The subdivision of the Drosophila wing imaginal disc into anterior and posterior compartments requires a transcriptional response to Hedgehog signaling. However, the genes regulated by Hedgehog signal transduction that mediate the segregation of anterior and posterior cells have not been identified. The previously predicted gene Cad99C has been molecularly characterized and shown to be regulated by Hedgehog signaling. Cad99C encodes a transmembrane protein with a molecular weight of approximately 184 kDa that contains 11 cadherin repeats in its extracellular domain and a conserved type I PDZ-binding site at its C-terminus. The levels of cad99C RNA and protein are low throughout the wing imaginal disc. However, in the pouch region, these levels are elevated in a strip of anterior cells along the A/P boundary where the Hedgehog signal is transduced. Ectopic expression of Hedgehog, or the Hedgehog-regulated transcription factor Cubitus interruptus, induces high-level expression of Cad99C. Conversely, blocking Hedgehog signal transduction by either inactivating Smoothened or Cubitus interruptus reduces high-level Cad99C expression. Finally, by analyzing mutant clones of cells, it was shown that Cad99C is not essential for cell segregation at the A/P boundary. It is concluded that cad99C is a novel Hedgehog-regulated gene encoding a member of the cadherin superfamily in Drosophila (Schlichting, 2005).
Cad99C is expressed at low levels throughout the wing, haltere, and leg imaginal discs, whereas elevated levels of Cad99C expression are confined to a strip of cells along the A/P boundary of the wing imaginal disc pouch that is known to respond to the Hh signal. Even though anterior cells along the A/P boundary of haltere and leg imaginal discs as well as cells outside the pouch region of wing imaginal discs also respond to the Hh signal, no elevated level of Cad99C was observed in these cells, indicating that cad99C is a region-specific Hh target gene. The Cad99C protein profile resembles cad99C RNA levels, indicating that the elevated expression of Cad99C is mainly due to transcriptional and not translational or posttranslational regulation. High-level Cad99C expression was reduced to the low level present in cells far away from the A/P boundary in clones of cells lacking Hh signal transduction due to mutations in either smo or ci. Conversely, ectopic expression of either Hh or Ci was sufficient to increase Cad99C expression in the wing imaginal disc pouch, indicating that high-level cad99C expression is controlled by Ci-mediated Hh signaling (Schlichting, 2005).
Different Hh-regulated genes respond differently to Ci[act] and Ci[rep]. For example, the expression of dpp is regulated both by Ci[act] and Ci[rep], whereas hh and ptc only respond to one form of Ci, Ci[rep] or Ci[act], respectively. Like ptc, cad99c appears to respond exclusively to Ci[act]. This is inferred from five observations: (1) ectopic expression in posterior cells of Ci, which under the influence of Hh is converted to Ci[act], induces high-level cad99C expression; (2) misexpression of a constitutively active form of Ci, CiPKA4, also induces high levels of cad99C expression; (3) ci null mutant clones in the anterior compartment close to the A/P boundary, where Ci[act] is the predominant form of Ci, fail to upregulate cad99C expression; (4) expression of a constitutive repressor form of Ci, CiCell, does not reduce the low-level expression of cad99C; (5) ci null mutant clones in the anterior compartment away from the A/P boundary, where Ci[rep] is the prevailing form of Ci, show no increase in the expression of cad99C. Taken together, it is concluded that cad99C expression is regulated by Ci[act] and not Ci[rep] (Schlichting, 2005).
The segregation of cells at compartment boundaries is thought to depend on the differential adhesiveness (affinity) of cells on both sides of the compartment boundaries. Based on thermodynamic considerations, it has been proposed that cells will maximize the total strength of their adhesive interactions with neighboring cells by replacing weak cell–cell interactions with stronger ones. Cells with strong adhesive interactions will thus associate preferentially with one another and will segregate from less avidly adhering cells. As predicted by this model, cells expressing different levels of the same adhesion molecule segregate from one another. However, few adhesion molecules have been identified that can promote the differential adhesiveness of cells at compartment boundaries (Schlichting, 2005).
The maintenance of the A/P boundary in the developing Drosophila wing requires Ci-mediated Hh signal transduction in anterior cells. This suggests that Hh signaling may regulate the transcription of one or more genes that in turn affect the adhesiveness of anterior cells. Members of the cadherin superfamily are known to mediate adhesion between cells and several cadherins have been shown to be involved in cell segregation. Even though most cadherins implicated so far in cell segregation contain cytoplasmic β-catenin binding sites, which are absent in Cad99C, several cadherins lacking β-catenin binding sites have also been shown to mediate cell segregation. The discovery of a gene that is both regulated by Hh signaling and encodes for a cadherin, therefore, provides an attractive candidate for mediating the segregation of anterior and posterior cells. However, cad99C expression is not elevated in cells along the A/P boundary of haltere and leg imaginal discs or outside the pouch region of wing imaginal discs, indicating that if the elevated expression of Cad99C were important for cell segregation, this could not be a general mechanism for segregating anterior and posterior cells. However, since wing imaginal disc pouch cells differ in their expression profile from wing imaginal disc cells outside of the pouch, it is not inconceivable that different molecules could operate to segregate cells at the A/P boundary in different regions of the wing imaginal disc or in different imaginal discs (Schlichting, 2005).
A mutant allele of cad99C, termed cad99C57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C57A appears to be a null allele of cad99C based on four criteria: (1) sequencing of the genomic DNA revealed that the predicted promoter region, the transcriptional start site, and the coding sequence for the first 101 amino acid residues were deleted; (2) an RNA probe recognizing the 3′ region of the cad99C transcript, outside of the deletion present in cad99C57A, does not show detectable staining in wing imaginal discs from homozygous cad99C57A mutant larvae, indicating that the cad99C transcript levels are highly reduced; (3) an antibody directed to the C-terminus of Cad99C does not recognize a protein of the predicted size for Cad99C in extracts from wing imaginal discs of homozygous cad99C57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C57A mutant clones within wing imaginal discs (Schlichting, 2005).
The identification of cad99C as an Hh-regulated gene provides a starting point to investigate a cell biological mechanism used by Hh signaling to control the development of the Drosophila wing. It also provides a further step towards the functional characterization of all remaining members of the cadherin superfamily present in Drosophila that have so far only been predicted based on the genomic sequence (Schlichting, 2005).
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.