Regenerative neurogenic response from glia requires insulin-driven neuron-glia communication

  1. Neale J Harrison
  2. Elizabeth Connolly
  3. Alicia Gascón Gubieda
  4. Zidan Yang
  5. Benjamin Altenhein
  6. Maria Losada Perez
  7. Marta Moreira
  8. Jun Sun
  9. Alicia Hidalgo

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Abstract

Understanding how injury to the central nervous system induces de novo neurogenesis in animals would help promote regeneration in humans. Regenerative neurogenesis could originate from glia and glial neuron-glia antigen-2 (NG2) may sense injury-induced neuronal signals, but these are unknown. Here, we used Drosophila to search for genes functionally related to the NG2 homologue kon-tiki (kon), and identified Islet Antigen-2 (Ia-2), required in neurons for insulin secretion. Both loss and over-expression of ia-2 induced neural stem cell gene expression, injury increased ia-2 expression and induced ectopic neural stem cells. Using genetic analysis and lineage tracing, we demonstrate that Ia-2 and Kon regulate Drosophila insulin-like peptide 6 (Dilp-6) to induce glial proliferation and neural stem cells from glia. Ectopic neural stem cells can divide, and limited de novo neurogenesis could be traced back to glial cells. Altogether, Ia-2 and Dilp-6 drive a neuron-glia relay that restores glia and reprogrammes glia into neural stem cells for regeneration.

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  1. Version published to 10.7554/elife.58756 on eLife
  2. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on June 17, 2020, follows.

    The reviewers feel that while many comments were adequately addressed, several essential points remain problematic and do not support the conclusions of the manuscript. These include experiments with technical difficulties or uninterpretable results. If you are willing and able to address the reviewers' concerns you may resubmit a revised manuscript with a detailed rebuttal letter.

    It is required that you address the following to be reconsidered for publication:

    1. Additional NSC markers must be optimized. Dpn staining in some figures is unconvincing and must be verified using additional markers.

    2. Cell markers routinely used by many labs must work (Wor, Ase, Elav).

    3. Dpn is a reagent that works well and should be clear.

    Please note that this decision letter does not guarantee that this manuscript will be accepted. At least one reviewer feels that their concerns were not addressed in the revised manuscript and that it is in everyone's best interests that the authors can prove that the inability to provide the appropriate expression patterns is not indicative of a deeper problem with the underlying hypothesis.

    1. Major comment. The main conclusion of the paper (glia transform into NSCs which produce neurons) but is not supported by data: only one NSC marker used out of many available. The authors tried two additional NSC markers but did not observe staining, despite these reagents working for many labs in many publications. "We did not consider these results satisfactory enough to present."

    This is a major flaw, especially how unusual the Dpn staining looks like in the ectopic Dpn+ cells (very speckly). Failure to show additional NSC markers very concerning is areal issue; also no evidence for asymmetric cell division at mitosis (a hallmark of these NSCs).

    1. There is no evidence for proliferation of the ectopic Dpn+ cells. The authors state that ectopic Dpn+ cells expressed the S phase marker PCNA:GFP and can be labeled with the mitotic marker pH3.

    However, only panes 8A-C show PCNA+ Dpn+ cells, which are increased following dilp-6 overexpression. No data in the figure shows ectopic Dpn+ cells that are pH3. The rest of the figure shows glial markers and PCNA or pH3, which is irrelevant to the question of whether ectopic Dpn+ cells can divide.

    1. To show evidence that ectopic Dpn+ cells produce neuronal progeny, the authors used the pros-Gal4 line to drive flybow expression, and observed a small cluster of cells that included one Dpn+ and one Elav+ cell. As the authors say "this does not prove these cells are related by lineage, but is consistent with it."

    This does not show Dpn+ cells are producing neurons.

    1. The authors also used "flip out" genetics to permanently mark glial cells.

    The genetics shown in the figure, legend, and reviewer response will not specifically label glia. The genotype is: actGAL4>y+>UASGFP/UAS-FLP; repoGAL4/Dilp-6. This would induce Flp widely, in all cells due to ubiquitous expression of actin-gal4. Most likely, the authors wrote down the wrong genotype in the figure, legend, methods, and reviewer response - it is probably actin promotor-FRT-stop-FRT-GFP. They cite Table 1 for more information on genotypes but there is no Table 1 provided.

    1. In order to call Kon and ia-2 partners, a direct physical interaction should be shown. The authors could not get the biochemical experiments to work for various reasons. Changed text from "partners" to "functional neuronal partner."

    The continued use of 'partner' is inappropriate. The most accurate description of their relationship is that they show 'genetic interactions' - so the first results header should be changed from "Ia-2 is a functional partner of Kon" to "Ia-2 and Kon show genetic interactions."

    1. Saying ectopic Pros+ cells are GMCs or neurons is premature and can be definitively resolved by staining for Wor or Dpn (neuroblast-specific), Ase (neuroblast and GMC), and Elav (neurons). All have been extensively used by many labs. The authors could not get the stains to work.

    This is unsatisfactory.

    1. Line 219 says loss of ia-2 "destabilizes cell fate" - which is a vague term that obscures the phenotype. The authors changed text to "... upregulated GMC and NSC markers."

    They looked at Dpn but no other NSC marker, and Pros is not a specific GMC marker, also being expressed in neuropile glia near the midline (which is worrying).

    1. Dpn staining in figure 3D is unconvincing; everything looks speckly. The authors state that Dpn staining is speckly in their hands.

    Many labs have used Dpn to mark neuroblasts, it is a very reliable reagent. The authors have good Dpn staining in other figures; this suggest to me that the ectopic Dpn+ cells are different from the normal Dpn+ NPCs, leading to different protein localization/levels. This concern is reinforced by the failure of the authors to show the ectopic Dpn+ cells express any other NSC marker.

    1. Ectopic Dpn+ cells were not quantified due to due to the disruption and variability of the abdominal crush procedure. The authors only counted the VNCs in which they could see ectopic Dpn+ (cells).

    Picking only VNCs that show ectopic Dpn+ cells is inappropriate.

    1. In response to InR-Gal4 expression concersn, the authors state "we do not know whether (InR-gal4) represents the endogenous expression pattern". It labels sparse patterns of neurons and sporadic glial cells.

    The authors directly state in the revised manuscript "we visualized InR expression using available GAL4 lines to drive his-YFP" but in the reviewer response they acknowledge this is not accurate.

  3. Version published to 10.1101/721498 on bioRxiv