Abstract
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.
Highlights
In multicellular organisms Hedgehog (Hh) morphogens play an essential role in tissue/organ development [1] and their subsequent maintenance [2]
6-well plates containing 2x106 cells in antibiotic-free GMEM were transfected with 4μg of pLKO-based, puromycin-expressing, Ubr5 shRNAi constructs (Sigma MISSION, TRCN0000003411 (Ubr5.1), TRCN0000226458 (Ubr5.2) and TRCN0000238587 (Ubr5.3) with Effectene (Qiagen) as per the manufacturer’s instruction. 24 hours later, transfected cells were selected in 0.1μg/ml puromycin (Sigma) and resistant colonies were pooled
Based upon the ability of Hyperplastic Discs (Hyd), the Drosophila orthologue of Ubiquitin Protein Ligase E3 Component N-recognin 5 (UBR5), to influence hh ligand expression we wished to examine whether murine UBR5 was capable of regulating Sonic hedgehog (Shh) expression
Summary
In multicellular organisms Hedgehog (Hh) morphogens play an essential role in tissue/organ development [1] and their subsequent maintenance [2]. Tight regulation of Hh ligand expression ensures temporal-spatial generation of morphogen gradients, which in turn ensure a well co-ordinated and appropriate cellular response [3]. Engagement of Hh ligands with one of their receptors Patched (Ptch1) [6,7,8] results in derepression of the Hedgehog signal transduction pathway, activation of the GLI family zinc finger (GLI) family of transcription factors and the subsequent transcription of GLI target genes. Activation of the Hh pathway (HhP) influences a wide range of cellular responses that include promotion of proliferation, differentiation and suppression of apoptosis[1]. Hedgehog signalling affects cell behaviour in multiple tissues and is heavily implicated in the communication between cells, including adult stem cells and their niches[2]
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