Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead

  1. Hideyuki Komori
  2. Qi Xiao
  3. Derek H Janssens
  4. Yali Dou
  5. Cheng-Yu Lee  Is a corresponding author
  1. University of Michigan Medical School, United States

Peer review process

This article was accepted for publication as part of eLife's original publishing model.

History

  1. Version of Record published
  2. Accepted Manuscript published
  3. Accepted
  4. Received

Decision letter

  1. Marianne E Bronner
    Reviewing Editor; California Institute of Technology, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “The Trithorax histone methyltransferase complex maintains the functional heterogeneity of neural stem cells” for consideration at eLife. Your article has been favorably evaluated by Fiona Watt (Senior editor), Marianne Bronner (Reviewing editor), and 2 reviewers, one of whom, Heinrich Reichert, has agreed to reveal his identity.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

This manuscript reports the role of Trithorax (Trx) in the maintenance of type II neuroblast identity. Type II neuroblasts mutant for Trx gradually convert to type I neuroblasts: they turn on Asense and lose PntP1, and they generate GMCs instead of intermediate neural progenitors (INPs). Neuroblasts mutant for a core component of the SET1/MLL complex also show the same phenotype. By sequencing brains enriched for type I or type II neuroblasts, the authors identify a transcription factor, buttonhead (btd) whose transcripts are up-regulated in mutant brains enriched with type II neuroblasts. They show by ChIP that Trx binds to the btd gene. Type II neuroblasts mutant for btd do not turn on Ase, but their INPs never mature. Further, the authors show that overexpression of btd in type I neuroblasts is sufficient to convert them into type II neuroblasts that generate INPs (although this phenotype is only observed in ∼28% of neuroblasts). Finally, they show that over-expressing btd in type II neuroblasts that are mutant for trx rescues type II neuroblast identity. These findings are very interesting because they show that Trx maintains type II neuroblast identity through a specific transcription factor, btd, and thus add to our knowledge of the molecular control of type II neuroblasts identity in the fly, which are emerging as a good model for comparable neural stem cell in vertebrates. The experiments are performed appropriately and the results are presented in a largely logical manner. The data are generally solid and well-quantified. However, there are several significant points that need to be addressed:

1) The discovery of the contribution of btd to Type II neuroblasts appears to be more important than that of Trx and it is suggested to mention btd in the title.

2) One important missing point is that the author do not show the expression of btd in type II neuroblasts through antibody staining, or the loss of btd in Trx mutants. Although an enhancer fragment of btd drives Gal4 in type II neuroblasts, this is only a good indication that btd is in the right place. It would be better to see protein expression by antibody staining, if available.

3) btd mutant type II neuroblasts do not have the same phenotype as Trx mutants, suggesting that other target genes of Trx are involved. Could it be PntP1? Although pntp1 mutants by themselves do not affect type II neuroblast identity, double mutant for btd and pntp1 might have more severe phenotype than btd or pntp1 mutants alone.

4) The reviewers strongly recommend deleting the discussion section on “Trx maintains a type II neuroblast functional identity independently of the Polycomb protein”. There is no mention or investigation of this issue at all in the Results section. Indeed in the Discussion section, the reader is confronted for the first time with largely undocumented data to support the section's conclusions. This is not acceptable, moreover it detracts from the quality of the experiments that are well described and documented in the Results section.

https://doi.org/10.7554/eLife.03502.016

Author response

1) The discovery of the contribution of btd to Type II neuroblasts appears to be more important than that of Trx and it is suggested to mention btd in the title.

We have modified the title to “The Trithorax histone methyltransferase complex maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead.”

2) One important missing point is that the author do not show the expression of btd in type II neuroblasts through antibody staining, or the loss of btd in Trx mutants. Although an enhancer fragment of btd drives Gal4 in type II neuroblasts, this is only a good indication that btd is in the right place. It would be better to see protein expression by antibody staining, if available.

Due to the lack of reagents, we were unable to perform the exact experiment that the reviewer suggested. We were unable to generate a specific antibody against the Btd protein for the immunofluorescent application. In addition, we were unable to recover any viable transgenic fly lines that carry a transgene containing a BAC clone covering the entire btd locus after two attempts. As the only option available, we examined the expression pattern of the btd-Gal4 driver in the rbbp5null mutant brain. We observed a dramatic reduction in the expression of btd-Gal4 in the rbbp5null mutant brain as indicated by the UAS reporter transgene expression. These data are consistent with our hypothesis that btd is a downstream target of Trx. We have now revised the text to include this result and added the image in Figure 4D.

3) Btd mutant type II neuroblasts do not have the same phenotype as Trx mutants, suggesting that other target genes of Trx are involved. Could it be PntP1? Although pntp1 mutants by themselves do not affect type II neuroblast identity, double mutant for btd and pntp1 might have more severe phenotype than btd or pntp1 mutants alone.

We were initially extremely enthusiastic about the possibility that pnt acts in parallel with btd to maintain the functional identity of a type II neuroblasts because Trx binds to the promoter region of the pntP1 transcription unit, and the expression of PntP1 was undetectable in trx mutant type II neuroblasts. Since the pnt locus encodes multiple alternatively spliced transcripts, we knocked down the function of all pnt isoforms in type II neuroblasts by over-expressing three distinct UAS-RNAi transgenes targeting the common exon. To our surprise, pnt mutant type II neuroblasts give rise to immature INP progeny that revert into supernumerary type II neuroblasts. This result strongly suggested that pnt functions in the immature INP to specify an INP identity. Consistently, the heterozygosity of pnt strongly enhanced the supernumerary neuroblast phenotype induced by genes required for proper specification of an INP identity, including brat and erm. Together, these data indicate that pnt functions in the immature INP rather than in the type II neuroblast. We attempted to knock down the function of both btd and pnt by over-expressing all possible combinations of available UAS-btdRNAi and UAS-pntRNAi transgenes in type II neuroblasts. We validated the effectiveness of the btdRNAi construct by inducing type II neuroblasts to assume the functional identity of a type I neuroblast in the larval brain. Larval brains co-expressing the UAS-btdRNAi and UASpntRNAi transgenes in type II neuroblasts possessed supernumerary neuroblasts that displayed a type II neuroblast marker expression profile. However, we were unable to interpret the identity of the supernumerary neuroblasts in the btd, pnt double mutant brain because btd mutant type II neuroblasts acquire a type I neuroblast functional identity without displaying a type I neuroblast marker expression profile. We have now revised the text and added a figure (Figure 4-figure supplement 2A) to describe the functional characterization of pnt in type II neuroblasts.

4) The reviewers strongly recommend deleting the discussion section on “Trx maintains a type II neuroblast functional identity independently of the Polycomb protein”. There is no mention or investigation of this issue at all in the Results section. Indeed in the Discussion section, the reader is confronted for the first time with largely undocumented data to support the section's conclusions. This is not acceptable, moreover it detracts from the quality of the experiments that are well described and documented in the Results section.

We have now deleted this paragraph from the Discussion section.

https://doi.org/10.7554/eLife.03502.017

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  1. Hideyuki Komori
  2. Qi Xiao
  3. Derek H Janssens
  4. Yali Dou
  5. Cheng-Yu Lee
(2014)
Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead
eLife 3:e03502.
https://doi.org/10.7554/eLife.03502

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https://doi.org/10.7554/eLife.03502