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Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

Tognon E, Kobia F, Busi I, Fumagalli A, De Masi F, Vaccari T - Autophagy (2016)

Bottom Line: In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development.Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity.Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

View Article: PubMed Central - PubMed

Affiliation: a IFOM - FIRC Institute of Molecular Oncology , Milan , Italy.

ABSTRACT
In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

No MeSH data available.


Related in: MedlinePlus

Misexpression of Mitf perturbs SOP development. (A and D) High magnification of the anterior part of the wing disc pouch of the indicated genotype stained as indicated. Note that Mitf and Mitf DN overexpression results in perturbation of the expression of ac protein (A), no perturbation of wg or ct expression at the D/V boundary (B and C), formation of misplaced or ectopic neur-GFP- ct -, (C) and neur-GFP- peb - (D) positive cells. Some of the ectopic ct and peb -positive cells are negative for neur-GFP and could represent incomplete SOP commitment. (E) Presence of normal and ectopic peb -positive cells is reduced to different extent in YFP::Vha55 discs overexpressing Mitf, when compared to YFP::GFP discs not overexpressing Mitf. A similar lack of peb -positive cells is observed in GFP::VhaSFD discs overexpressing Mitf. Note that the genetic  tagged forms of these genes are highly expressed, due to induction by Mitf. (F) High magnification of the antero-distal dorsal area of the margin of adult wings of the indicated genotypes. The stereotypic position of sensory margin bristles is shown by black arrows. Expression of the indicated constructs in wing discs results in loss (red arrowheads) or misplacement of sensory bristles (red arrows).
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f0005: Misexpression of Mitf perturbs SOP development. (A and D) High magnification of the anterior part of the wing disc pouch of the indicated genotype stained as indicated. Note that Mitf and Mitf DN overexpression results in perturbation of the expression of ac protein (A), no perturbation of wg or ct expression at the D/V boundary (B and C), formation of misplaced or ectopic neur-GFP- ct -, (C) and neur-GFP- peb - (D) positive cells. Some of the ectopic ct and peb -positive cells are negative for neur-GFP and could represent incomplete SOP commitment. (E) Presence of normal and ectopic peb -positive cells is reduced to different extent in YFP::Vha55 discs overexpressing Mitf, when compared to YFP::GFP discs not overexpressing Mitf. A similar lack of peb -positive cells is observed in GFP::VhaSFD discs overexpressing Mitf. Note that the genetic tagged forms of these genes are highly expressed, due to induction by Mitf. (F) High magnification of the antero-distal dorsal area of the margin of adult wings of the indicated genotypes. The stereotypic position of sensory margin bristles is shown by black arrows. Expression of the indicated constructs in wing discs results in loss (red arrowheads) or misplacement of sensory bristles (red arrows).

Mentions: To determine whether Mitf might influence the proneural differentiation cascade that leads to SOP formation, we overexpressed Mitf and Mitf DN in the wing pouch and assessed for possible alteration of patterning of the PNCs and SOPs straddling the anterior D/V boundary that will give rise to the mechano-sensory bristles of the adult wing margin. 34 We found perturbation of PNC patterning as revealed by broadening of expression of the PNC marker Ac, compared to control discs (Fig. 5A, Fig. S2F). This is unlikely to be due to changes in wg or N signaling because overexpression of Mitf did not change expression of wg and of the N target cut (ct) at the D/V margin, compared to control wing discs (Fig. 5B and C, Fig. S2F). To assess SOP differentiation, we analyzed discs expressing neur-GFP or stained for peb or ct, which marks sense organs and non-neuronal cells in the hinge and notum.36 Interestingly, misexpression of Mitf or Mitf-DN led to loss and ectopic peb-, neur- or ct-positive cells (Fig. 5C and D, Fig. S2F). Strikingly, in YFP::Vha55 or GFP::VhaSFD or GFP::Vha16-1 discs overexpressing Mitf, ectopic and normal peb-positive cells were not present. This is not the case in YFP::Vha55 discs, which showed a normal pattern (Fig. 5E). Thus, when high amounts of mutant tagged forms of V-ATPase components are present, the ability of Mitf to generate normal and ectopic SOPs observed in overexpressing discs is prevented, suggesting that Mitf requires V-ATPase to support SOP development. Consistent with this, the wing margin of adult animals displayed missing or ectopic mechano-sensory bristles, similar to animals in which the activity of N target genes of the E(Spl) cluster was modulated (Fig. 5F). To test whether this is the case also upon impairment of Vha16-1, we used in vivo RNAi (see Fig. S1F for details). Expression of a Vha16-1 RNAi hairpin in the whole wing pouch led to specific reduction of endogenous Vha16-1 mRNA expression and of GFP expression in GFP::Vha16-1 wing disc, indicating that the RNAi line is on target (Fig. S2G and H). Expression of Vha16-1 RNAi also led to the formation of an adult wing margin with ectopic SOPs (Fig. 5F), indicating that the Vha16-1 is involved in correct SOP development.Figure 5.


Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

Tognon E, Kobia F, Busi I, Fumagalli A, De Masi F, Vaccari T - Autophagy (2016)

Misexpression of Mitf perturbs SOP development. (A and D) High magnification of the anterior part of the wing disc pouch of the indicated genotype stained as indicated. Note that Mitf and Mitf DN overexpression results in perturbation of the expression of ac protein (A), no perturbation of wg or ct expression at the D/V boundary (B and C), formation of misplaced or ectopic neur-GFP- ct -, (C) and neur-GFP- peb - (D) positive cells. Some of the ectopic ct and peb -positive cells are negative for neur-GFP and could represent incomplete SOP commitment. (E) Presence of normal and ectopic peb -positive cells is reduced to different extent in YFP::Vha55 discs overexpressing Mitf, when compared to YFP::GFP discs not overexpressing Mitf. A similar lack of peb -positive cells is observed in GFP::VhaSFD discs overexpressing Mitf. Note that the genetic  tagged forms of these genes are highly expressed, due to induction by Mitf. (F) High magnification of the antero-distal dorsal area of the margin of adult wings of the indicated genotypes. The stereotypic position of sensory margin bristles is shown by black arrows. Expression of the indicated constructs in wing discs results in loss (red arrowheads) or misplacement of sensory bristles (red arrows).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836007&req=5

f0005: Misexpression of Mitf perturbs SOP development. (A and D) High magnification of the anterior part of the wing disc pouch of the indicated genotype stained as indicated. Note that Mitf and Mitf DN overexpression results in perturbation of the expression of ac protein (A), no perturbation of wg or ct expression at the D/V boundary (B and C), formation of misplaced or ectopic neur-GFP- ct -, (C) and neur-GFP- peb - (D) positive cells. Some of the ectopic ct and peb -positive cells are negative for neur-GFP and could represent incomplete SOP commitment. (E) Presence of normal and ectopic peb -positive cells is reduced to different extent in YFP::Vha55 discs overexpressing Mitf, when compared to YFP::GFP discs not overexpressing Mitf. A similar lack of peb -positive cells is observed in GFP::VhaSFD discs overexpressing Mitf. Note that the genetic tagged forms of these genes are highly expressed, due to induction by Mitf. (F) High magnification of the antero-distal dorsal area of the margin of adult wings of the indicated genotypes. The stereotypic position of sensory margin bristles is shown by black arrows. Expression of the indicated constructs in wing discs results in loss (red arrowheads) or misplacement of sensory bristles (red arrows).
Mentions: To determine whether Mitf might influence the proneural differentiation cascade that leads to SOP formation, we overexpressed Mitf and Mitf DN in the wing pouch and assessed for possible alteration of patterning of the PNCs and SOPs straddling the anterior D/V boundary that will give rise to the mechano-sensory bristles of the adult wing margin. 34 We found perturbation of PNC patterning as revealed by broadening of expression of the PNC marker Ac, compared to control discs (Fig. 5A, Fig. S2F). This is unlikely to be due to changes in wg or N signaling because overexpression of Mitf did not change expression of wg and of the N target cut (ct) at the D/V margin, compared to control wing discs (Fig. 5B and C, Fig. S2F). To assess SOP differentiation, we analyzed discs expressing neur-GFP or stained for peb or ct, which marks sense organs and non-neuronal cells in the hinge and notum.36 Interestingly, misexpression of Mitf or Mitf-DN led to loss and ectopic peb-, neur- or ct-positive cells (Fig. 5C and D, Fig. S2F). Strikingly, in YFP::Vha55 or GFP::VhaSFD or GFP::Vha16-1 discs overexpressing Mitf, ectopic and normal peb-positive cells were not present. This is not the case in YFP::Vha55 discs, which showed a normal pattern (Fig. 5E). Thus, when high amounts of mutant tagged forms of V-ATPase components are present, the ability of Mitf to generate normal and ectopic SOPs observed in overexpressing discs is prevented, suggesting that Mitf requires V-ATPase to support SOP development. Consistent with this, the wing margin of adult animals displayed missing or ectopic mechano-sensory bristles, similar to animals in which the activity of N target genes of the E(Spl) cluster was modulated (Fig. 5F). To test whether this is the case also upon impairment of Vha16-1, we used in vivo RNAi (see Fig. S1F for details). Expression of a Vha16-1 RNAi hairpin in the whole wing pouch led to specific reduction of endogenous Vha16-1 mRNA expression and of GFP expression in GFP::Vha16-1 wing disc, indicating that the RNAi line is on target (Fig. S2G and H). Expression of Vha16-1 RNAi also led to the formation of an adult wing margin with ectopic SOPs (Fig. 5F), indicating that the Vha16-1 is involved in correct SOP development.Figure 5.

Bottom Line: In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development.Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity.Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

View Article: PubMed Central - PubMed

Affiliation: a IFOM - FIRC Institute of Molecular Oncology , Milan , Italy.

ABSTRACT
In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

No MeSH data available.


Related in: MedlinePlus