Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila (2021)
Figure 6

Figure 6 Hh and lipid metabolism regulators mediate the effects of glial htlACT on NB proliferation

A) Pan-glial (repo-GAL4) htlACT overexpression causes upregulation of hh, fasn1 and lsd2 transcripts (n = 3 biological replicates pooled from 20 brains for each genotype; for each biological replicate, we ran 3 technical replicates for each PCR reaction). The lipogenesis genes (acc, lipin), and lipolysis gene bmm transcripts are not significantly altered. We utilised rpl32 as a reference gene in these experiments, as it is not altered by htlACT overexpression. The data are represented by log2 fold change relative to the control (repo-GAL4> w1118).

B) Schematic depicting lipogenesis and lipolysis. Lipogenesis begins with de novo synthesis of fatty acids by carboxylation of cytosolic acetyl-CoA via acetyl-CoA carboxylase (ACC) and elongation of fatty chain via fatty acid synthase (Fasn, red).

Dietary-derived and de novo-generated fatty acids are converted into fatty acylCoA, which re-localizes to ER and participates in triglyceride (TAG) synthesis with glycerol-3 phosphate (G3P). This process is mediated by a series of enzymes: glycerol-3-phosphate acyltransferase (GPAT), acylCoA acylglycerol-3-phasphte acyltransferases (AGPAT), Lipin (a phosphatidate phosphatase), diacylglycerol acyltransferase (DGAT, red). TAG is translocated from the ER to the core of the intracellular organelles called LDs. On the surface of LDs, a triglyceride lipase called Brummer (Bmm), and its inhibitor Lsd-2, antagonistically control TAG storage.

C-G) Representative images showing that Hh staining normally localised to a ring like structure (yellow arrows), becomes delocalises to the glial cytoplasm upon htlACT overexpression, quantified in (E) (n=4, 6 brain lobes). Glial cells are marked with repo-GAL4>GFP. (E-F') are zoomed in images of (C-D').

H) Cortex glial (NP2222-GAL4>) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression (n=10, 16; 7, 12; 14, 11 brain lobes). The NP2222-GAL4> w1118 vs htlACT columns depict the same data as those in Fig 5E.

I) Knockdown of NB Hh signalling pathway (dnab-GAL4> UAS-ciRNAi) rescues NB EdU incorporation defects induced by glial htlACT overexpression (repo-LexA> LexAop-htlACT). Induction of ciRNAi in NBs alone increases NB EdU incorporation (n=8, 13; 10, 10; 30, 26; 12, 7 brain lobes).

J) The NB EdU incorporation defects due to cortex glial (NP2222-GAL4>) overexpression of htlACT, is significantly rescued by overexpression of RNAis against fasn1 and lsd2, compared to corresponding control RNAis (n=10, 16; 25, 17; 14, 8 brain lobes). The NP2222-GAL4> w1118 vs htlACT columns depict the same data as those in Fig 5E. The control column for htlACT; lsd2 RiKK102269 depicts the same data as the control column for htlACT; hh RiBL25794 in Fig 6H.

Data information: Scale bar=50 μm. Error bar represents SEM. In (A), t test was conducted to compare log transformed fold change: rpl32: Welch's t test, (ns) p=0.9355; hh: unpaired t test, (**) p=0.0061; fasn1: unpaired t test, (*) p=0.0242; acc: unpaired t test, (ns) p=0.7570; lipin: unpaired t test, (ns) p=0.1222; lsd2: unpaired t test, (**) p=0.0074; bmm: unpaired t test, (ns) p=0.9356. In (G): Man-Whitney test, (**) p=0.0095. In (H): unpaired t test, (****) p<0.0001; Mann-Whitney test, (**) p=0.0031; unpaired t test, (**) p=0.0012. In (I): unpaired t test, (**) p=0.0019; unpaired t test, (****) p<0.0001; Mann-Whitney test, (***) p=0.0006; unpaired t test, (***) p=0.0003. In (J): unpaired t test, (****) p<0.0001; unpaired t test, (****) p<0.0001; Welch's t test, (***) p=0.0004.