Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway.

During cerebellum development, Sonic hedgehog (Shh)-induced proliferation of cerebellar granular neuronal precursors (CGNPs) is potently inhibited by bone morphogenetic proteins (BMPs). We have previously reported the upregulation of TIEG-1 and Mash1, two antimitotic factors that modulate MYCN transcription and N-Myc activity, in response to BMP2. To gain further insight into the BMP antimitotic mechanism, we used microRNA (miRNA) arrays to compare the miRNAs of CGNPs proliferating in response to Shh with those of CGNPs treated with Shh plus BMP2. The array analysis revealed that miRNA 11 (miR-22) levels significantly increased in cells treated with BMP2. Additionally, in P7 mouse cerebellum, miR-22 distribution mostly recapitulated the combination of BMP2 and BMP4 expression patterns. Accordingly, in CGNP cultures, miR-22 overexpression significantly reduced cell proliferation, whereas miR-22 suppression diminished BMP2 antiproliferative activity. In contrast to BMP2, miR-22 did not induce neural differentiation but instead significantly increased cell cycle length. Consistent with the central role played by N-myc on CGNP proliferation, Max was revealed as a direct target of miR-22, and miR-22 expression caused a significant reduction of Max protein levels and N-myc/Max-dependent promoter activity. Therefore, we conclude that, in addition to the previously described mechanisms, miR-22 plays a specific role on downstream BMPs through cerebellum growth.

Speciation of Hg2+ and MeHg+ has been achieved by in vitro approaches with fluorogenic probes supported in cultured cells.

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway.

The neural tube forms when neural stem cells arrange into a pseudostratified, single-cell–layered epithelium, with a marked apico-basal polarity, and in which adherens junctions (AJs) concentrate in the subapical domain. We previously reported that sustained β-catenin expression promotes the formation of enlarged apical complexes (ACs), enhancing apico-basal polarity, although the mechanism through which this occurs remained unclear. Here, we show that β-catenin interacts with phosphorylated pro-N-cadherin early in its transit through the Golgi apparatus, promoting propeptide excision and the final maturation of N-cadherin. We describe a new β-catenin–dependent interaction of N-cadherin with Drebrin-like (Dbnl), an actin-binding protein that is involved in anterograde Golgi trafficking of proteins. Notably, Dbnl knockdown led to pro-N-cadherin accumulation and limited AJ formation. In brief, we demonstrate that Dbnl and Drebrin-like β-catenin assist in the maturation of pro-N-cadherin, which is critical for AJ formation and for the recruitment AC components like aPKC and, consequently, for the maintenance of apico-basal polarity.

Phosphatidylinositol 3-kinases (PI3Ks) are signal transducers of many biological processes. Class 1 A PI3Ks are hetero dimers formed by a regulatory and a catalytic subunit. We have used the developing chicken neural tube (NT) to study the roles played by PI3K during the process of cell proliferation and differentiation. Notably, we have observed that in addition to its well characterized anti apoptotic activity, PI3K also plays a crucial role in intra epithelial cell positioning, and unlike its role in survival that mainly depends on AKT, the activity in cell positioning is mediated by Rho GTPase family members. Additionally, we have observed that activating mutations of PI3K that are remarkably frequent in many human cancers, cause an unrestrained basal migration of the neuroepithelial cells that end up breaking through the basal membrane invading the surrounding mesenchymal tissue. The mechanism described in this work contribute not only to acquire a greater knowledge of the intraepithelial cell positioning process, but also give new clues on how activating mutations of PI3K contribute to cell invasion during the first stages of tumour dissemination.

No abstract

SUMMARY STATEMENTDuring neural tube development, PI3K pathway promotes cell survival and provides the apical-basal navigation clues that define the final location of neurons in the epithelium.SUMMARYPhosphatidylinositol 3-kinases (PI3Ks) are signal transducers of many biological processes. Class 1A PI3Ks are hetero dimers formed by a regulatory and a catalytic subunit. We have used the developing chicken neural tube (NT) to study the roles played by PI3K during the process of cell proliferation and differentiation. Notably, we have observed that in addition to its well characterized anti apoptotic activity, PI3K also plays a crucial role in intra epithelial cell positioning, and unlike its role in survival that mainly depends on AKT, the activity in cell positioning is mediated by Rho GTPase family members. Additionally, we have observed that activating mutations of PI3K that are remarkably frequent in many human cancers, cause an unrestrained basal migration of the neuroepithelial cells that end up breaking through the basal membrane invading the surrounding mesenchymal tissue. The mechanism described in this work contribute not only to acquire a greater knowledge of the intraepithelial cell positioning process, but also give new clues on how activating mutations of PI3K contribute to cell invasion during the first stages of tumour dissemination.