Cavefish cope with environmental hypoxia by developing more erythrocytes and overexpression of hypoxia-inducible genes

  1. Corine M van der Weele
  2. William R Jeffery

  • Curated by eLife

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    Evaluation Summary:

    This article provides insight into how Astyanax mexicanus cavefish may have adapted to the hypoxic waters present in the cave environment. How the extreme environmental pressure of low oxygen has shaped cavefish evolution has been understudied compared to other pressures like absence of light or low nutrients. This is the first study to look for changes in early cavefish development that may provide hypoxia tolerance. The claims that cavefish have expanded erythrocyte development and increased hypoxia gene expression are strongly supported by the data. Demonstrating that these traits are adaptive and provide hypoxia tolerance requires further assessment of the current results and would be strengthened by additional experiments. Overall, this work is an important first step in understanding the evolution of hypoxia tolerance in A. mexicanus cavefish.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax , potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory-induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 ( hif1 ) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.

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  1. Version published to 10.7554/elife.69109 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The manuscript: "Cavefish adapt to hypoxia by constitutive overexpression of hypoxia-inducible factor genes and increased erythrocyte development" by Corine van der Weele and William Jeffery addresses an interesting topic. The authors find that cavefish of the species Astyanax mexicanus develop more red blood cells than surface fish of the same species when raised in the laboratory under normoxic conditions. The authors perform a detailed analysis of the developmental origins of hematopoiesis and find an expansion of the hematopoietic domains in cavefish embryos. Further the authors test how cavefish respond to chemically induced hemolitic anemia and how growth is affected under artificial hypoxic conditions. Finally, they look at transcriptional regulation of known hypoxia response genes.

    Major comments:

    While the authors are performing a careful and conclusive developmental analysis of the hematopoiesis in these fish,there are a few inconsistences. It would be nice to see comparable timepoints in both the insitu and the qPCR analysis. For example, the authors show in Figure 1G/H 36 and 84 hpf timepoints while the qPCR is performed at different stages. This is especially relevant as the authors make quantitative statements from whole mount in situ analysis which are not necessarily suited to do quantitative comparisons. Furthermore, they are not using the same genes as readout as they use hbb2 in the insitu and hbbe2 in the qPCR analysis.

    We thank the reviewer for this comment. We now report gene expression experiments done at the same developmental stages and with same genes in revised Figure 2. More specifically: (1) the hbb2, hbbe2, and gfi1aa qPCR results are shown at 24 and 60 hpf, (2) the hbb2 in situ hybridization results are shown at 24 and 60 hpf in parallel with the qPCR results for this gene, (3) in situ hybridization results for hbb2 are still included at 36 hpf because this stage most obviously illustrates expression enhancement in the cavefish yolk mass, (4) in situ results for hbbe2 are not presented because after many attempts using probes made from the coding or UTR regions of this gene, we were unable to obtain a reactive RNA probe, and (5) gfi1aa in situ is shown only for the 24 hpf stage because this was the stage in which this gene was strongly expressed according to qPCR data. The overall conclusion that hemoglobin and gfi1aa transcription factor genes are upregulated in cavefish compared to surface fish remains the same.

    While the growth study is conceptually interesting, it is unclear why the authors average all 12 larvae even though they keep them individually. They mention that this is to avoid "pseudoreplication", however I am not sure why that would be the case. It would be important to see all the data. Also, in Figure 4B the statistics for the comparison between surface normoxic and hypoxic are not shown, even though the text mentions it as significant.

    Because the growth study has now been repeated using a hypoxia chamber to better control oxygen levels, these comments are no longer specifically relevant. New data is presented in revised Figure 6. In this figure, data changes for individual larvae and their statistical significance are included.

    Reviewer #3 (Public Review):

    In this manuscript van der Weele and Jeffery propose two evolutionary mechanisms, via which cavefish (Mexican tetra Astyanax mexicanus) have adapted to a hypoxic environment based on direct comparison with hypoxia-sensitive surface dwellers of the same species. These adaptations are increased erythrocyte production and heightened transcription of hypoxia-inducible factor 1 (hif1). Using a combination of time lapse imaging, bright field microscopy and in situ hybridization with a battery of hematopoietic markers, they provide clear and compelling evidence that cavefish have a larger number of erythrocytes than surface dwellers and that this increase stems from an expansion of two erythropoiesis domains during early development. To address the functional relevance of increased erythrocytes they induced hemolytic anemia using the drug phenylhydrazine (PHT) and showed that cavefish are less sensitive to this disorder than surface fish, possibly as a result of the increase in erythrocytes. Nevertheless, both types of fish exhibited developmental anomalies post-treatment, including reduced tail length. They further propose that the larger number of erythrocytes may promote adaptation to hypoxia by countering the negative impact of this environmental stressor on growth. Indeed, surface fish appear to be more susceptible to hypoxia-induced stunted growth of the post-anal tail than cavefish, although the numbers are quite variable. Lastly, the authors go on to show that cave dwellers express constitutively high transcript levels of hif1 genes and downstream targets of Hif1. One of these targets is the growth suppressor IGFBP1a, which could explain growth restriction of surface fish under hypoxia. Overall, even though increased erythrocyte production is a well-documented response to life in hypoxic environments, this study provides an interesting perspective on this adaptation seen through the lens of evolutionary biology.

    The data support the main conclusions that erythropoiesis and hif1 transcription are enhanced in cavefish but do not convincingly identify the functional relevance of these traits to hypoxia adaptation in cavefish. In this regard:

    1. The induction of hemolytic lysis using PHT and associated developmental defects raises concerns about lack of drug specificity.

    We appreciate this concern and cannot entirely exclude non-specific effects of PHZ. However, the PHZ approach has been used successfully in other systems, including zebrafish, to “ablate” erythrocytes and check development. In the zebrafish case (see Pelster and Bruggren,1996 in the reference list), similar concentrations of PHZ resulted in no effects on development (blood cells were not determined), which seems inconsistent with any non-specific effects. In addition, we have now performed a new experiment (see revised Figure 5D) in which treatment with a very high level of PHZ resulting in hemolysis of all erythrocytes reduced tail growth without producing notochord abnormalities, suggesting that non-specific effects on the latter cannot explain PHZ effects on growth.

    1. The connection between erythrocyte number and organismal growth is not clearly established. The reduced tail length defect that is observed following PHT treatment, even if specific, is likely to be an indirect consequence of abnormal notochord morphology rather than arrested tail growth.

    As also described immediately above, to address this concern, we have performed a new experiment in which PHZ effects on tail length have been measured for a shorter period and at higher PHZ concentration resulting in the virtual absence of red blood cells (revised Figure 5D). The results showed significant reduction in tail growth compared to controls but no effects on notochord morphology, providing evidence that effects on growth are not related to notochord defects.

    1. Because the above connection was not clearly established, the lack of growth inhibition in cavefish following exposure to hypoxia (thought to be offset by the elevated number of erythrocytes in cavefish) is not readily explainable. The data itself showing a difference in tail growth between cavefish and surface dwellers exposed to hypoxia is not strong due to high variability and low numbers.

    New results using a hypoxia chamber have now more clearly established a differential relationship between hypoxia and growth in surface fish and cavefish.

    1. Increased hif1 transcript levels may contribute to enhanced hypoxia adaptation in cavefish, however Hif1 is known to be primarily regulated at the post-translational level, resulting in its enhanced stability and activity. It is unclear whether the activity of Hif1 is elevated in cavefish relative to surface fish as several known Hif1 targets are not up-regulated in cavefish relative to surface fish.

    Thank you for reminding us about the known regulation of Hif1 at the post-translational level. We do not address this point in the revised manuscript because all of our work has been carried out at the transcriptional level. In this, we show significant upregulation of all of the hif1 genes, and two downstream target genes. Three downstream target genes are not upregulated by hypoxia, but we do not currently understand why. Nothing is known about Hif1 regulation of downstream targets in Astyanax.

    In addition, there are a few technical concerns

    1. The manner in which hypoxic water was generated (bubbling of nitrogen gas) is unlikely to maintain a constant value over time. Furthermore, covering the tank with foil will not prevent gas exchange. Hence there is a concern that the hypoxic values may be variable between trials and even within the same trial.

    Thank you for this suggestion. To address the concerns about stability of oxygen depletion within and between trials, we have now repeated all of the hypoxia experiments using a hypoxia chamber.

    1. It is important that transcript levels for the q-PCR reference gene be stable across normoxic and hypoxic conditions. A discussion about the considerations that went into selecting RPL13a as a reference gene was not provided.

    We have provided discussions justifying the use rpl13a and other genes as references for qPCR analysis.

  3. eLife

    Evaluation Summary:

    This article provides insight into how Astyanax mexicanus cavefish may have adapted to the hypoxic waters present in the cave environment. How the extreme environmental pressure of low oxygen has shaped cavefish evolution has been understudied compared to other pressures like absence of light or low nutrients. This is the first study to look for changes in early cavefish development that may provide hypoxia tolerance. The claims that cavefish have expanded erythrocyte development and increased hypoxia gene expression are strongly supported by the data. Demonstrating that these traits are adaptive and provide hypoxia tolerance requires further assessment of the current results and would be strengthened by additional experiments. Overall, this work is an important first step in understanding the evolution of hypoxia tolerance in A. mexicanus cavefish.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript: "Cavefish adapt to hypoxia by constitutive overexpression of hypoxia-inducible factor genes and increased erythrocyte development" by Corine van der Weele and William Jeffery addresses an interesting topic. The authors find that cavefish of the species Astyanax mexicanus develop more red blood cells than surface fish of the same species when raised in the laboratory under normoxic conditions. The authors perform a detailed analysis of the developmental origins of hematopoiesis and find an expansion of the hematopoietic domains in cavefish embryos. Further the authors test how cavefish respond to chemically induced hemolitic anemia and how growth is affected under artificial hypoxic conditions. Finally, they look at transcriptional regulation of known hypoxia response genes.

    Major comments:

    While the authors are performing a careful and conclusive developmental analysis of the hematopoiesis in these fish,there are a few inconsistences. It would be nice to see comparable timepoints in both the insitu and the qPCR analysis. For example, the authors show in Figure 1G/H 36 and 84 hpf timepoints while the qPCR is performed at different stages. This is especially relevant as the authors make quantitative statements from whole mount in situ analysis which are not necessarily suited to do quantitative comparisons. Furthermore, they are not using the same genes as readout as they use hbb2 in the insitu and hbbe2 in the qPCR analysis.

    While the growth study is conceptually interesting, it is unclear why the authors average all 12 larvae even though they keep them individually. They mention that this is to avoid "pseudoreplication", however I am not sure why that would be the case. It would be important to see all the data. Also, in Figure 4B the statistics for the comparison between surface normoxic and hypoxic are not shown, even though the text mentions it as significant.

  5. eLife

    Reviewer #2 (Public Review):

    The authors aim to gain an understanding of how cavefish have evolved to thrive in an environment where the oxygen content of the water is low. They provide strong evidence that cave-adapted fish, compared to their river-adapted counterparts, have more blood cells and higher constitutive expression of hypoxia-inducible genes. Evidence that these traits provide cavefish with an advantage in low oxygen is weak however. The authors achieve their aim of identifying possible mechanisms that would allow cavefish to survive low oxygen, but do not go far enough in my opinion to show the traits they identified are adaptive. The work will be of great interest to researchers in the fields of evolution, physiology, and developmental biology. Details about each claim and the data in support are provided below.

    The authors claim that cavefish (CF) have enlarged embryonic hematopoietic domains compared to surface fish (SF). This is supported by in situ hybridization using several markers of hematopoiesis. It is further supported by quantitative PCR that shows elevated expression levels of the same markers. The authors also report that compared to other teleost, both SF and CF have expanded hematopoietic domains. This is an interesting finding of more broad relevance as it could provide insight into how A. mexicanus SF, and not other fish, were able to invade caves initially.

    The authors claim that CF develop more erythrocytes than surface fish and this is a maternally controlled trait. This is supported by quantification of blood cell number in live images from CF, SF, and reciprocal hybrid crosses. The authors also show the qualitative difference in blood cell number with o-dianisidine staining.

    The authors use a method to reduce oxygen concentration in the laboratory water to levels that they claim are closer to those observed in the cave (5.0-5.4 mg/L). It should be noted that lower (4.43 mg/L) and much lower (2.97 mg/L) levels of oxygen have been recorded in the rivers and caves, respectively. The authors claim that SF respond to the reduction by increasing insulin growth factor binding protein 1a (igfbp1a) expression while cavefish do not. This is supported by the qPCR data.

    The authors claim that SF respond to hypoxic environments (5.0-5.4 mg/L) by reducing growth. While some of the SF exhibited lower axial growth rate compared to the control group, the results are not significant and the number of biological replicates in low (N = 6). The claim is therefore not strongly supported by the data. Repeating this experiment with additional biological replicates would strengthen the claims. Extending the amount of time the fish are exposed to hypoxia to measure a larger change in growth could also strengthen the claims.

    The authors claim that CF exposure to hypoxia in the laboratory does not affect igfbp1a expression or retard growth. These results are supported by the qPCR data and the finding that there is not a significant difference in growth between CF in "normoxic" vs "hypoxic" conditions. The results would be strengthened by increasing the number of fish as suggested for the SF samples.

    The authors claim that CF show elevated expression of Hypoxia Inducible Factor (HIF) regulatory genes relative to surface fish. This claim is strongly supported by quantitative PCR.

    The authors carry out one functional experiment; they expose SF and CF to increasing concentrations of phenylhydrazine (PHZ) to reduce red blood cell number and examine the impact on development and growth. They claim that "cavefish are less sensitive to hemolytic anemia". I don't agree with this interpretation of the data. Instead, cavefish are less sensitive to developing hemolytic anemia with increasing concentrations of PHZ because they have more blood cells. This experiment can be used to ask what happens to CF when the number of red blood cells they contain is reduced to levels that are similar to SF. It appears that when CF are experimentally manipulated to have the same amount of red blood cells as SF, they develop abnormally. The results would be easier for the reader to interpret if the actual number of red blood cells in the treatment conditions were reported (instead of as a percentage of treatment) and if statistical analysis were performed on treatment conditions where the number of red blood cells in SF and CF are roughly equal.

    Based on the data presented the authors conclude that the developmental and expression differences they observed in CF represent adaptations to the hypoxic cave environment. The evidence that these traits provide CF with an advantage in low oxygen waters is weak; the results showing that SF growth is restricted in low oxygen is not significant and the amount of time and oxygen concentration the CF are challenged with is minimal.

    The existence of a trait in CF is not strong evidence that the trait is adaptive. I think the authors would need to provide more experimental evidence to support the major claim (the title of the paper) that "Cavefish adapt to hypoxia by constitutive overexpression of hypoxia-inducible factor genes and increased erythrocyte development."

    Finally, there are several independent A. mexicanus CF populations and many different species of cavefish around the world; the authors would need to discuss whether the traits they observed are present in other cavefish or other cave animals before applying their conclusions more broadly.

  6. eLife

    Reviewer #3 (Public Review):

    In this manuscript van der Weele and Jeffery propose two evolutionary mechanisms, via which cavefish (Mexican tetra Astyanax mexicanus) have adapted to a hypoxic environment based on direct comparison with hypoxia-sensitive surface dwellers of the same species. These adaptations are increased erythrocyte production and heightened transcription of hypoxia-inducible factor 1 (hif1). Using a combination of time lapse imaging, bright field microscopy and in situ hybridization with a battery of hematopoietic markers, they provide clear and compelling evidence that cavefish have a larger number of erythrocytes than surface dwellers and that this increase stems from an expansion of two erythropoiesis domains during early development. To address the functional relevance of increased erythrocytes they induced hemolytic anemia using the drug phenylhydrazine (PHT) and showed that cavefish are less sensitive to this disorder than surface fish, possibly as a result of the increase in erythrocytes. Nevertheless, both types of fish exhibited developmental anomalies post-treatment, including reduced tail length. They further propose that the larger number of erythrocytes may promote adaptation to hypoxia by countering the negative impact of this environmental stressor on growth. Indeed, surface fish appear to be more susceptible to hypoxia-induced stunted growth of the post-anal tail than cavefish, although the numbers are quite variable. Lastly, the authors go on to show that cave dwellers express constitutively high transcript levels of hif1 genes and downstream targets of Hif1. One of these targets is the growth suppressor IGFBP1a, which could explain growth restriction of surface fish under hypoxia. Overall, even though increased erythrocyte production is a well-documented response to life in hypoxic environments, this study provides an interesting perspective on this adaptation seen through the lens of evolutionary biology.

    The data support the main conclusions that erythropoiesis and hif1 transcription are enhanced in cavefish but do not convincingly identify the functional relevance of these traits to hypoxia adaptation in cavefish. In this regard:

    1. The induction of hemolytic lysis using PHT and associated developmental defects raises concerns about lack of drug specificity.

    2. The connection between erythrocyte number and organismal growth is not clearly established. The reduced tail length defect that is observed following PHT treatment, even if specific, is likely to be an indirect consequence of abnormal notochord morphology rather than arrested tail growth.

    3. Because the above connection was not clearly established, the lack of growth inhibition in cavefish following exposure to hypoxia (thought to be offset by the elevated number of erythrocytes in cavefish) is not readily explainable. The data itself showing a difference in tail growth between cavefish and surface dwellers exposed to hypoxia is not strong due to high variability and low numbers.

    4. Increased hif1 transcript levels may contribute to enhanced hypoxia adaptation in cavefish, however Hif1 is known to be primarily regulated at the post-translational level, resulting in its enhanced stability and activity. It is unclear whether the activity of Hif1 is elevated in cavefish relative to surface fish as several known Hif1 targets are not up-regulated in cavefish relative to surface fish.

    In addition, there are a few technical concerns

    1. The manner in which hypoxic water was generated (bubbling of nitrogen gas) is unlikely to maintain a constant value over time. Furthermore, covering the tank with foil will not prevent gas exchange. Hence there is a concern that the hypoxic values may be variable between trials and even within the same trial.

    2. It is important that transcript levels for the q-PCR reference gene be stable across normoxic and hypoxic conditions. A discussion about the considerations that went into selecting RPL13a as a reference gene was not provided.

  7. Version published to 10.1101/2019.12.12.874230v2 on bioRxiv
  8. Version published to 10.1101/2019.12.12.874230v1 on bioRxiv