Abstracts - Society for Developmental Biology

10
address the implications of our findings for genomics-based and computational approaches to understanding transcriptional
networks.
Program/Abstract # 29
Maternal mRNA retention as a mechanism for maintaining totipotency in primordial germ cells
Swartz, S. Zachary, Brown University, Providence, United States; Raz, Tal; Milos, Patrice (Helicos Biosciences);
Hamdoun, Amro (Scripps Inst of Oceanography); Wessel, Gary (Brown U)
The germ line contains the stem cells responsible for transmitting all heritable information between generations. Unlike the
soma, which lacks reproductive potential and will ultimately die, germ line stem cells (GSCs) retain the capacity for
totipotency and create the sperm and eggs necessary for developing a new organism. A widely conserved strategy for
segregating primordial germ cells (PGCs) involves maternally loaded and spatially localized determinants, often called a
“germ plasm,” which direct the cells that inherit it toward GSC fate. Our investigations have not revealed a localized germ
plasm in the early embryo of the sea urchin S. purpuratus. However, several mRNAs commonly found in germ plasm are
maternally loaded and ubiquitously distributed in the early embryo, followed by later restriction to the PGCs. To better
understand the initial segregation of PGCs in the sea urchin, we performed deep sequencing and differential expression
analysis of FACS isolated PGCs. We identified a set of genes whose mRNAs are ubiquitous in eggs and early embryos, but
are later restricted to the PGCs. This suggests a general post-transcriptional mechanism that retains mRNAs in the PGCs,
and degrades them in somatic cells. The 3’UTRs of these mRNAs contain shared sequence motifs which may accelerate
their destruction in somatic cells. In addition, we found that the CCR4-NOT deadenylase complex member Cnot6, a poly-
A nuclease, is absent in the PGCs but present in all other cells of the embryo. This observation suggests a mechanism for
the stabilization of germ line mRNAs in the PGCs but turnover elsewhere. Finally, the 3’UTR of Cnot6 contains three
putative Nanos response elements (NREs), which may be targeted for destruction by Pumilio and its partner Nanos, which
is selectively expressed in the PGCs. Through this work we are uncovering a new mechanism by which PGCs are protected
from differentiation by the retention of mRNAs of the early totipotent embryo.
Program/Abstract # 30
Specification of the proximodistal axis by Irx3 and Irx5 homeobox genes prior to limb bud initiation
Hui, Chi-Chung (Hosp Sick Children, Toronto, Canada)
Pattern formation requires coordination of growth in three dimensions. The anteroposterior (AP) and proximodistal (PD)
axes of the developing limb are linked when Shh from the zone of polarizing activity (ZPA) and Fgfs from the apical
ectodermal ridge (AER) form a positive feedback loop in the limb bud. It is unknown whether the AP and PD axes are
coordinated before limb bud formation. In this study, we show that both axes are regulated by Iroquois homeobox (Irx)
genes Irx3 and Irx5 prior to the establishment of Shh and Fgf8 signaling centers. Through interactions with Gli3, Irx3 and
Irx5 regulate AP prepattern as well as PD specification of Shh-independent progenitors in the hindlimb. These findings
suggest that AP and PD limb axes are coordinated among limb progenitors prior to outgrowth. Our data also provide the
first genetic evidence in support of the early specification of PD pattern.
Program/Abstract # 31
Identifying Hox targets by transcriptional profiling of the mouse hindbrain
Yurieva, Marina; De Kumar, Bony; Krumlauf, Robb, Stowers Institute for Medical Research, Kansas City, United States
The vertebrate hindbrain forms the medulla, pons and cerebellum which play a crucial role in regulating functions such as
sleep, respiration and heart rate. During development regional diversity in hindbrain is established through the
segmentation of the neural tube into seven morphological discrete domains termed rhombomeres. A network of
transcription factors including Hoxa1, Hoxa2, Hoxb1 and Krox20 are involved in establishing the segmental cellular
organization critical for hindbrain function. Genetics studies in a number of vertebrates have shown that mutations in these
regulatory genes cause dramatic changes in segmentation and rhombomere identity, resulting in severe neuronal defects
and lethality in adult animals. Our research has utilized transcriptional profiling and functional validation to identify and
characterize rhombomeric-specific patterns of gene expression regulated by these key transcription factors. Using laser
capture microscopy to isolate individual rhombomeres from 9.5 dpc mouse embryos, we performed transcriptional
profiling on either single rhombomeres from the hindbrains of wild type embryos or whole hindbrains of wild type and
Hoxa1, Hoxa2, Hoxb1 and Krox20 mutant embryos. Computational analyses and validation of these results have
uncovered several novel downstream targets and pathways associated with their roles in hindbrain specification. For
example, Hox genes which are induced by retinoids in turn modulate multiple aspects of retinoid metabolism and
catabolism. These feedback loops are important for hindbrain patterning.