Abstracts - Society for Developmental Biology

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tube was aberrant. Our data indicate that Foxa1 and Foxa2 are required for the proper formation of the IVD, perhaps by
activating the Hedgehog pathway, which is known to be required for IVD development.
Program/Abstract # 318
Irx3 and Irx5 homeobox genes link the anteroposterior and proximodistal axes prior to hindlimb formation
Li, Danyi, University of Toronto, Toronto, Canada; Sakuma, Rui (The Hospital for Sick Children, Toronto, Canada);
Vakili, Niki (University of Toronto, Toronto, Canada); Mo, Rong; Hopyan, Sevan (The Hospital for Sick Children,
Toronto, Canada)
Development of organs and appendages requires coordination of pattern formation and growth in three dimensions. The
anteroposterior (AP) and proximodistal (PD) axes of the embryonic limb are linked when sonic hedgehog (Shh) from the
zone of polarizing activity (ZPA) and fibroblast growth factors (Fgfs) from the apical ectodermal ridge (AER) form a
positive feedback loop in the developing limb bud. It is unknown whether the AP and PD axes are coordinated before the
feedback loop formation. Here we show that both axes are regulated by Iroquois homeobox (Irx) genes Irx3 and Irx5 prior
to the establishment of the ZPA and the AER. Analysis of Irx3/5 double knockout (DKO) mutants suggested that Irx3/5 are
required early for the formation of Shh-independent elements during hindlimb development. Marker analysis and qRT-
PCR data suggested that Irx3/5 promote Gli3 expression in the initiating hindlimb bud to prepattern the AP axis and
restrict ZPA-Shh activation posteriorly. Using conditional knockout mutants, we demonstrate that the early function of
Irx3/5 in the hindlimb field is essential to specify distal structures. Interestingly, Irx3/5 genetically interact with Gli3 to
promote expression of the PD outgrowth signal Fgf8, further indicating their function in limb development along the PD
axis. Therefore, AP and PD limb axes are coordinated by these transcription factors prior to establishment of signalling
centres.
Program/Abstract # 319
Hyaluronic Acid Synthase 2 expression in the limb mesenchyme is regulated by Shh and plays an essential role in
joint pattern formation
Liu, Jiang, Vanderbilt University, Nashville, United States; Li, Qiang (The University of Texas at Austin, United States);
Litingtung, Ying (Vanderbilt University, Nashville, United States); Vokes, Steven (The University of Texas at Austin,
United States); Chiang, Chin (Vanderbilt University, Nashville, United States)
Sonic hedgehog (Shh) signal generated from the zone of polarizing activity plays an essential role in growth and patterning
of the autopods. However, major gaps remain in understanding the molecular mechanism by which Shh activity regulates
limb development. Through analysis of early limb bud transcriptome, we identified a posteriorly-enriched gene,
Hyaluronan Acid Synthase 2 (Has2), as a potential effector of Shh signaling during early mouse limb development.
BothRNA in situ hybridization and chromatin immunoprecipitation experiments indicate that Has2 is transcriptionally
regulated by Gli3 transcription factor. After analyzing Has2 conditional mutant (Has2cko) mice, we found that Has2 itself
is dispensable for A-P digit patterning. However, Has2cko mice displayed a profound phalange patterning defect in which
joints have been shifted perpendicularly. Associated with the joint patterning defect is misexpression of joint markers such
as Gdf5, Wnt4, Chrd. Further analysis indicates that p-Erk1/2 and cleaved-caspase 3 are ectopically distributed in the
center of mutant digits, suggesting that Has2 may prevent p-Erk-induced apoptosis in condensed chondrocytes. Our results
reveal Has2 as a downstream target of Shh signaling and an essential regulator in early joint patterning.
Program/Abstract # 320
Characterizing the role of Pitx1, Tbx4 and Tbx5 genes in regulation of limb growth, patterning and identity
Nemec, Stephen; Drouin, Jacques, Institut de Recherches Cliniques de Montréal, Montreal, PQ, Canada
The genetic programs involved in limb development direct the growth and patterning of complex and heterogeneous
anatomical structures. While a conserved, generic limb development program underpins the development of all limbs,
recent research regarding specification of limb identity has focused on three transcription factor genes with limb-specific
expression patterns: Pitx1, Tbx4 and Tbx5. Tbx5 is expressed in forelimbs (FL), while Tbx4 and Pitx1 are expressed in
hindlimbs (HL). Tbx5 is necessary for FL bud growth, as Tbx5 -/- mice fail to develop FL at all. Pitx1, meanwhile, has a
profound role in HL development: Pitx1 -/- mice develop HL with FL-like anatomical features. Pitx1 expression also partly
controls Tbx4 expression: gain-of-function experiments in mouse show that Tbx4 has a more ambiguous role than Tbx5 or
Pitx1, directing both growth and patterning. Tbx4 can replace Tbx5 in FL as a growth promoter, rescuing growth in the
Tbx5 -/- mutant FL without affecting patterning. Expressing Tbx4 in the Pitx1 -/- mouse HL, however, rescues several HL
patterning characteristics, an effect that cannot be replicated by Tbx5. This ambiguous activity has been attributed to the
fact that, while Tbx4 and Tbx5 share a transcriptional activation domain that may control growth, Tbx4 has aC-terminal
repressor domain that is not present in Tbx5. We are currently analyzing the targets of each of these transcription factors