In this work, we identify physical and genetic interactions that implicate E3 identified by differential display (EDD) in promoting spindle assembly checkpoint (SAC) function. During mitosis, the SAC initiates a mitotic checkpoint in response to chromosomes with kinetochores unattached to spindle pole microtubules. Similar to Budding uninhibited by benzimidazoles-related 1 (BUBR1) siRNA, a bona fide SAC component, EDD siRNA abrogated G₂/M accumulation in response to the mitotic destabilizing agent nocodazole. Furthermore, EDD siRNA reduced mitotic cell viability and, in nocodazole-treated cells, increased expression of the promitotic progression protein cell division cycle 20 (CDC20). Copurification studies also identified physical interactions with CDC20, BUBR1, and other components of the SAC. Taken together, these observations highlight the potential role of EDD in regulating mitotic progression and the cellular response to perturbed mitosis.

Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling.

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.

This review highlights the essential role of Hedgehog (Hh) signalling in the developmental steps of temporomandibular joint (TMJ) formation. We review evidence for intra- and potentially inter-tissue Hh signaling as well as Glioma-Associated Oncogene Homolog (GLI) dependent and independent functions. Morphogenesis and maturation of the TMJ’s individual components and the general landscape of Hh signalling is also covered. Comparison of the appendicular knee and axial TMJ also reveals interesting differences and similarities in their mechanisms of development, chondrogenesis and reliance on Hh signalling.