Abstracts - Society for Developmental Biology

103
Sonic Hedgehog (Shh) is a secreted protein that plays a critical role in ventral CNS development. In the spinal cord, there
are two tissue sources of Shh: the notochord (ShhNOTO) and floor plate (ShhFP). Prior studies showed a critical role for
Shh in the patterned expression of bHLH and HD proteins; however, the requirement of each source has not been clearly
defined. To address this, we employed cre-loxp mediated recombination to selectively inactivate ShhFP while preserving
ShhNOTO (ShhΔFP). Using double labeled ShhΔFP/ΔFP mutant tissue at E10.5, we found fewer Nkx2.2+ and Olig2+
cells marking the p3 and pMN domains, respectively, but progenitor domain patterns remain largely intact. At E11 during
the transition from neurogenesis to gliogenesis, the notochord is initially in contact with the neural tube but then separates,
leaving ShhFP as the only intrinsic source. Strikingly, ShhΔFP/ΔFP mutants at E12.5 show a complete loss of Olig2+ cells.
We examined the consequences of this on oligodendrocyte (OL) development at E15.5 and E18.5 and found a decrease in
both OL precursor cells (OPC) and OL marker protein. Shh controls target gene expression by promoting Gli activators
and inhibiting Gli repressor functions. To address the genetic mechanism of how ShhFP controls OPC protein marker
expression, we generated mice mutant to both ShhFP and Gli3, the sole Gli repressor. Significantly, double mutants show a
partial recovery of Olig2+ OPCs, showing that ShhFP is specifically required to suppress the formation of Gli3 repressors
and allow Olig2 expression. Taken together, our results demonstrate a specific requirement of ShhFP for OL specification
and differentiation.
Program/Abstract # 313
PTCH1, PTCH2, and HHIP1 feedback antagonism is required for Hedgehog-dependent vertebrate neural
patterning
Holtz, Alexander M., University of Michigan Cell and Molecular Biology, Ann Arbor, United States; McMahon, Andrew
P. (Harvard University, Cambridge, MA, United States); Allen, Benjamin L. (University of Michigan, Ann Arbor, MI,
United States)
Hedgehog (HH) signaling plays critical roles in both invertebrate and vertebrate embryogenesis. Sonic Hedgehog (SHH)
ligand specifies distinct ventral neuronal identities within the developing vertebrate neural tube. Precise mechanisms exist
to limit the range of HH signaling during development, including feedback upregulation of the HH receptor, PTCH1, and
an additional HH-binding protein, HHIP1. While feedback antagonism of PTCH1 or HHIP1 alone is dispensable for
normal neural patterning, combined loss of PTCH1 and HHIP1 inhibition produces a dramatic expansion of the HHresponsive
domain. This suggests that restraining the HH response during vertebrate embryogenesis is governed by
overlapping activities of multiple cell surface HH pathway antagonists. To determine whether additional cell surface
antagonists control HH signaling during ventral neural patterning, we have examined the vertebrate-specific protein,
PTCH2. The contribution of PTCH2 to HH pathway antagonism during ventral neural patterning is unknown. Using cellbased
functional assays and chick in ovo neural tube electroporations, we show that PTCH2 can function as a HH-pathway
antagonist. Intriguingly, PTCH2 localizes to the primary cilium, an organelle required for HH pathway function. Although
neural patterning is normal in the absence of PTCH2, combined loss of PTCH2 and PTCH1-feedback upregulation
increases the range of HH signaling in the developing mouse neural tube. Strikingly, combined loss of PTCH1, PTCH2,
and HHIP1 feedback antagonism results in complete ventralization of the neural tube, revealing an essential role for these
vital antagonists during vertebrate embryogenesis.
Program/Abstract # 314
Endodermal requirement for Prdm1 in mouse craniofacial development
Lamonica, Kristi, UC Denver Dept of Craniofacial Biology, Aurora, United States; Clouthier, David; Artinger, Kristin
(UC Denver Department of Craniofacial Biology, Aurora, CO, United States)
Cranial neural crest cells (CNCCs) populate the pharyngeal arches (PAs) during development and subsequent interactions
with the endoderm and ectoderm of the PAs is critical for formation of the adult craniofacial skeleton. Prdm1 (Blimp1) is a
zinc finger containing transcription factor with a known role in zebrafish neural crest and craniofacial development. Loss
of prdm1a in Danio rerio leads to a loss of posterior arch derived ceratobranchials 2-5 and functions downstream of FGF
and RA signaling. To address tissue specific requirements of Prdm1 in mammalian craniofacial development, we are
utilizing tissue specific Cre- mediated recombination to knockout Prmd1 in the mouse model system. Unlike in zebrafish
where prdm1ais expressed in all tissues of the arches, mouse Prdm1 is expressed in the pharyngeal endoderm and ectoderm
and Prdm1fl/fl; Foxa2-Cre endoderm deletion leads to morphological defects in the craniofacial region. Gene expression
analysis at E10.5 shows increased Fgf8 mRNA expression in the cleft between mandibular and maxillary arch 1, along
with decreased expression of Nkx3.2 and Eya, suggesting a defect in the formation of the temporal mandibular joint
(TMJ). In the posterior arches, we observe decreased Eya1 and Tbx1 throughout the arches and we hypothesize that Prdm1
expression in the endoderm is necessary for appropriate patterning of CNCCs and their derivatives. Taken together, our