Fas2EB112 : a tale of two chromosomes

  1. Tara M Finegan
  2. Christian Cammarota
  3. Oscar Mendoza Andrade
  4. Audrey M Garoutte
  5. Dan T Bergstralh

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Abstract

The cell–cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

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  1. Version published to 10.1093/g3journal/jkae047
  2. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors reveal the genetic basis for why two different null alleles of Fasiclin II (Fas2), a member of the Igg superfamily of cell surface proteins, result in very different phenotypes in the follicular epithelia of the developing Drosophila egg chamber. One null allele - Fas2G0336 - results in rare occurrences of follicular cells being found outside of and apical to the plane of the epithelia in mutant clones, whereas the other null allele - Fas2EB112 - results in frequent occurrences of cells apical to the plane of the epithelia in mutant clones. Using recombination, they map a second mutation on the Fas2EB112 chromosome and demonstrate that it is a known allele of another Igg superfamily protein member - Neuroglian (Nrg). Thus, the severe phenotypes associated with only one of the two Fas2 null alleles can be explained by the additional absence of Nrg function, supporting previous studies that, in the follicular epithelia, the functions of Fas2 and Nrg are overlapping and compensatory.

    The authors fully support this finding by several carefully done experiments, all of which are thoroughly described and nicely illustrated in the figures in the main body of the paper, with helpful information provided in the supplemental figures. Indeed, the authors explore a number of possible explanations for the phenotypic differences with the two null Fas2 alleles and their conclusion of there being a known mutation in Nrg on one of the two null chromosomes is supported by several independent approaches. No additional experiments are necessary, and the findings should be easily reproducible by others using the reagents described in the study.

    Minor point: On the second page of the results, the last paragraph starts with "The second chromosome" in the first sentence. This is a bit confusing - could be considered chromosome 2, when the authors do not mean chromosome 2. It would be better to refer to this as "The other chromosome generated by recombination".

    Referees cross-commenting

    The other two reviewers have picked up some minor issues that should be addressed by the authors (quantitation of the Western, labels on all figure panels, etc). These changes would definitely improve the manuscript and should be done. I'm not convinced that the overall findings are important for a significant part of the Drosophila community - seems more of a specialty audience who needs to know that there is a second mutation in Neuroglian on one of the Fas2 null chromosomes that are available through the stock centers.

    Significance

    The findings are particularly relevant to anyone who has used the Fas2EB112 allele for their studies or who plans to do so. Otherwise, it serves as a cautionary tale to all to examine and report phenotypes using multiple different alleles as a control for what other mutations may also exist on mutant chromosomes of interest.

    I am a Drosophila developmental biologist who uses most of the same tools used in this manuscript on a regular basis. I am also quite familiar with the experimental system used for this work.

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    Referee #2

    Evidence, reproducibility and clarity

    Epithelial integrity is fundamental to organ function and development. There are mechanisms to reintegrate cells that divide outside of the cell layer that depend on cell adhesion. In many cases, including the Drosophila follicular epithelium, this adhesion and reintegration is dependent on partially redundant functions of the IgCAMs Fas2, Fas3, and Neruoglian (Nrg).

    Here, Finegan et al have identified discrepancies in phenotypic strength between two null alleles of Fas2 and show that this is due to an additional mutation in Nrg on the phenotypically stronger chromosome (likely due to a submission of a double mutant rather than a single mutant to the stock center, as this Nrg allele seems to be the same aberration as the Nrg14 null alle used in this study as well). Overall, the data is sound and of interest to people working with these genes in the adhesion field, but not of a broader interest. Over passages the paper is lengthy (such as a description of recombinations separating the mutations (or not) on p5 or the extensive description of the Nrg splice variants on p7.

    Points to address:

    Major:

    Fig 1C and Fig 4B: Why do the shRNAs used for Fas2 and Nrg have stronger phenotypes than the mutants? As clones were used, the argument of adaptation is harder to make. Specificity of shRNAs may have to be shown in a mutant background. Therefore, actually, this is particularly puzzling/worrisome in case of Fig. 4B where Nrg RNAi is stronger than the Nrg14 null allele (i.e. e(Fas2)mut) background.

    P8/Fig 3F Western blot: Despite description of quantification of blots in methods, nothing is quantified, and the arguments of dosage compensation thus cannot be made. Comes up again in the discussion. Furthermore, would the resolution of the blot allow distinction of Nrg180-YFP from Nrg167-YFP to really exclude that only one of them is expressed from the tagged locus?

    Discussion: Similarly, Mannheim Fas2EB112 has a lower average number of...., though this difference is not significant. Thus, is does not have a lower average!

    Discussion: Sentence ending in '...that E(Fas2)mut is suppressed' is confusing. Suppressed with repressed to what by what?

    Minor:

    • P2 second paragraph: insert 'Drosophila' before 'follicular epithelium'.
    • P4: Dr. Riechmann's first name is misspelled.
    • P8 last line: Fig. X should be Fig. 4A.
    • P12, legend of Fig. 2: description of panel B missing.
    • Tables are supplementary and should be labeled as such in the methods.
    • Fig. S3C: the non-disjunction aspect is not straight forward to see in the figure (e.g. XXY genotype and its correlation to eye shape from balancers).

    Significance

    Overall, the data is sound and of interest to people working with these genes in the adhesion field, but not of a broader interest.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript reports an intriguing genetic puzzle and its solution that is very relevant to Drosophila researchers studying the Immunoglobulin-superfamily cell adhesion molecules (IgCAMs) Fasciclin 2 (Fas2) and Neuroglian (Nrg), homologs of vertebrate NCAM and L1-CAM, respectively. In their earlier work, the authors described the roles of these IgCAMs in the Drosophila follicle epithelium, where Fas2 and Nrg are required for cell reintegration after mitosis. During cell division, errors in spindle positioning can cause newly born cells to get displaced from the epithelial sheet. Such apically extruded cells normally re-integrate with high efficiency into the epithelium. As the authors previously showed through genetic studies in the Drosophila follicle epithelium, mutations in Fas2 and Nrg act synergistically to disrupt reintegration, leading to the accumulation of extruded ("popped out") cells. A puzzle arose when the authors compared the phenotypes of two different presumed protein-null alleles of Fas2 (Fas2(EB112) and Fas2(G0336)), which showed different strengths of epithelial cell reintegration defects. They systematically examined the phenotypes of Fas2(G0336) and two different fly strains carrying the Fas2(EB112) allele and discovered that the more severe defects seen in one of the Fas2(EB112) strains are due to the presence of an additional mutation in Nrg on the same chromosome. Intriguingly, they identified the mutation in the Nrg locus as the previously characterized Nrg(14) allele. This suggests that the Fas2 Nrg double mutant chromosome did not arise naturally through random appearance of a second-site mutation, but that it was generated intentionally and then apparently got mixed up with the Fas2(EB112) strain (however, the authors do not comment on this point). The authors spared no effort to resolve this genetic puzzle in a clear and convincing manner. The used clever genetics, genome sequencing and polytene chromosome preparations to unveil the Nrg mutation as Nrg(14). The results are very clearly documented, and the text is well written, almost in the style of a criminal investigation. I have only minor comments to be addressed by the authors before the manuscript should be published. p. 5: " The second chromosome that we generated through recombination did not demonstrate decreased anti-Fas2 immunoreactivity or the presence of popped-out cells (Figure 2A,B)..." The latter finding is surprising, as the authors previously showed that the Nrg(14) mutation leads to an increase in the number of popped out cells (Cammarota et al. 2020). They discuss this issue later in the text, but the reason for this discrepancy remains unclear. In the same context, p. 9: " Consistent with this, we find that E(Fas2)mut does not increase the number of popped-out cells in Nrg knockdown tissue (Figure 4B)." But Fig. 4B shows that Nrg knockdown increases the number of popped out cells in E(Fas2) mutant clones. Assuming that Nrg14 is a true null allele, this result would suggest that the effect of Nrg knockdown on the frequency of popped out cells is an RNAi artifact (off-target effect)? The authors should comment on this issue.

    Fig. 3F: The Nrg(167) band in the Fas2(EB112) Mannheim lane is stronger than in the control (w1118), suggesting that Nrg(167) expression is upregulated when Fas2 levels are reduced. Please comment.

    The authors refer to the enhancer of Fas2 mutation interchangeably as e(Fas2) and E(Fas2). As I understand, the mutation is recessive, and should therefore be referred to as e(Fas2).

    p. 4: "Both Fas2EB112 (Grenningloh, Rehm, and Goodman 1991) and Fas2G0336 are thought to be protein null (Bergstralh, Lovegrove, and St Johnston 2015)." The authors should explain why these mutations are "thought to be protein null". They show that Nrg immunoreactivity is lost in Nrg(14) mutants, and likewise for Fas2. If both anti-Fas2 (1D4) and anti-Nrg antisera detect all isoforms of the respective proteins, the authors should state this clearly and modify their rather vague statement on p. 4 ("thought to be protein null").

    Fig. 4C: Please add labels to indicate the gene locus (Fas2, Nrg) analyzed in each panel.

    Fig. 4D: The chromosomes shown are from heterozygous flies carrying the respective mutation in trans to the w1118 X-chromosome. Correct genotypes should be indicated.

    For the genome sequencing, what was the exact genotype of the flies that were sequenced? Hemizygous (lethal) embryos or heterozygotes? If DNA from heterozygotes was analyzed, how were short reads assigned to one of the parental (mutation-bearing or wild-type) chromosomes?

    p. 8/9: "Figure X".

    Figure legends: scale bars are in µm, not µM.

    Referees cross-commenting

    I agree that the results are important for a specialty audience concerned with the specific mutant strains described here.

    Significance

    Although the manuscript does not report a conceptual advance, the findings are very important for a significant part of the Drosophila community, particularly those studying Fas2 and Nrg. These proteins are involved in development of the nervous system, in synapse formation, as well as in epithelial morphogenesis and barrier formation. The Fas2(EB112) mutant has been widely used in at least 37 publications (dating back to 1991), several of which will need to be revisited in the light of the new findings reported here. This reviewer is a cell and developmental biologist with expertise in Drosophila genetics.

  6. Version published to 10.1101/2024.01.03.574100v1 on bioRxiv