Genetic compensation in podocalyxin-like mutants during zebrafish liver development

Reviewer #1 (Public review):

Summary:

The manuscript by Ross, Miscik, and others describes an intriguing series of observations made when investigating the requirement for podxl during hepatic development in zebrafish. Podxl morphants and CRISPants display a reduced number of hepatic stellate cells (HSCs), while mutants are either phenotypically wild type or display an increased number of HSCs.

The absence of observable phenotypes in genetic mutants could indeed be attributed to genetic compensation, as the authors postulate. However, in my opinion, the evidence provided in the manuscript at this point is insufficient to draw a firm conclusion. Furthermore, the opposite phenotype observed in the two deletion mutants is not readily explainable by genetic compensation and invokes additional mechanisms.

Major concerns:

(1) Considering discrepancies in phenotypes, the phenotypes observed in podxl morphants and CRISPants need to be more thoroughly validated. To generate morphants, authors use "well characterized and validated ATG Morpholino" (lines 373-374). However, published morphants, in addition to kidney malformations, display gross developmental defects including pericardial edema, yolk sack extension abnormalities, and body curvature at 2-3 dpf (reference 7 / PMID: 24224085). Were these gross developmental defects observed in the knockdown experiments performed in this paper? If yes, is it possible that the liver phenotype observed at 5 dpf is, to some extent, secondary to these preceding abnormalities? If not, why were they not observed? Did kidney malformations reproduce? On the CRISPant side, were these gross developmental defects also observed in sgRNA#1 and sgRNA#2 CRISPants? Considering that morphants and CRISPants show very similar effects on HSC development and assuming other phenotypes are specific as well, they would be expected to occur at similar frequencies. It would be helpful if full-size images of all relevant morphant and CRISPant embryos were displayed, as is done for tyr CRISPant in Figure S2. Finally, it is very important to thoroughly quantify the efficacy of podxl sgRNA#1 and sgRNA#2 in CRISPants. The HRMA data provided in Figure S1 is not quantitative in terms of the fraction of alleles with indels. Figure S3 indicates a very broad range of efficacies, averaging out at ~62% (line 100). Assuming random distribution of indels among cells and that even in-frame indels result in complete loss of function (possible for sgRNA#1 due to targeting the signal sequence), only ~38% (.62*.62) of all cells will be mutated bi-allelically. That does not seem sufficient to reliably induce loss-of-function phenotypes. My guess is that the capillary electrophoresis method used in Figure S3 underestimates the efficiency of mutagenesis, and that much higher mutagenesis rates would be observed if mutagenesis were assessed by amplicon sequencing (ideally NGS but Sanger followed by deconvolution analysis would suffice). This would strengthen the claim that CRISPant phenotypes are specific.

(2) In addition to confidence in morphant and CRISPant phenotypes, the authors' claim of genetic compensation rests on the observation that podxl (Ex1(p)_Ex7Δ) mutants are resistant to CRISPant effect when injected with sgRNA#1 (Figure 3L). Considering the issues raised in the paragraph above, this is insufficient. There is a very straightforward way to address both concerns, though. The described podxl(-194_Ex7Δ) and podxl(-319_ex1(p)Δ) deletions remove the binding site for the ATG morpholino. Therefore, deletion mutants should be refractive to the Morpholino (specificity assessment recommended in PMID: 29049395, see also PMID: 32958829). Furthermore, both deletion mutants should be refractive to sgRNA#1 CRISPant phenotypes, with the first being refractive to sgRNA#2 as well.

Reviewer #2 (Public review):

In this manuscript, Ross and Miscik et. al described the phenotypic discrepancies between F0 zebrafish mosaic mutant ("CRISPants") and morpholino knockdown (Morphant) embryos versus a set of 5 different loss-of-function (LOF) stable mutants in one particular gene involved in hepatic stellate cells development: podxl. While transient LOF and mosaic mutants induced a decrease of hepatic stellate cells number stable LOF zebrafish did not. The authors analyzed the molecular causes of these phenotypic differences and concluded that LOF mutants are genetically compensated through the upregulation of the expression of many genes. Additionally, they ruled out other better-known and described mechanisms such as the expression of redundant genes, protein feedback loops, or transcriptional adaptation.

While the manuscript is clearly written and conclusions are, in general, properly supported, there are some aspects that need to be further clarified and studied.

(1) It would be convenient to apply a method to better quantify potential loss-of-function mutations in the CRISPants. Doing this it can be known not only percentage of mutations in those embryos but also what fraction of them are actually generating an out-of-frame mutation likely driving gene loss of function (since deletions of 3-6 nucleotides removing 1-2 aminoacid/s will likely not have an impact in protein activity, unless that this/these 1-2 aminoacid/s is/are essential for the protein activity). With this, the authors can also correlate phenotype penetrance with the level of loss-of-function when quantifying embryo phenotypes that can help to support their conclusions.

(2) It is unclear that 4.93 ng of morpholino per embryo is totally safe. The amount of morpholino causing undesired effects can differ depending on the morpholino used. I would suggest performing some sanity check experiments to demonstrate that morpholino KD is not triggering other molecular outcomes, such as upregulation of p53 or innate immune response.

(3) Although the authors made a set of controls to demonstrate the specificity of the CRISPant phenotypes, I believe that a rescue experiment could be beneficial to support their conclusions. Injecting an mRNA with podxl ORF (ideally with a tag to follow protein levels up) together with the induction of CRISPants could be a robust manner to demonstrate the specificity of the approach. A rescue experiment with morphants would also be good to have, although these are a bit more complicated, to ultimately demonstrate the specificity of the approach.

(4) In lines 314-316, the authors speculate on a correlation between decreased HSC and Podxl levels. It would be interesting to actually test this hypothesis and perform RT-qPCR upon CRISPant induction or, even better and if antibodies are available, western blot analysis.

(5) Similarly, in lines 337-338 and 342-344, the authors discuss that it could be possible that genes near to podxl locus could be upregulated in the mutants. Since they already have a transcriptomic done, this seems an easy analysis to do that can address their own hypothesis.

(6) Figures 4 and 5 would be easier to follow if panels B-F included what mutants are (beyond having them in the figure legend). Moreover, would it be more accurate and appropriate if the authors group all three WT and mutant data per panel instead of showing individual fish? Representing technical replicates does not demonstrate in vivo variability, which is actually meaningful in this context. Then, statistical analysis can be done between WT and mutant per panel and per set of primers using these three independent 3-month-old zebrafish.

Reviewer #3 (Public review):

Summary:

Ross et al. show that knockdown of zebrafish podocalyxin-like (podxl) by CRISPR/Cas or morpholino injection decreased the number of hepatic stellate cells (HSC). The authors then generated 5 different mutant alleles representing a range of lesions, including premature stop codons, in-frame deletion of the transmembrane domain, and deletions of the promoter region encompassing the transcription start site. However, unlike their knockdown experiment, HSC numbers did not decrease in podxl mutants; in fact, for two of the mutant alleles, the number of HSCs increased compared to the control. Injection of podxl CRISPR/Cas constructs into these mutants had no effect on HSC number, suggesting that the knockdown phenotype is not due to off-target effects but instead that the mutants are somehow compensating for the loss of podxl. The authors then present multiple lines of evidence suggesting that compensation is not exclusively due to transcriptional adaptation - evidence of mRNA instability and nonsense-mediated decay was observed in some but all mutants; expression of the related gene endoglycan (endo) was unchanged in the mutants and endo knockdown had no effect on HSC numbers; and, expression profiling by RNA sequencing did not reveal changes in other genes that share sequence similarity with podxl. Instead, their RNA-seq data showed hundreds of differentially expressed genes, especially ECM-related genes, suggesting that compensation in podxl mutants is complex and multi-genic.

Strengths:

The data presented is impressively thorough, especially in its characterization of the 5 different podxl alleles and exploration of whether these mutants exhibit transcriptional adaptation.

Weaknesses:

RNA sequencing expression profiling was done on adult livers. However, compensation of HSC numbers is apparent by 6 dpf, suggesting compensatory mechanisms would be active at larval or even embryonic stages. Although possible, it's not clear that any compensatory changes in gene expression would persist to adulthood.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript by Ross, Miscik, and others describes an intriguing series of observations made when investigating the requirement for podxl during hepatic development in zebrafish. Podxl morphants and CRISPants display a reduced number of hepatic stellate cells (HSCs), while mutants are either phenotypically wild type or display an increased number of HSCs.

The absence of observable phenotypes in genetic mutants could indeed be attributed to genetic compensation, as the authors postulate. However, in my opinion, the evidence provided in the manuscript at this point is insufficient to draw a firm conclusion. Furthermore, the opposite phenotype observed in the two deletion mutants is not readily explainable by genetic compensation and invokes additional mechanisms.

Major concerns:

(1) Considering discrepancies in phenotypes, the phenotypes observed in podxl morphants and CRISPants need to be more thoroughly validated. To generate morphants, authors use "well characterized and validated ATG Morpholino" (lines 373-374). However, published morphants, in addition to kidney malformations, display gross developmental defects including pericardial edema, yolk sack extension abnormalities, and body curvature at 2-3 dpf (reference 7 / PMID: 24224085). Were these gross developmental defects observed in the knockdown experiments performed in this paper? If yes, is it possible that the liver phenotype observed at 5 dpf is, to some extent, secondary to these preceding abnormalities? If not, why were they not observed? Did kidney malformations reproduce? On the CRISPant side, were these gross developmental defects also observed in sgRNA#1 and sgRNA#2 CRISPants? Considering that morphants and CRISPants show very similar effects on HSC development and assuming other phenotypes are specific as well, they would be expected to occur at similar frequencies. It would be helpful if full-size images of all relevant morphant and CRISPant embryos were displayed, as is done for tyr CRISPant in Figure S2. Finally, it is very important to thoroughly quantify the efficacy of podxl sgRNA#1 and sgRNA#2 in CRISPants. The HRMA data provided in Figure S1 is not quantitative in terms of the fraction of alleles with indels. Figure S3 indicates a very broad range of efficacies, averaging out at ~62% (line 100). Assuming random distribution of indels among cells and that even in-frame indels result in complete loss of function (possible for sgRNA#1 due to targeting the signal sequence), only ~38% (.62*.62) of all cells will be mutated bi-allelically. That does not seem sufficient to reliably induce loss-of-function phenotypes. My guess is that the capillary electrophoresis method used in Figure S3 underestimates the efficiency of mutagenesis, and that much higher mutagenesis rates would be observed if mutagenesis were assessed by amplicon sequencing (ideally NGS but Sanger followed by deconvolution analysis would suffice). This would strengthen the claim that CRISPant phenotypes are specific.

The reviewer points out some excellent caveats regarding the morphant experiments. We agree that at least some of the effects of the podxl morpholino may be related to its effects on kidney development and/or gross developmental defects that impede liver development. Because of these limitations, we focused our experiments on analysis of CRISPant and mutant phenotypes, including showing that podxl (Ex1(p)_Ex7Δ) mutants are resistant to CRISPant effects on HSC number when injected with sgRNA#1. We did not observe any gross morphologic defects in podxl CRISPants. Liver size was not significantly altered in podxl CRISPants (Figure 2A). We will add brightfield images of podxl CRISPant larvae to the supplemental data for the revised manuscript.

We agree with the reviewer that HRMA is not quantitative with respect to the fraction of alleles with indels and that capillary electrophoresis likely underestimates mutagenesis efficiency. Nonetheless, even with 100% mutation efficiency, podxl CRISPant knockdown, like most CRISPR knockdowns, would not represent complete loss of function: ~1/3 of alleles will contain in-frame mutations and likely retain at least some gene function, so ~1/3*1/3 = 1/9 of cells will have no out-of-frame indels and contain two copies of at least partially functional podxl and ~2/3*2/3 = 4/9 of cells will have one out-of-frame indel and one copy of at least partially functional podxl. Thus, the decreased HSCs we observe with podxl CRISPant likely represents a partial loss-of-function phenotype in any case.

(2) In addition to confidence in morphant and CRISPant phenotypes, the authors' claim of genetic compensation rests on the observation that podxl (Ex1(p)_Ex7Δ) mutants are resistant to CRISPant effect when injected with sgRNA#1 (Figure 3L). Considering the issues raised in the paragraph above, this is insufficient. There is a very straightforward way to address both concerns, though. The described podxl(-194_Ex7Δ) and podxl(-319_ex1(p)Δ) deletions remove the binding site for the ATG morpholino. Therefore, deletion mutants should be refractive to the Morpholino (specificity assessment recommended in PMID: 29049395, see also PMID: 32958829). Furthermore, both deletion mutants should be refractive to sgRNA#1 CRISPant phenotypes, with the first being refractive to sgRNA#2 as well.

The reviewer proposes elegant experiments to address the specificity of the morpholino. For the revision, we plan to perform additional morpholino studies, including morpholino injections of podxl mutants and assessment of tp53 and other immune response/cellular stress pathway genes in podxl morphants.

Reviewer #2 (Public review):

In this manuscript, Ross and Miscik et. al described the phenotypic discrepancies between F0 zebrafish mosaic mutant ("CRISPants") and morpholino knockdown (Morphant) embryos versus a set of 5 different loss-of-function (LOF) stable mutants in one particular gene involved in hepatic stellate cells development: podxl. While transient LOF and mosaic mutants induced a decrease of hepatic stellate cells number stable LOF zebrafish did not. The authors analyzed the molecular causes of these phenotypic differences and concluded that LOF mutants are genetically compensated through the upregulation of the expression of many genes. Additionally, they ruled out other better-known and described mechanisms such as the expression of redundant genes, protein feedback loops, or transcriptional adaptation.

While the manuscript is clearly written and conclusions are, in general, properly supported, there are some aspects that need to be further clarified and studied.

(1) It would be convenient to apply a method to better quantify potential loss-of-function mutations in the CRISPants. Doing this it can be known not only percentage of mutations in those embryos but also what fraction of them are actually generating an out-of-frame mutation likely driving gene loss of function (since deletions of 3-6 nucleotides removing 1-2 aminoacid/s will likely not have an impact in protein activity, unless that this/these 1-2 aminoacid/s is/are essential for the protein activity). With this, the authors can also correlate phenotype penetrance with the level of loss-of-function when quantifying embryo phenotypes that can help to support their conclusions.

Reviewer #2 raises an excellent point that is similar to Reviewer #1’s first concern. Please see our response above. In general, we agree that correlating phenotype penetrance with level of loss-of-function is a very good way to support conclusions regarding specificity in knockdown experiments. Unfortunately, because the phenotype we are examining (HSC number) has a relatively large standard deviation even in control/wildtype larvae (for example, 63 ± 19 (mean ± standard deviation) HSCs per liver in uninjected control siblings in Figure 1) it would be technically very difficult to do this experiment for podxl.

(2) It is unclear that 4.93 ng of morpholino per embryo is totally safe. The amount of morpholino causing undesired effects can differ depending on the morpholino used. I would suggest performing some sanity check experiments to demonstrate that morpholino KD is not triggering other molecular outcomes, such as upregulation of p53 or innate immune response.

Reviewer #2 raises an excellent point that is similar to Reviewer #1’s second concern. Please see our response above. We acknowledge that some of the effects of the podxl morpholino may be non-specific. To address this concern in the revised manuscript, we plan to perform additional morpholino studies, including morpholino injections of podxl mutants and assessment of tp53 and other immune response/cellular stress pathway genes in podxl morphants.

(3) Although the authors made a set of controls to demonstrate the specificity of the CRISPant phenotypes, I believe that a rescue experiment could be beneficial to support their conclusions. Injecting an mRNA with podxl ORF (ideally with a tag to follow protein levels up) together with the induction of CRISPants could be a robust manner to demonstrate the specificity of the approach. A rescue experiment with morphants would also be good to have, although these are a bit more complicated, to ultimately demonstrate the specificity of the approach.

(4) In lines 314-316, the authors speculate on a correlation between decreased HSC and Podxl levels. It would be interesting to actually test this hypothesis and perform RT-qPCR upon CRISPant induction or, even better and if antibodies are available, western blot analysis.

We appreciate the reviewer’s acknowledgement of the controls we performed to demonstrate the specificity of the CRISPant phenotypes. The proposed experiments (rescue, assessment of Podxl levels) would help bolster our conclusions but are technically difficult due to the relatively large standard deviation for the HSC number phenotype even in wildtype larvae and the lack of well-characterized zebrafish antibodies against Podxl.

(5) Similarly, in lines 337-338 and 342-344, the authors discuss that it could be possible that genes near to podxl locus could be upregulated in the mutants. Since they already have a transcriptomic done, this seems an easy analysis to do that can address their own hypothesis.

Thank you for this suggestion. We were referring in these sections to genes that are near the podxl locus with respect to three-dimensional chromatin structure; such genes would not necessarily be near the podxl locus on chromosome 4. We will clarify the text in this paragraph for the revised manuscript. At the same time, we will examine our transcriptomic data to check expression of mkln1, cyb5r3, and other nearby genes on chromosome 4 as suggested and include this analysis in the revised manuscript.

(6) Figures 4 and 5 would be easier to follow if panels B-F included what mutants are (beyond having them in the figure legend). Moreover, would it be more accurate and appropriate if the authors group all three WT and mutant data per panel instead of showing individual fish? Representing technical replicates does not demonstrate in vivo variability, which is actually meaningful in this context. Then, statistical analysis can be done between WT and mutant per panel and per set of primers using these three independent 3-month-old zebrafish.

Thank you for this suggestion. We will modify these figures to clarify our results.

Reviewer #3 (Public review):

Summary:

Ross et al. show that knockdown of zebrafish podocalyxin-like (podxl) by CRISPR/Cas or morpholino injection decreased the number of hepatic stellate cells (HSC). The authors then generated 5 different mutant alleles representing a range of lesions, including premature stop codons, in-frame deletion of the transmembrane domain, and deletions of the promoter region encompassing the transcription start site. However, unlike their knockdown experiment, HSC numbers did not decrease in podxl mutants; in fact, for two of the mutant alleles, the number of HSCs increased compared to the control. Injection of podxl CRISPR/Cas constructs into these mutants had no effect on HSC number, suggesting that the knockdown phenotype is not due to off-target effects but instead that the mutants are somehow compensating for the loss of podxl. The authors then present multiple lines of evidence suggesting that compensation is not exclusively due to transcriptional adaptation - evidence of mRNA instability and nonsense-mediated decay was observed in some but all mutants; expression of the related gene endoglycan (endo) was unchanged in the mutants and endo knockdown had no effect on HSC numbers; and, expression profiling by RNA sequencing did not reveal changes in other genes that share sequence similarity with podxl. Instead, their RNA-seq data showed hundreds of differentially expressed genes, especially ECM-related genes, suggesting that compensation in podxl mutants is complex and multi-genic.

Strengths:

The data presented is impressively thorough, especially in its characterization of the 5 different podxl alleles and exploration of whether these mutants exhibit transcriptional adaptation.

Thank you very much for appreciating the hard work that went into this manuscript.

Weaknesses:

RNA sequencing expression profiling was done on adult livers. However, compensation of HSC numbers is apparent by 6 dpf, suggesting compensatory mechanisms would be active at larval or even embryonic stages. Although possible, it's not clear that any compensatory changes in gene expression would persist to adulthood.

This reviewer makes an excellent point. Our finding that the largest changes in gene expression were in extracellular matrix (ECM) genes and ECM modulation is a major function of HSCs supports the hypothesis that genetic compensation is occurring in adults. Nonetheless, we agree that compensatory changes in adults may not fully reflect the compensatory changes during development, so it would bolster the conclusions of the paper to perform the RNA sequencing and qPCR experiments on zebrafish larval livers.

We tried very hard to do this experiment proposed by Reviewer #3. In our hands, obtaining sufficient high-quality RNA for robust gene expression analysis typically requires pooling of ~10-15 larval livers. These larvae need to be obtained from a heterozygous in-cross in order to have matched wildtype sibling controls. Livers must be dissected from freshly euthanized (not fixed) zebrafish. Thus, this experiment requires genotyping live, individual larvae from a small amount of tissue (without sacrificing the larvae) before dissecting and pooling the livers. Unfortunately we were unable to confidently and reproducibly genotype individual live podxl larvae with these small amounts of tissue despite trying multiple approaches. Therefore we were not able to perform gene expression analysis on podxl mutant larval livers.