Happenchance and hedgehogs

Link to Ingham lab website Philip Ingham biosketch	Link to McMahon lab website	Link to Tabin lab website Clifford Tabin biosketch

In 2013, the European Medicines Agency approved the use of a new anti-cancer drug, Erivedge, developed by Genentech in collaboration with the Cambridge (Mass) based biotech company Curis Inc, for the treatment of Basal Cell Carcinoma (BCC), the most common cancer amongst Caucasians. This decision followed similar approval for its clinical use in the USA by the FDA in 2012. The drug works by targeting a transmembrane protein called Smoothened that is a pivotal component of the intracellular pathway that transduces the activity of a secreted signaling protein known as Sonic hedgehog (Shh;1). Shh plays a key role in initiating the development of hair follicles, being the signal by which epidermal cells of the hair placode induce the dermal papilla in the underlying mesenchyme. BCCs arise when the Shh signaling pathway is inappropriately activated in the basal progenitor cells of the interfollicular epidermis, causing them to behave like constitutively stimulated hair follicle progenitors.

The role of Shh in hair development was originally reported in one of a steady stream of papers that followed the discovery of the SHH gene in the labs of Philip Ingham, Andrew McMahon and Cliff Tabin, the result of a three way transatlantic collaboration funded by HFSP in 1993.

The original motivation for the research that led to this discovery could not have been further removed from its ultimate application: Ingham and McMahon had been studying the expression of two other genes Engrailed, which encodes a transcription factor, and Wnt-1, a proto-oncogene that encodes another secreted signaling protein, in the embryos of Drosophila and mice respectively. Struck by the similar spatial relationships between the domains of expression of the two genes in each species, McMahon speculated that they might form part of a regulatory loop that maintains the midbrain-hindbrain boundary in the vertebrate brain, by analogy with their well established role in maintaining segmental boundaries in the Drosophila embryo.

Ingham’s work had already shown that the Drosophila hedgehog (hh) gene was another key component of this regulatory loop in the fly embryo (1) – so the pair agreed to collaborate in the search for vertebrate homologues of the Drosophila gene to test McMahon’s idea. Aware of Tabin’s interest in identifying vertebrate homologues of hh that might function in the vertebrate limb, McMahon approached his Harvard-based colleague with an invitation to join the team: the motivation for this invitation was not entirely scientific – though based in the US, McMahon like Ingham was a UK citizen so a non-Brit had to be included to comply with HFSP rules¹! This bureaucratic expedient proved highly fortuitous: working with the sequence of the first mouse hh homologue isolated in the McMahon lab (2), the Tabin lab revealed the presence of not one but three distinct hh-like genes through PCR amplification of chicken DNA (3). At the same time the Ingham lab identified a full-length version of one of the Tabin PCR fragments in the zebrafish, and examined its expression by the still relatively novel technique of non-radioactive in situ hybridization.

The results were spectacular: while not expressed where Ingham and McMahon had anticipated at the mid-hindbrain boundary, the Sonic hedgehog gene (as it would become known) was expressed along the length of the notochord (4), a structure underlying the neural tube and known to be the source of a mysterious and elusive signal that specifies the identity of different neuronal progenitors. Intriguingly, it was known from experimental manipulation in the chick embryo that transplantation of pieces of notochord into the developing limb bud could also mimic the effects of transplantation of a region of the limb bud known as the Zone of Polarising Activity or ZPA, causing duplications of digits in the manipulated limb. Within a matter of days, the Tabin lab had repeated the expression analysis of Shh, this time in the chick embryo – the limb buds of which are an order of magnitude bigger than the zebrafish’s rather puny fin buds – and found that its expression corresponds precisely to the location of the ZPA. And within a few more weeks they had shown that misexpression of Shh in the anterior limb bud mimics the effects of ZPA grafts (3).

At the same time, the McMahon and Ingham labs had misexpressed Shh in the neural tubes of mouse and fish embryos to uncover its ability to specify ventral identity on naïve neural progenitors (2, 4). The significance of these findings for the fledgling field of Regenerative Medicine was not lost on the three collaborators and a patent application covering the use of Hh proteins in directing the differentiation of stem or progenitor cells for therapeutic use was filed by their respective institutions. This and a number of other follow-up patents helped facilitate the establishment of a biotech start-up company named Ontogeny that would ultimately merge into Curis Inc. And it was at Curis Inc. that a simple high-throughput screen for Hh pathway agonists and antagonists paved the way for the development of Erivedge in partnership with Genentech.

In the two decades since the landmark discovery of Shh, there has been an explosion of interest in the Hh pathway – hedgehog is now recognized as one of the half dozen or so key signaling pathways underlying animal development. Hh signaling plays pivotal roles in a plethora of developmental and regenerative processes and has been implicated in many cancers besides BCC. The discoveries catalyzed by the outcome of the initial HFSP funded curiosity driven research program have thus had profound effects on the understanding of human development and disease and hold great promise for a range of therapeutic applications in the not too distant future.

By HFSP alumnus Philip Ingham

¹Currently HFSP grant applications only require that participating laboratories are located in different countries. The nationality of team members is no longer relevant.

Papers arising from work supported by the HFSP grant

Krauss, S., Concordet, J-P., and Ingham, P.W. (1993) A Functionally Conserved Homolog of the Drosophila Segment Polarity Gene hedgehog is Expressed in Tissues with Polarising Activity in Zebrafish Embryos. Cell 75 1431-1444

Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA, McMahon AP.  (1993)  Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75:1417-1430

Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993 Dec 31;75(7):1401-16.

Fietz MJ, Concordet JP, Barbosa R, Johnson R, Krauss S, McMahon AP, Tabin C, Ingham PW. The hedgehog gene family in Drosophila and vertebrate development. Dev Suppl. 1994:43-51.

Concordet, J-P., Lewis, K.E.,Moore, J. Goodrich, L.V.Johnson, R., Scott, M.P. and Ingham, P.W. (1996) Spatial Regulation of a Zebrafish patched Homologue Reflects the Roles of Sonic hedgehog and Protein Kinase A in Neural Tube and Somite Patterning.  Development 122: 2835-2846

Currie, P. and Ingham, P. W. (1996) Induction of a specific muscle cell type by a novel hedgehog gene in the zebrafish, Nature 382: 452-455

Johnson RL, Riddle RD, Laufer E, Tabin C. Sonic hedgehog: a key mediator of anterior-posterior patterning of the limb and dorso-ventral patterning of axial embryonic structures. Biochem Soc Trans. 1994 Aug;22(3):569-74

Chan DC, Laufer E, Tabin C, Leder P. Polydactylous limbs in Strong's Luxoid mice result from ectopic polarizing activity. Development. 1995 Jul;121(7):1971-8.

Marigo, V., Roberts, D.J., Lee, S.M.K., Tsukurov, O., Levi, T., Gastier, J.M., Epstein, D.J., Gilbert, D.J., Copeland, N.G., Seidman, C.E., Jenkins, N.A., Seidman, J.G., McMahon, A.P. and Tabin, C. Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog. Genomics 28:44-51 (1995).

Marigo V, Johnson RL, Vortkamp A, Tabin C. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. J. Dev Biol. 1996 Nov 25;180(1):273-83.

Marigo V, Tabin CJ. Regulation of patched by sonic hedgehog in the developing neural tube. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9346-51.

Marigo V, Scott MP, Johnson RL, Goodrich LV, Tabin CJ. Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development. 1996 Apr;122(4):1225-33.

Marigo V, Laufer E, Nelson CE, Riddle RD, Johnson RL, Tabin C. Sonic hedgehog regulates patterning in early embryos. Biochem Soc Symp. 1996;62:51-60

Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin C. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. J.Science. 1996 Aug 2;273(5275):613-22.

Quirk J, van den Heuvel M, Henrique D, Marigo V, Jones TA, Tabin C, Ingham PW. The smoothened gene and hedgehog signal transduction in Drosophila and vertebrate development. Cold Spring Harb Symp Quant Biol. 1997;62:217-26.

Bitgood MJ, McMahon AP.  (1995)  Hedgehog and Bmp genes are co-expressed at many diverse sites of cell-cell interaction in the mouse embryo.  Developmental Biology 172:126-138.

Münsterberg AE, Kitajewski J, Bumcrot DA, McMahon AP, Lassar AB.  (1995)  Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite. Genes and Development 9:2911-2922.

Bitgood MJ, Shen L, McMahon AP.  (1996)  Sertoli cell signaling by Desert hedgehog regulates the male germ line. Current Biology, 6:298-304.  

Bumcrot DA, Takada R, McMahon AP.  (1995)  Proteolytic Processing Yields Two Secreted Forms of Sonic hedgehog. Molecular and Cellular Biology 15:2294-2303.

Marti E, Takada R, Bumcrot DA, Sasaki H, McMahon AP.  (1995)  Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121:2537-2547.

Norris W, Neyt C, Ingham PW, Currie PD. (2000) Slow muscle induction by Hedgehog signalling in vitro. J Cell Sci. 113:2695-703.