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Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila

['Dnyanesh Dubal', 'Biology', 'Indian Institute Of Science Education', 'Research', 'Pune', 'Prachiti Moghe', 'Rahul Kumar Verma', 'Bhavin Uttekar', 'Richa Rikhy']

Date: 2022-04

Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. Our results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage.

Mitochondrial morphology and function are coupled to stem cell differentiation and organism development. It is of interest to examine the mechanisms of interaction of mitochondrial dynamics with signaling pathways during stem cell differentiation. We have assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial fusion proteins Opa1 and Marf led to mitochondrial fragmentation, loss of mitochondrial activity and proliferation, thereby causing a decrease in the numbers of differentiated cells in each type II NB lineage. Mutants in mitochondrial fission protein Drp1 led to mitochondrial fusion but did not cause any differentiation defects. Decreased Notch signaling by the canonical pathway led to mitochondrial fragmentation and a decrease in differentiated cells in each type II NB lineage. Expression of Drp1 mutants in type II NB lineages depleted of Opa1 and Marf suppressed their proliferation and differentiation defects. Expression of Drp1 mutant in type II NB lineages depleted of Notch also led to a rescue of differentiated progeny in each lineage. Our results implicate crosstalk between Notch signaling, mitochondrial activity and fusion as important steps for proliferation and differentiation in the type II NB lineage.

Funding: RR received the following funding for the study. This study is funded by the Department of Biotechnology, India ( http://dbtindia.gov.in/ ) by the project code BT/PR18898/MED/122/19/2016. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Dubal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we have assessed the role of mitochondrial morphology proteins Opa1, Marf and Drp1 in regulating type II NB differentiation. RNAi mediated knockdown of mitochondrial fusion proteins Opa1 and Marf led to mitochondrial fragmentation, loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage while NB number and polarity remained unaffected. Opa1 depletion led to decreased proliferation of the type II NBs and mature INPs and decrease in mature INPs, GMCs and neurons in the type II NB lineage. Marf depletion led to decreased proliferation of GMCs and loss of neurons in the type II NB lineage. On the other hand there was no defect in differentiation in NBs overexpressing a mutant form of mitochondrial fission protein Drp1. Inhibition of mitochondrial fragmentation in Opa1 and Marf depletion in combination with Drp1 mutant overexpression suppressed the differentiation defects suggesting that fused mitochondria are essential for differentiation in the type II NB lineage. Further, Notch depletion led to fragmented mitochondria and loss of differentiation. Increased Notch activity showed mitochondrial clustering. Mitochondrial fusion in the type II NB lineage deficient of Notch led to differentiation. Our results show that mitochondrial fusion interacts with Notch signaling to drive differentiation in the type II NB lineage.

A: Schematic of larval CNS (left) containing the central brain (CB) lobes and ventral nerve cord (VNC) and type I (blue) and type II NB (purple) distribution and lineages of type I (left) and type II NB (right). The type I NB lineage has nuclear Dpn (red) and Ase (green) and cytoplasmic Pros (light blue) expressing NBs, nuclear Ase (green) and Pros (light blue) expressing GMCs and high levels of nuclear Pros (dark blue) expressing neurons. The type II NB lineage has Dpn positive NBs (red nuclei), Dpn negative, Ase negative and Pros negative immature INPs (black and white), only Ase expressing immature INPs, nuclear Dpn (red) and cytoplasmic Pros (light blue) expressing mature INPs, nuclear Ase and Pros expressing GMCs (green nuclei) and high levels of nuclear Pros (dark blue) expressing young neurons. B-D: Mitochondrial morphology and distribution in type II NBs expressing pnt-Gal4, mCD8-GFP (white dotted line, magnified area shown in the panel on the right) stained with ATPβ (red) antibody using confocal imaging and STED super resolution microscopy is shown in representative single optical plane images with zoomed inset in the right panel (B). Single plane confocal images from mature INPs from each image are marked with Dpn and stained with mito-GFP (red). mCherry RNAi (32 type II NBs, 8 brains: 100% intermediate morphology; 40 mature INPs, 5 brains, 8 lineages, 75% intermediate, opa1 RNAi (46 type II NBs, 8 brains: 100% fragmented; 47 mature INPs, 3 brains, 9 lineages, 78% fragmented), marf RNAi (45 type II NBs, 8 brains: 100% fragmented; 21 mature INPs, 3 brains, 4 lineages, 61% fragmented), Drp1 SD (80 type II NBs, 10 brains: 85% clustered; 34 mature INPs, 3 brains, 6 lineages, 79% clustered). Graph shows the distribution of mitochondria into fragmented, intermediate and clustered in the form of a stacked histogram (C). The percentage documented on each bar in the histogram is for the group that is seen at the maximum extent. Average mitochondrial area quantification from type II NBs (D) in mCherry RNAi (6 type II NBs, 3 brains), opa1 RNAi (6,3), marf RNAi (19,3), Drp1 SD (6,3). Graph shows mean ± sd. Statistical analysis is done using an unpaired t-test. *** p<0.001. Scale bar- 5μm for the type II NB in B, 2.7μm for the mature INP in B.

The Drosophila neural stem cell or neuroblast (NB) differentiation model has been used effectively to identify regulators of steps of differentiation such as stem cell renewal, asymmetric cell division, polarity formation and lineage development. NBs rely on glycolysis and ETC activity for their energy production during differentiation and tumorigenesis [ 22 – 24 ]. Mitochondrial fusion has recently been found to be essential for tumorigenesis [ 24 ]. It remains to be studied whether mitochondrial morphology is also regulated to provide appropriate mitochondrial activity for differentiation in NBs. The type I NB lineage in the larval brain consists of 90 NBs marked by the expression of the transcription factors Deadpan (Dpn) and Asense (Ase). These NBs divide asymmetrically to give rise to a ganglion mother cells (GMCs) marked by the expression of Prospero (Pros) in the nucleus [ 25 , 26 ]. NBs of the type II lineage express Dpn and are 8 in number per optic lobe [ 27 ]. Like the mammalian neural stem cell differentiation type II NBs undergo multiple steps of differentiation by forming transit amplifying cells called intermediate neural precursor cells (INPs). Newly formed INPs are smaller in size as compared to type II NBs and do not express Ase and Dpn. Some immature INPs express Ase. Immature INPs undergo a defined series of transcriptional changes to form mature INPs. Mature INPs express Dpn and Ase and proliferate to form GMCs that express nuclear Pros and Ase. GMCs in both the type I and type II lineages finally differentiate into neurons or glia ( Fig 1A ). Young neurons are present at the base of the lineage and express increased nuclear Pros. Elav is expressed in all neurons [ 28 ]. Notch signaling regulates number and differentiation in the type II NB lineage [ 29 , 30 ].

Recent studies show that alteration of mitochondrial dynamics affects signaling pathways such as the Notch signaling pathway during stem cell differentiation. The Notch receptor is a transmembrane protein activated by ligands such as Delta. The Delta-Notch interaction is followed by cleavage of the Notch intracellular domain (NICD) in the signal receiving cell. NICD enters the nucleus and regulates gene expression along with Suppressor of hairless (Su(H)) by the canonical pathway thereby providing a signal for proliferation or differentiation [ 17 ]. Fragmented mitochondrial morphology maintained by Drp1 in ovarian follicle cells in Drosophila is crucial for activating Notch signaling [ 18 , 19 ]. Similarly, loss of Opa1 and Mfn leading to mitochondrial fragmentation in mouse embryonic stem cells causes hyperactivation of Notch and reduces differentiation of ESCs into functional cardiomyocytes due to loss of calcium buffering [ 20 ]. On the other hand, activation of Notch signaling by depletion of mitochondrial fusion and increasing reactive oxygen species (ROS) enhances differentiation in mammalian neural stem cells [ 21 ]. Thus mitochondrial fragmentation along with elevated calcium and reactive oxygen species increase has been found to be involved in Notch signaling in these contexts. It is of interest to understand whether Notch signaling induces appropriate mitochondrial morphology in differentiation.

Mitochondria are sparse and fragmented in stem cells and are an elaborate network in differentiated cells [ 1 – 3 ]. Stem cells largely depend upon glycolysis as an energy source, whereas differentiated cells produce a large amount of ATP by electron transport chain (ETC) activity [ 1 , 4 , 5 ]. Fused mitochondrial morphology is associated with high membrane potential, increased ETC activity and high ATP production, while low membrane potential, reduced ETC activity and low ATP production is seen in fragmented mitochondria [ 1 ]. Mitochondrial architecture is regulated by a balance of fusion and fission events [ 6 ]. Proteins belonging to the family of large GTPases are involved in mitochondrial fusion and fission. Optic atrophy 1 (Opa1, or Opa1-like in Drosophila) and Mitofusin or Mitochondrial assembly regulatory factor (Marf in Drosophila or Mitofusin, Mfn in mammals) facilitate inner and outer mitochondrial membrane fusion respectively while Dynamin related protein 1 (Drp1) is required for mitochondrial fragmentation [ 7 – 10 ]. A balance of levels and activity of these proteins regulates mitochondrial shape in the cell [ 6 ]. Further, Opa1 plays a significant role in regulating cristae organization in addition to inner membrane fusion [ 11 ]. Opa1 oligomerization and inner membrane cristae organization is important for ETC activity. The presence of elaborate cristae leads to organization of ETC complexes as super complexes and enhances their activity [ 12 ]. Hyperfusion of mitochondria protects them from degradation in autophagy and also loss of ETC activity [ 13 – 16 ].

Results

Depletion of mitochondrial morphology proteins Opa1, Marf and Drp1 leads to change in mitochondrial organization in type II NBs and mature INPs We depleted Opa1, Marf and Drp1 to investigate the effect of perturbation of mitochondrial morphology on NB numbers and differentiation. We expressed multiple RNAi lines against Opa1, Marf and an RNAi line against Drp1 and a dominant negative Drp1 mutation with neuronal Gal4 drivers in different stages of NB differentiation to analyze their effect on lethality and behavior (S1 Fig). inscuteable-Gal4, worniu-Gal4, scabrous-Gal4 and prospero-Gal4 were used to deplete Opa1, Marf and Drp1 using RNAi expression and Drp1 using overexpression of a dominant negative mutant in all NBs. Opa1, Marf and Drp1 depletion by multiple RNAi lines and a dominant negative mutant showed survival of animals until the pupal stage with inscuteable-Gal4 and worniu-Gal4 and were lethal or showed behavioral defects as adults. The RNAi lines for Opa1 and Marf that gave a stronger defect with inscuteable-Gal4 and worniu-Gal4 and overexpression of the dominant negative mutant of Drp1, (Drp1SD) [31] were used to deplete these proteins in the type II NB lineage using pointedP1-Gal4, mCD8-GFP (pnt-Gal4, mCD8-GFP) for further experiments (Figs 1A and S1). opa1 RNAi, marf RNAi and Drp1SD expression in the type II NB lineage gave normal adults at 25°C. opa1 RNAi and marf RNAi expression in type II NB lineage when performed at a higher temperature of 29°C with pnt-Gal4 gave sluggish adults. To characterize mitochondrial morphology in type II NBs, we performed super-resolution Stimulated Emission Depletion microscopy (STED). Mitochondria were stained with an antibody against ATPβ subunit of complex V in the third instar larval brain. STED microscopy allowed better separation of mitochondria in these small cells as compared to confocal microscopy (Figs 1B and S2A). We observed mitochondria in a bead-like organization, often present as spheres evenly distributed all around the nucleus in control type II NBs (Figs 1B and S2A). The mitochondrial morphology was similar to previous observations in type I NBs [32]. We used pnt-Gal4, mCD8-GFP to identify the type II NBs using mCD8-GFP and deplete Opa1 and Marf and express the Drp1SD mutant. Pnt expresses in type II NBs, immature INPs and mature INPs [33]. Partial loss of pnt leads to a decrease in mature INPs and GMCs in the type II NB lineage. Pnt-Gal4, mCD8-GFP shows GFP expression in type II NBs, brighter GFP in cells closer towards the type II NB and lighter GFP in cells towards the base of the lineage. RNAi against mitochondrial fusion proteins Opa1 and Marf has been previously shown to deplete the corresponding mRNA and lead to mitochondrial fragmentation in electron microscopy studies [34–37]. We classified mitochondrial morphology into fragmented, intermediate and clustered in different genotypes in type II NBs and mature INPs by analyzing their distribution in different optical planes (Fig 1B and 1C). We observed an increase in numbers of type II NBs containing mitochondria in a fragmented form often organized as small spheres on depletion of Opa1 and Marf using two different RNAi lines as compared to an intermediate mitochondrial morphology in mCherry RNAi controls, confirming the requirement of these proteins for mitochondrial fusion (Figs 1B, 1C, and S2A). NBs depleted of Opa1 and Marf showed a significant decrease in mitochondrial area as compared to controls (Figs 1D and S2B). The extent of decrease in mitochondrial area was similar upon depletion of either Opa1 or Marf. Mature INPs (Dpn+ cells in the lineage) expressing Opa1 and Marf RNAi also showed increased numbers of cells containing fragmented mitochondria as compared to an intermediate state in controls (Fig 1B and 1C). Overexpression of mitochondrial fission mutant Drp1SD resulted in clustering of mitochondria on one side of the NB suggesting that mitochondria were fused (Fig 1B). We observed a similar clustering of mitochondria in mitotic clones of type II NBs depleted of Drp1 using the null allele of drp1, drp1KG [38] (S2C Fig). Mitochondrial clustering on Drp1 depletion has been seen earlier in mammalian cells [39,40] and also in Drosophila spermatocytes, neurons, follicle cells and embryos [18,19,31,41,42]. Mitochondrial clusters have inter connected mitochondrial tubes in electron microscopy studies in mammalian cells [40]. Drp1 depletion led to an increase in numbers of type II NBs showing clustered mitochondria and a significant increase in mitochondrial area as compared to control NBs (Fig 1C and 1D). Mature INPs expressing Drp1SD also showed an increased number of cells with clustered mitochondria (Fig 1B and 1C). We confirmed the effect of mitochondrial morphology protein depletion on mitochondrial morphology in muscle cells using mhc-Gal4, mito-GFP. In line with the previous studies [34,43,44] and NB data, we found that mitochondria were relatively smaller upon depletion of Opa1 and Marf in muscle cells. Expression of Drp1SD resulted in relatively large mitochondria compared to control (S2D Fig). Whereas our analysis of mitochondrial distribution could capture changes in mitochondrial morphology at the qualitative level, it could not ascertain changes in mitochondrial size and density of mitochondria in the type II NBs and mature INPs. In summary, depletion of Opa1 and Marf led to mitochondrial fragmentation and depletion of Drp1 led to mitochondrial clustering consistent with mitochondrial fusion in the type II NBs.

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