Abstracts - Society for Developmental Biology

51
malformations in murine limb buds
Paradis, France Helene; Hales, Barbara, McGill University, Montreal, Canada
Valproic acid (VPA), a common anticonvulsant and antidepressant, is an established human teratogen, causing spina bifida
and limb malformations. It is also a known inhibitor of histone deacetylases (HDACs) which are involved in chromatin
remodeling and cellular signaling. Other HDAC inhibitors have been shown to cause apoptosis in cancer cell lines. We
hypothesize that VPA-induced HDAC inhibition triggers cellular apoptosis, leading to limb teratogenesis. To test this
hypothesis, we compared the effects of VPA and its inactive analog, valpromide (VPD), using an in vitro limb bud culture
system. GD12 murine embryonic forelimbs were cultured in the absence or presence of VPA or VPD(0.6, 1.8 or 3.6 mM)
for 6 days and stained with toluidine blue. Limbs were cultured for 1, 3, 6 or 12h and used for Western blot quantification
of histone4 acetylation, p53 acetylation and cleaved-caspases 9 and 3 or for the mRNA quantification of p53 target genes,
Bcl2 and Survivin, by qRT-PCR. VPA significantly increased limb malformations that included oligodactyly and missing
digits. At 3h, VPA induced the hyper acetylation of both histone 4 and p53. At 6h, both Bcl2 and Survivin were
downregulated and at 12h caspases 9 and3 were both activated. In contrast, VPD caused a small significant effect on limbs
only in the highest concentration group; no changes were observed in the acetylation of histone 4 or the cleavage of
caspase 3 at any concentration.Together, these data show a sequential activation of the intrinsic apoptotic pathway and
suggest that VPA induces apoptosis via p53 acetylation and activation of the downstream pathway. We propose this plays
a role in VPA-induced teratogenesis. These studies were supported by CIHR and FRSQ.
Program/Abstract # 156
Renal lineage and self-renewing potential of GDNF-expressing cells
Cebrian, Cristina, Columbia University, New York, United States; Asai, Naoya (Nagoya University Graduate School of
Medicine, Nagoya, Japan); D'Agati, Vivette; Costantini, Frank (Columbia University, New York, United States)
Nephrogenesis initiates when the ureteric bud (UB), invades the metanephric mesenchyme (MM), inducing it to
epithelialize and differentiate into nephrons. GDNF, expressed in the MM, is a critical factor for renal development and
signals to the UB tips via Ret receptors. We generated a mouse line for inducible Cre-mediated recombination in GDNFexpressing
cells, and used it to show that these cells are self-renewing progenitors that also give rise to the condensing
mesenchyme, nephron tubules, Bowman’s capsule and podocytes. Interestingly, between E7 and E9, GDNF-expressing
cells contribute to the UB lineage, while after E9 they contribute exclusively to the MM lineage. To test whether
progenitor number is important for nephron number and kidney size, GDNF-expressing cells were depleted by mating
GDNF-Cre-ERT2 and R26DTA mice followed by Tamoxifen injection. When the vast majority of progenitors are ablated
(Tam injection at E9.5), the kidneys fail to form. If recombination is induced later, at E12.5, only a subset of progenitors is
ablated. At birth, these animals present a significant reduction in kidney size and nephron number. By adulthood, kidney
size recovers to ~80%of normal, but the nephron number fails to recover, remaining