11.5 the expression pattern of genes that could be de-regulated by the loss of genomic structure due to
lack of CTCF. More specifically, we are studying genes that are organized in tandem on the genome, that show divergent expression
pattern in the developing heart, and that are separated by stable CTCF binding sites. Results will be presented for genes that show
these features, including transcription factors of the Irx and Tbx families.
Program/Abstract # 460
DNA demethylation confers competence on the genome for zygotic genome activation in zebrafish embryos
Meng, Anming; Wu, Di; Jia, Shunji (Tsinghua U, China)
The zygotic genome of animal embryos is transcriptionally inactive upon fertilization, and becomes active after a certain period of
development. As transcription can be controlled by the presence of 5-methylcytosines (5mC) in DNA, the global DNA methylation
level in an embryo may play a role in the zygotic genome activation (ZGA). In mammals, the paternal genome is rapidly demethylated
immediately after fertilization through 5mC hydroxymethylation, resulting in a decrease of the global DNA methylation level of the
zygote genome. We found that the genome-wide erasure of DNA methylation upon fertilization in zebrafish embryos is not associated
with 5mC hydroxymethylation. Instead, one of DNA glycosylases, hypothesized as XDG, appeared to mediate global DNA
demethylation. xdg knockdown in zebrafish embryos causes an increase of the global DNA methylation level concomitantly with a
reduction of the nuclear transcription level, ultimately resulting in embryonic lethality; conversely, xdg overexpression in embryos is
sufficient to reduce the global DNA methylation level and induce earlier activation of zygotic genome transcription. Thus, XDGmediated
DNA demethylation through base excision and repairing is crucial for development of vertebrate embryos.
Program/Abstract # 461
MicroRNA-30a regulates zebrafish myogenesis via targeting the Six1 homeoprotein
O'Brien, Jenean H.; Hernandez-Lagunas, Laura; Artinger, Kristin Bruk; Ford, Heide L. (UColorado-Denver,USA)
Six1 is a homeodomain containing transcription factor that functions in embryonic muscle development, muscle regeneration after
injury, and muscle tumor promotion. In mouse and zebrafish, knockdown of Six1 results in decreased myogenic progenitor cell
activation and decreased fast-twitch fiber differentiation. However, overexpression of Six1 can also inhibit early differentiation. These
seemingly paradoxical functions of Six1 suggest that precise control of Six1 expression levels is critical for directing proper
myogenesis. Throughout embryogenesis, microRNAs (miRs) coordinate complex temporal patterns of protein expression,
highlighting miRs as potential regulators of Six1. Several miRs have been demonstrated to be important for muscle development,
however most target genes that act downstream of Six1. Here, we investigated miR-mediated regulation of Six1 in myogenesis.
Prediction algorithms identify zebrafish paralogs six1a/b as potential targets of miR-30a, which is supported by a reciprocal
expression pattern in the somites at 24 and 48 hours post fertilization. Morpholino-mediated miR-30a knockdown results in
upregulation of endogenous Six1 levels, and phenocopies Six1 overexpression. Further, miR-30a overexpression leads to decreased
six1a/b mRNA and protein levels, and directly represses GFP-six1a 3’UTR reporter expression. Importantly, abnormal somite
morphology and increased cell death are observed with miR-30a overexpression, phenocopying six1a/b inhibition, and these
phenotypes can be rescued with six1a RNA lacking a 3’UTR. Together, these data indicate that miR-30a regulates zebrafish
myogenesis via targeting Six1, and provide a framework to examine whether miR dysregulation contributes to muscle pathologies.
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