Abstracts - Society for Developmental Biology

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in the Shh pathway. A strong allele of Pericentrin, which greatly reduces the amount ofcentrosomal pericentrin, causes
lethality at e14. Pericentrin mutants at this stage show incompletely penetrant polydactyly, suggesting a mild gain of Shh
signaling. Shh-dependent dorsal-ventral patterning is only mildly affected in the pericentrin mutant, and PKA appears to
localize to the pericentriolar region in pericentrin mutants, suggesting that additional proteins mediate PKA localization to
the centrosome. Akap9 null mutants are viable and male sterile, and do not exhibit defects in Shh signaling. We are
currently analyzing the phenotypes of Pericentrin and Akap9 double mutants to test whether these proteins have
overlapping functions in the localization of PKA to the centrosome. Through this study we will establish whether the
centrosomal localization of PKA is important for Shh pathway output and thus mouse embryonic development.
Program/Abstract # 270
The role of hedgehog signaling pathway in the development of the mouse patellar tendon
Liu, Chia-Feng; Aschbacher-Smith, Lindsey (Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United
States); Butler, David (University of Cincinnati, Cincinnati, OH, United States); Wylie, Christopher (Cincinnati
Children's Hospital Medical Center, Cincinnati, OH, United States)
Tendon can be divided into two anatomical areas: midsubstance and insertion site. Using a mouse reporter line, we found
Gli1 was expressed in the tendon-to-bone insertion site but not in the midsubstance. GLI1 is a downstream effector of
hedgehog (Hh) signaling pathway. Thus, its expression in the insertion site suggests Hh pathway may be involved in the
differentiation of insertion site. To test this hypothesis, we first activated Hh pathway by expressing constitutively active
smoothened (SmoM2), the Hh signal transducer, using CRE recombinase driven by the promoter of scleraxis(ScxCre), a
transcription factor expressed in developing tenocytes. SmoM2 animals died shortly after birth due to respiratory
problems. However, we observed that several insertion site markers were expressed ectopically in the midsubstance during
the fetal period. We confirmed these findings in organ cultures in vitro. Next, we targeted the Smo gene in the tenocyte
population using ScxCre. There was a reduced development of the tendon-to-bone insertion site in mutant. These data
suggest that differentiation of the insertion site is controlled by Hh signaling in the mouse. Our studies provide new insight
into the role of Hh signaling during the development of tendon.
Program/Abstract # 271
Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling
Maier, Dominic; Cheng, Shuofei; Hipfner, David, IRCM, Montreal, Canada
G-protein-coupled receptor kinases (GRKs) play a conservedrole in Hedgehog (Hh) signaling. In several systems, GRKs
are required forefficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the
intracellular signal generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein
family. In Drosophila, a GRK called Gprk2 is needed for internalization and down regulation of activated Smo, consistent
with thetypical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly
impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To
investigate its function in signaling, we analyzed several different readouts of Hh pathway activity in animals or cells
lackingGprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream
components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a
role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a
level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by
stimulating cAMP production or activating the cAMP-dependent Protein kinase A (PKA). Our results suggest that direct
regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP
regulation, and thus has an indirect effect on the activity of PKA-regulated components of the Hh pathway, including Smo
itself.
Program/Abstract # 272
Do Mek1 and Mek2 regulate distinct functions during mouse development?
Aoidi, Rifdat, Centre de recherche en cancérologie de l'Université Laval, CRCHUQ, Québec, Canada; Catling, Andrew D
(LSU Health Sciences Center, New Orleans, United States); Charron, Jean (Centre de recherche en cancérologie de
l'Université Laval, CRCHUQ, Quebec, Canada)
The mammalian genome contains two ERK/MAP kinase kinase genes, Mek1 and Mek2, encoding dual-specificity kinases
responsible for ERK/MAP kinase activation. Mek1-/-embryo die of placental defects at E10.5, while Mek2-/- and Mek1+/-
micesurvive with a normal lifespan. However most of Mek1+/- Mek2+/-embryos die during gestation of underdevelopment
of the placenta indicatingthat both Mek genes contribute to placental development. Spatio-temporal expression profile, or
difference in MEK1 and MEK2 properties, could explain the specific role of each Mek genes in placental development. To