Research

Current Projects

Research Overview

Research in the Allen Lab is broadly focused on understanding the mechanisms of growth factor and morphogen signaling in development and disease. Specifically, we study the regulation of Hedgehog (Hh) signaling during vertebrate embryogenesis using a wide range of approaches, including mouse developmental genetics, chick in ovo electroporation, biochemistry, and cell biology. The long-term goal of our research is to use insights gained from the study of Hh signaling in normal development in the treatment of a broad spectrum of developmental diseases and childhood and adult cancers.

Shh-dependent specification of the ventral neural tube during mouse embryogenesis

In the developing mouse neural tube, Sonic Hedgehog (Shh) is initially expressed in the notochord (NC) underlying the ventral neural tube; as development progresses, Shh auto-induces a secondary domain of Shh production within the floor plate (FP)  of the neural tube at the ventral midline.  Several lines of evidence indicate that Shh acts in a concentration dependent manner to specify all ventral cell types of the developing neural tube (PV3, PMN, PV2, PV1, PV0).  Importantly, markers exist for these cell types (center panel), allowing for both qualitative and quantitative assesment of alterations in Shh signaling during neural patterning.  A representative example of such marker analysis is shown in the right panel.  Yellow, FoxA2; purple, Nkx2.2; green, Olig2; Blue, Nkx6.1; Red, Pax7.

Current model of cell surface regulation of the vertebrate Hh signaling pathway

Top panel: In the absence of Hh, Ptch1 inhibits the activity of Smo. Gas1, Cdo and Boc expression sensitizes cells to low levels of ligand and permits full Hh pathway activation.   Bottom panel: Hh binding to Ptch1 results in de-repression of Smo and activation of a signal transduction cascade that culminates in the transcriptional modulation of Hh target genes, including upregulation of Hh antagonists (Ptch1, Ptch2, Hhip1), and downregulation of positive acting Hh pathway components (Gas1, Cdo, Boc).

Current projects in the lab include:

Functional analysis of three novel Hh co-receptors, Gas1, Cdo and Boc.

Recent work from our lab has identified Gas1, Cdo, and Boc as novel Hh co-receptors that are essential to transduce the Hh signal during numerous developmental processes, including neural patterning, craniofacial development, and digit specification.  Despite strong genetic evidence that these proteins are essential components of the Hh pathway, little data exist to explain the mechanisms of action of these molecules.  There are currently opportunities in the lab for detailed examination of the mode of action of these novel pathway components and their roles in different Hh-dependent developmental processes.  

Gas1, Cdo and Boc promote Shh-dependent cell fate specification in the chick neural tube

HH stage 22 chick neural tubes electropolated with either control vector, Gas1, Cdo, Boc or SmoM2 were sectioned at the forelimb level and stained with antibodies raised against Nkx.2(red). GFP expression (green) denotes cells expressing the indicated contstruct. Arrows indicate extopic expression of Shh-dependent markers. Note that ectopic expression of SmoM2 result in epression of ectopic Nkx2.2 in the dorsal Neural tube, while the effects of ectopic Gas1, Cdo and Boc are restriced ventrally.

Complete loss of Shh signaling in the ventral neural tue of E9.5 Gas1-/-; Cdo;-/-; Boc-/- embryos

Antibody detection of Shh(top row; green0, Foxa (middle row; red) and Nkx2.2 and Olig2 (bottom row; red and green respecively) in forelimb level sections of E9.5 mouse embryos. Embryo genotypes are listed at the top of each column.

 

 

 

 

 

 

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Characterization of the cell surface Hh antagonists Ptch1, Ptch2 and Hhip1.

In addition to its essential role as the canonical Hh receptor, Ptch1 also functions as a critical antagonist of the Hh pathway in multiple tissues.  Previous studies also identified Hhip1 as a second Hh antagonist, while recent studies in our lab indicate that Ptch2 also functions to limit activation of the Hh pathway.  In fact, the simultaneous removal of Ptch1, Ptch2 and Hhip1 antagonist functions result in constitutive Hh signaling.  Similar to Gas1, Cdo and Boc, opportunities exist for detailed functional studies to explain the mechanisms of action of these vital antagonists of the Hh pathway.

Complete ventralization of the neural tube in E10.5 Ptch2-/-; MtPtch1; Ptch1-/-; Hhip1-/- embryos

Antibody detection of FoxA2(yellow), Nkx2.2(green), Olig2(blue) and Nkx6.1 (red) in forelimb level sections of E10.5 mouse embyos. Merged images of all markers are shown at the far right. Note that Olig2 expression in restricted dorsally in embryos lacking all cell surface antagonism of Shh signaling(bottom middle panel). Also note the mixing of Nkx6.1 single positive cells throughout the neural tube(bottom right panel).

 

 

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Identification of novel cell surface Hh pathway components and characterization of the Hh receptorsome.

The recent identification of several novel Hh co-receptors (Gas1, Cdo and Boc) and the existence of multiple Hh patthway antagonists (Ptch1, Ptch2, and Hhip1) raise the questions of what constitutes the complete Hh signal reception complex and whether additional cell surface regulators of the Hh pathway await discovery.  We currently seek someone to pursue a mass-spectrometry based approach to identify the Hh receptorsome, and to search for novel general and tissue-specific components of the Hh receptor complex.

Identification of the Hh receptorsome.

Recent studies have identified multiple novel cell surface components of the Hh pathway, including the Hh co-receptors Gas1, Cdo, and Boc. These studies raise several questions, including: What proteins comprise the complete Hh receptor complex? Do the compnents of the Hh receptor complex differ between different Hh-responsive tissues? Does the composition of the receptor complex change in response to Hh signaling? To address this, we are using a mass spectrometry-based approach in both cells and tissues to identify novel pathway components.

 

 

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Examination of the role of cilia in Hh signaling.

Recenty, the primary cilium, a previously underappreciated cellular organelle, has emerged as an essential part of the Hh signal transduction cascade.  Importantly, a number of Hh pathway components, including the cell surface proteins Ptch1 and Smo dynamically localize to this structure during the Hh response. We seek people interested in examining the role of the primary cilium in the function of other cell surface Hh components.

Ciliary localization of cell-surface Hh-binding proteins.

Several Hh pathway components, including Smo (top left), Ptch1, and Gli2 (top right) dynamicaly localize to the primary cilium (center) in response to Hh signaling. We are currently exploring whether the cell surface Hh co-receptors Gas1, Cdo and Boc (left), as well as the Hh antagonists Hhip1 and Ptch2 (right) also localize to cilia, or regulate the localization of other Hh pathway components to cilia.

 

 

 

 

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