Congress Abstracts - Society for Developmental Biology

Program/Abstract # 340
Mapping the functional domains in LRP2, an auxiliary SHH receptor in the developing neuroepithelium
Christa, Anna; Christ, Annabel ; Hammes, Annette; Willnow, Thomas (Max-Delbrueck-Center for Molecular Medicine, Germany)
Sonic hedgehog (SHH) is a key morphogen in mammalian brain development. It acts by binding to its cognate receptor patched 1,
resulting in down-stream signal transduction through smoothened and GLI transcription factors. Intriguingly, several membraneassociated
proteins have been identified recently that act as SHH binding proteins and as co-receptors to patched 1 in the developing
nervous system. LRP2, a member of the LDL receptor gene family, is such an auxiliary SHH receptor expressed on the apical surface
of the neural tube. Loss of LRP2 expression in mouse models results in the inability of the neuroepithelium to respond to SHH despite
proper expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increase SHH signaling. Using
biochemical studies and cell-based reporter assays, we now have characterized the functional domains in LRP2 required to promote
SHH signaling. Thus, we mapped several bindings sites for SHH to the complement-type repeats in the extracellular domain of LRP2,
suggesting multivalent ligand interaction. In addition, we document the ability of LRP2 to directly interact with patched 1 through its
EGF precursor homology domains, an interaction that proceeds in the absence of SHH. Generation of an LRP2 mini receptor variant
that carries a single SHH binding site and one EGF precursor homology domain for patched 1 interaction is sufficient to enhance SHH
signaling in cells. Our data suggest a mechanistic model whereby LRP2 and patched 1 exist as a preformed co-receptor complex at the
neuroepithelial cell surface and that this co-receptor complex initially interacts with SHH through multiple bindings sites located in
the extracellular domain of LRP2.
Program/Abstract # 341
LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline
Christ, Annabel; Christa, Anna; Willnow, Thomas; Hammes, Annette (Max-Delbrueck-Centrum, Germany)
Sonic hedgehog (SHH) is a regulator of forebrain development and genetic defects in Shh and in components of its cellular signaling
machinery lead to a broad spectrum of brain malformations. Anomalies observed in patients and in rodent models include
holoprosencephaly (HPE), a failure in midline induction resulting in the lack of forebrain separation into two hemispheres. Still little
is known about the mechanisms at early neurulation whereby SHH from the prechordal plate governs specification of the rostral
diencephalon ventral midline (RDVM), a major forebrain organizer. Here, we identified LRP2, a member of the LDL receptor gene
family as intricate component of the SHH signaling machinery in the RDVM. LRP2 acts as apical SHH binding protein that sequesters
the morphogen in this target field. Binding to LRP2 at the base of the primary cilium enables interaction of SHH with its receptor
patched 1, resulting in internalization of SHH/patched 1 complexes and subsequent signal transduction through smoothened. As well
as mediating SHH and patched 1 interaction, LRP2-dependent internalization delivers SHH molecules to the recycling compartment
of the cell suggesting re-secretion as means to further increase local morphogen concentrations. In line with a critical role for LRP2 in
SHH trafficking and signaling in the RDVM, lack of this receptor in mice and in patients results in failure to respond to SHH, and in
forebrain formation defects and HPE. Our data substantiate the emerging concept that auxiliary receptors are critical modulators of
morphogen signal reception in target tissues, and identified an important role for LRP2 in SHH action in the forebrain.
Program/Abstract # 342
Transcriptional regulation of Shh target genes in the developing spinal cord
Kurdija, Sanja, KI; Oosterveen, Tony; Alekseenko, Zhanna; Uhde, Christopher; Sandberg, Magnus; Andersson, Elisabet; Bergsland,
Maria; Dias, José; Muhr, Jonas; Ericson, Johan (Karolinska Institutet, Sweden)
Secreted protein Sonic hedgehog (Shh) acts in a graded fashion at long-range to establish cell pattern in several tissues but little is
known about how these concentration dependent mechanisms function on a transcriptional level. We have identified the cis-regulatory
modules (CRM) of neural Shh-target genes, which we use as tools to elucidate the mechanisms imposed by Gli proteins, the
bifunctional transcriptional mediators of Shh gradient. We find that Gli activators have a non-instructive role in long-range patterning
and in synergy with SoxB1 proteins activate Shh target genes in a largely concentration independent manner. Instead, Gli repressors
are interpreted at transcriptional level into precise spatial gene patterns in combination with regional homeodomain co-repressors.
Moreover, the local interpretation of Shh displays lower CRM context sensitivity and requires Gli activators to accumulate to a
threshold level sufficient to counteract Gli repressors. Thus our data propose a novel mechanism for transcriptional interpretation of
Shh gradient.
Program/Abstract # 343
Following a transient dose, Sonic Hedgehog function in normal digit formation is dispensable and can be substituted entirely
by enforced cell survival
Mackem, Susan; Zhu, Jianjian (National Cancer Institute, USA)
Different approaches have led to very different models of how Sonic Hedgehog (Shh) functions as a limb morphogen.
Pharmacological studies in chick and some genetic analysis in mouse support a requirement for sustained Shh activity integrated over
time to specify digit pattern. However, timed genetic deletion of Shh in mouse indicates only a very transient requirement in
patterning. In both models, Shh is required for limb bud expansion to produce a normal number of digits; they differ in how these two
roles are integrated. To directly evaluate the contribution of temporal integration to Shh function in digit patterning, we restored cell
98