Getting in shape: ATP pumps up the volume in Hh signalling

Glycolysis is a major metabolic process which ensures the break down of glucose into pyruvate via multiple enzymatic steps, but if and how this catabolism can impact on developmental patterning is unclear. In this issue, Spannl et al (2020) demonstrate a novel link between energy metabolism and tissue formation in the fly imaginal discs. They show that ATPs generated via glycolysis maintain active transport of a smoothened inhibitor, which keeps Hh signalling in check to preserve the correct shape and proportion of the developing wing.

New work demonstrates a direct link between glycolysis, membrane potential and developmental patterning in the fly wing.

Pattern formation is the developmental process by which cells acquire different identities, specified by their relative positions within the developing animal. The wing imaginal disc of the fruit fly Drosophila has been extensively utilised to study the molecular interactions between patterning regulators, where morphogens along the anterior–posterior and dorsoventral axes intersect to specify the correct shape and proportions of the wing. The anterior (A) and posterior (P) cells in the wing imaginal disc are segregated by a compartment boundary, where P cells produce a morphogen called Hedgehog (Hh), that binds to its receptor Patched (Ptc) in the A cells. Hh ligand exerts its effect not only on its immediate neighbouring cells, but also over considerable distances to trigger the activation of their target genes [reviewed by Varjosalo and Taipale (2008)]. In the absence of Hh, Ptc inhibits the seven‐transmembrane protein Smoothened (Smo) to prevent target gene activation. Hh activates signalling via its binding to Ptc, which in turn alleviates repressive effect of Ptc on Smo, which then blocks proteasomal processing of Cubitus interruptus (Ci), and changes its activity from a transcriptional repressor (Ci75) to a transcriptional activator (Ci155) to activate downstream target genes (Fig 1).

Figure 1. ATP drives ATPase pump, membrane potential and Hh patterning.

In the wing imaginal disc, the diffusible ligand Hh exerts its function by binding to its receptor Ptc, which relieves the inhibitory effects of Ptc on Smo. Smo signalling then activates the transcription factor Cubitus interruptus (Ci) and its target genes. The work by Spannl et al (2020) demonstrates that the splicing factor Ecd directly splices several enzymes of the glycolytic pathway, GAPDH,PYK and PDK to drive glycolysis, ATP production and membrane potential via the Na⁺ (blue)/K⁺ (orange)‐ATPase pump. The sodium/potassium pump in addition to importing nutrients into the cell is also responsible for transporting a small lipoprotein (LPP)‐associated inhibitor called N‐acylethanolamides (purple) that destabilises Smo to repress Hh signalling and maintain the correct shape of the developing wing imaginal disc.

Energy and metabolism have emerged to be central regulators of growth and proliferation. Glycolysis and oxidative phosphorylation (OxPhos) are two key metabolic pathways for energy production. Much effort has focused on understanding how glycolysis and OxPhos support proliferation and tumour progression; however, if and how metabolism drives a tissue's response to morphogens and identity is less well understood (Pegoraro et al, 2015; Bulusu et al, 2017; Oginuma et al, 2017). Notably, previous work has hinted at this connection, as inhibition of the pyruvate metabolism pathway has been linked to overgrowth phenotypes reminiscent of Notch signalling activation (Saj et al, 2010). In this issue of The EMBO Journal, Spannl et al (2020) further investigate this association, through elegant genetic studies and the use of bio‐sensors. Together, their data suggest that energy metabolism affects wing patterning, through ATP‐driven membrane import of negative regulators of the Hh signalling pathway.

The authors approached the contribution of glycolysis to wing development by first developing a versatile ATP‐FRET sensor. Notably, downregulation of key enzymes of the glycolytic pathway caused a reduction in the steady state of ATP and altered wing proportions. They further showed, that in this context, glycolysis is regulated by a splicing factor called Ecdysoneless (Ecd), which directly splices several glycolysis enzymes including GAPDH and PFK. As Hh pathway is an important regulator of wing patterning, the authors went on to examine the effect of disruption of glycolysis on the accumulation of G protein‐coupled receptor Smo, a key signal transducer of the Hh pathway. They found that Smo accumulated in the basolateral membrane of fly wings, as well as at the primary cilium of mammalian cells upon glycolysis inhibition. Consequently, this led to the stabilisation of Smo‐dependent transcriptional activator Ci155, both in the presence and the absence of Hh ligand.

To find out how glycolysis affects Smo accumulation, the authors hypothesised that ATPs generated by glycolysis might be responsible for generating differences in plasma membrane potential, that in turn affects the transportation of small inhibitors of Hh signalling. Membrane potential has recently been shown to be patterned in the wing imaginal disc, and disruption of membrane potential alone is sufficient to lead to the stabilisation of Smoothed and Ci155 (preprint: Emmons‐Bell & Yasutomi, 2020). Na⁺/K⁺‐ATPase, or the sodium‐potassium pump, is primarily responsible for utilising ATP to export three sodium ions and import two potassium ions to generate membrane potential. Export of sodium out of the cell provides the driving force for import of glucose, amino acids and other nutrients into the cell. Consistent with this, knockdown of glycolytic enzymes GAPDH and PFK caused a change in membrane potential. But how does the sodium–potassium pump cause Smo accumulation? Could it be that other than nutrients, these pumps also transport small inhibitors of Hh signalling? To follow up on this idea, the authors tested whether glycolysis impeded the transport of a lipid‐associated N‐acylethanolamides (NAE), which are known to prevent Smo‐mediated Hh activation in the absence of the ligand (Khaliullina et al, 2015). Utilising a photoactivated NAE analogue (PAC‐NAE), the authors showed that indeed the transport of NAE across the plasma membrane was affected by interference with glycolytic genes, resulting in an accumulation of Smo and the activation of Hh signalling.

Based on these results, the authors concluded that changes in energy production affected the import of Hh‐inhibitors into the cell, which in turn enhanced the activity of the Hh pathway. Interestingly, in the wing imaginal disc, in addition to glycolysis, OxPhos is also highly active. It would be interesting to test whether the ATPs generated by OxPhos also play roles in modulating Hh signalling. Furthermore, diet and the metabolic status of the animal are the main determinants of a cell's decision to utilise glycolysis or OxPhos. In the future, it would be important to understand how the overall nutritional and metabolic status of the animal impacts on wing patterning; whether in addition to Hh, other signalling pathways are also impacted by energy metabolism, and if so, whether they are also modulated via membrane transport of mediators or inhibitors. Interestingly, wing‐specific glycolytic manipulations affect wing proportion, whereas nutrient restriction reduces wing size, but does not alter its proportion (Britton & Edgar, 1998; Cheng et al, 2011). These intriguing but important differences could be accounted for by local versus systemic effects of energy metabolism on wing patterning, and it would be interesting to explore whether systemic metabolism also directly impacts Hh activity. Finally, the authors showed that the ultimate consequence of altered patterning is an increase in growth along the A‐P axis, resulting in altered wing proportions. How energy metabolism differentially affects growth versus patterning, and whether these features are linked, remains to be explored in future work.

In summary, the work by Spannl et al (2020) suggests that energy metabolism drives changes in membrane transport of regulators of developmental signalling pathways and presents a tantalising link in joining metabolic status of a tissue with its ability to form the right shape.

The EMBO Journal (2020) 39: e106564