Congress Abstracts - Society for Developmental Biology

expression was observed in the limb buds close to but not within the cartilage elements, we focused on the effect of EVI-1 on
chondrogenesis in stage 24 limb micromass cultures. Cells were transfected with an siRNA to target chicken EVI-1 at the time of
plating. After six days of cultivation, the level of chondrogenesis was evaluated in cultures by Alcian blue staining. Perinodular
inhibition of chondrogenesis in cultures was observed. Downregulation of EVI-1 after siRNA treatment was confirmed by qPCR.
Since EVI-1 was expressed adjacent to the apical ectodermal ridge and this structure is a source of FGFs, we wanted to analyse the
influence of FGFR inhibitors on EVI-1 gene expression. Local injection of FGF inhibitor PD161570 into the limb bud at stages 20–22,
downregulated EVI-1 expression was determined by qPCR. Next, we performed gain-of-function experiment, where FGF2 beads were
implanted into the right wings at stage 20. We observed upregulation of EVI-1 expression 16h after treatment. In summary, it was
found that EVI-1 levels need to be maintained in order for chondrogenesis to occur. In addition EVI-1 may be a novel gene mediating
the effects of FGF on chondrogenesis. This work was supported by GACR (grant 304/09/0725 ) to MB and CIHR grant to JMR.
Program/Abstract # 244
Effects of homocysteine on mesenchymal cells during limb development on chick embryos
Bourckhardt, Gilian; Kobus, Karoline; Cecchini, Manuela; Müller, Yara; Ammar, Dib; Nazari, Evelise (Universidade Federal de
Santa Catarina (UFSC), Brazil)
Hyperhomocysteinemia is a metabolic condition resultant of folic acid dietary deficiency. This condition is related with the occurrence
of congenital anomalies that include limb defects. High levels of homocysteine (Hcy) can induce DNA damage and cell cycle arrest
due to non-remethylation of Hcy to methionine. The aim of this study was to investigate whether high levels of Hcy can affect the
mesenchymal cell dynamics d uring limb development. Chick embryos were treated with 20 µmol D-L Hcy/50 µL saline at E2 and
analyzed at E6. Control embryos were treated with 50 µL saline. To identify cells in proliferation and proteins involved in cell cycle
we performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (mitosis marker), anti-p53,
anti-p21 and anti-PCNA. No significant differences on cell proliferation rate were observed between Hcy-treated and control embryos.
Thus, we observed a downregulation of proliferating cell nuclear antigen (PCNA) and the p21 protein, both involved in the G1 phase
of cell cycle progression. On the other hand, the Hcy induces in mesenchymal cells of the limbs, an upregulation in expression of p53
protein, which can be activated by DNA damage. Additionally, we observed an increase of apoptosis rates. Our results indicate that
the Hcy-treatment changes the mesenchymal cell dynamics during limb development of the G. domesticus.
Program/Abstract # 245
Inhibition of Hedgehog Signaling is Necessary for ß-Catenin-Regulated Interzone Differentiation and Joing Morphogenesis
Rockel, Jason; Yu, Chunying; Whetstone, Heather (The Hospital for Sick Children, Canada); Craft, April (University Health Network,
Canada); Reilly, Katherine; Alman, Benjamin (The Hospital for Sick Children, Canada)
The mechanisms responsible for articular chondrocyte (AC) development are incompletely elucidated. ACs derive from Gdf5-
expressing interzone cells and differentiate through a distinct pathway compared growth plate chondrocytes (GPCs), which do not
derive from interzone cells . Hedgehog (HH) signalling is active in chondrocytes, primarily in GPCs. In osteoarthritis, a degenerative
disease of articular cartilage, the HH-regulated GPC developmental program is r ecapitulated in ACs. Thus inhibition of HH
signalling may be necessary for normal interzone cell differentiation, joint morphogenesis, and the maintenance of ACs. Using
transgenic mice and ex vivo cultured embryos, we found that inhibition of HH signalling maintained interzone populations cell
autonomously but had no effect on joint or skeletal morphogenesis. In contrast, activation of HH signalling inhibited interzone cell
differentiation and maintenance in a cell non-autonomous manner. Interestingly, transgenic mice that had activated HH signalling in
interzone cells developed osteochondrodysplasias and morphological abnormalities including ectopic joint cartilage, reduced AC
differentiation and undifferentiated cells within the joint space. HH signalling also reduced Wnt/β -catenin activity in interzone
progeny. Constitutive activation of β-catenin rescued HH-induced knee joint abnormalities and partially rescued the
osteochondrodysplasias. Treatment of hindlimb organ cultures with FGF18, a β-catenin target gene, also rescued HH-induced joint
abnormalities. These data indicate that HH signalling needs to be downregulated in interzone cells for β-catenin-regulated AC
differentiation and joint morphogenesis.
Program/Abstract # 246
Characterizing gene expression dynamics between Shox2 and Hox genes during limb development
Neufeld, Stanley John (University of Calgary, Canada), Scott, Alexandra; Wang, Fan (Durham, USA); Cobb, John (University of
Calgary, Canada)
The proper development of the vertebrate limb relies on homeobox genes of both the Hox and Shox families of genes. In mice and
humans, mutation of certain members of either gene family results in similar phenotypes, such as malformed or shortened limb
segments. We have previously established that Shox2 and Hox genes genetically interact in the mouse limb, supporting the view that
these genes function together. To gain further insight, we are analyzing their relative expression dynamics through double mRNA
FISH in whole embryos. This analysis reveals an intriguing pattern where Shox2 expression prominently overlaps with proximalacting
Hox genes, and is complementary to the expression of distal-acting Hox genes. This dichotomy is established as early as E10.5,
suggesting that these relative dynamics could be important for the proper development of the discrete segments of the limb. We are
currently assessing the possibility of cross-regulatory control between Hox genes and Shox2, and also the possibility that these genes
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