Regulation of Hedgehog signaling: a complex story

Abstract

The Hedgehog (Hh) signal transduction pathway plays critical instructional roles during development. Activating mutations in human Hh signaling components predispose to a variety of tumor types, and have been observed in sporadic tumors occurring in a wide range of organs. Multiple insights into the regulation of Hh signaling have been achieved through studies using Drosophila melanogaster as a model organism. In Drosophila, regulation of the transcription factor Cubitus interruptus (Ci) is the ultimate target of the Hh pathway. Ci is regulated through communication of the membrane proteins Patched (Ptc) and Smoothened (Smo) to the intracellular Hedgehog Signaling Complex (HSC) in response to a graded concentration of Hh ligand. The HSC consists of the Kinesin Related Protein, Costal2 (Cos2), the serine-threonine protein kinase. Fused (Fu) and Ci. In the absence of Hh stimulation, the HSC is involved in processing of Ci to a truncated repressor protein. In response to Hh binding to Ptc, processing of Ci is blocked to allow for accumulation of full-length Ci activator protein(s). Differential concentrations of Hh ligand stimulate production of Ci transcriptional activators of varying strength, which facilitate activation of distinct subsets of target genes. The mechanism(s) by which Ptc and Smo communicate with the HSC in response to differential ligand concentrations to regulate Ci function are not yet fully elucidated. Here, we review what is known about regulation of individual Hh signaling components, concentrating on the mechanisms by which the Hh signal is propagated through Smo to the HSC.

Fig. 1

Differing levels of Hedgehog stimulation specify distinct cell fates. Hh diffusing into a field of cells specifies differing cell fates in cells near and far from the Hh source. In the Drosophila wing imaginal disc, the level of Hh stimulation controls target gene activation by regulating the relative amounts of Ci transcriptional repressors (Ci₇₅) and activators (Ci^act and Ci*). Ci transcription factors then control expression of specific subsets of genes to establish cell fate for development of specific adult structures. iro: iroquois; col: collier; kn: knot; ptc: patched; en: engrailed.

Fig. 2

Functional domains of Hh signaling components. Indicated are the consensus interaction domains for each of the trimeric HSC components. Additional low affinity interactions that are not indicated may exist, and be involved in maintenance of HSC integrity. (A) Cubitus interruptus (Ci). Schematic of the transcription factor Ci, diagramming the Zinc finger domain [54], putative cleavage site [32] and activation domain. The putative nuclear localization sequence (amino acids 581–616 [96]) is indicated in by a green bar. The putative nuclear export sequence (amino acids 675–860 [97]) is indicated by the red bar. Interaction domains for binding partners CBP [70], Su(fu) and Cos2 [83,84] are also indicated. (B) Costal2 (Cos2). Schematic of the KRP Cos2, diagramming the motor domain, coiled-coil and cargo domains. Also indicated are interaction domains for Hh pathway binding partners Smo [75,76,94,95], Fu and Ci [83,84]. (C) Fused (Fu). Schematic of the serine/threonine protein kinase Fu, diagramming the kinase and carboxyl-terminal functional domains [82]. Also indicated are interaction domains for Hh pathway binding partners Cos2 and Su(fu) [83-85].

Fig. 3

Hedgehog sensitive processing of Cubitus interruptus. (A) Schematic of Ci₁₅₅ and Ci₇₅ depicting the epitope of the 2A1 monoclonal antibody. (B) The Ci gradient in the Drosophila wing imaginal disc. Late third instar wing imaginal discs were immuno-stained with 2A1 monoclonal antibody. Hedgehog diffuses from the Posterior (P) compartment to affect production of Ci activator species in Anterior (A) compartment cells. Cells near the A/P compartment boundary, receiving the highest level of Hh stimulation convert Ci₁₅₅ to the enhanced transcriptional activator Ci*. It is evidenced by decreased detection of Ci by the 2A1 monoclonal antibody. Cells further into the A compartment accumulate Ci^act, as evidenced by more intense Ci staining, which results from decreased Ci proteolysis. Cells far into the A compartment contain higher amounts of Ci₇₅, which is not detected by the 2A1 antibody. (C) Hedgehog sensitive Ci processing is dependent on multiple Hedgehog signaling components. Factors listed in green promote conversion to the indicated Ci species. Factors listed in red prevent conversion.

Fig. 4

Model depicting how the Hh gradient regulates Hedgehog Signaling Complex activator and repressor functions. In the cell receiving no Hh (left cell), HSC-R is producing Ci₇₅ and HSC-A is inactive. In the middle cell, HSC-A is producing Ci^act. However, Ci^act may be acting in the presence of a lower amount of Ci₇₅. In the cell receiving the greatest amount of Hh (right cell), HSC-A is maximally activated by Smo multimerization, and HSC-R is completely off.