A unifying theme of cave habitats is complete darkness (Culver and Pipan, 2009) and the absence of primary productivity can subject cave-dwelling animals to oxygen deficiency (Malard and Hervant, 2001). We have used the teleost Astyanax mexicanus, which consists of surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs, as a model to study adaptation to hypoxia in caves. Surface fish colonized subterranean waters in the Sierra de El Abra region of Mexico (Mitchell et al., 1977; Gross, 2012) during the late Pleistocene (Fumey et al., 2018; Herman et al., 2018), and their cavefish descendants evolved numerous traits for adaptation to perpetual darkness. Cavefish adaptations include more taste buds and cranial neuromasts, increased olfactory capacity, and fat deposits, which could facilitate feeding and survival during times of low food input, and decreased metabolic rates and loss of eyes, which may be important in energy conservation (Yamamoto et al., 2004; Moran et al., 2014; Moran et al., 2015; Varatharasan et al., 2009; Blin et al., 2018; Yoshizawa et al., 2014; Hüppop, 1986; Xiong et al., 2018). Hypoxia may be a deterrent to the survival of colonizing teleosts in the Sierra de El Abra, where oxygen reductions of 50% or more have been measured in cave waters (Boggs and Gross, 2021; Rohner et al., 2013; Ornelas-García et al., 2018). Astyanax cavefish thrive under these conditions, but little is known about how they have adapted to hypo-oxygenated waters.

Oxygen is bound to hemoglobin and transported through the body by red blood cells. Increased erythrocyte production is a well-documented strategy for coping with hypoxia at high altitude (Haase, 2013), but the role of red blood cells in adaptation to hypoxic caves is unknown. Hematopoiesis, the process in which erythrocytes and other blood cells develop during embryogenesis, occurs in two waves in vertebrates: the primitive and definitive waves (Davidson and Zon, 2004; Paik and Zon, 2010). In the zebrafish Danio rerio and other teleost embryos, the first or primitive wave occurs in the anterior and posterior lateral mesoderm beginning during the tailbud stage. The anterior lateral mesoderm (ALM) undergoes myelopoiesis to form macrophages, and some of these later differentiate into neutrophils and microglia (Herbomel et al., 1999; Herbomel et al., 2001; Le Guyader et al., 2008), whereas the posterior lateral mesoderm (PLM) undergoes erythropoiesis to form primitive erythrocytes. Subsequently, the PLM converges into the intermediate cell mass, where the precursors of endothelial cells lining the first blood vessels, primitive erythrocytes, and myeloid cells will ultimately differentiate. The second or definitive wave of hematopoiesis begins later in development when hematopoietic stem and progenitor cells are formed in the aorta-gonad-mesonephros (AGM), which seed the caudal hematopoietic tissue and form the posterior blood islands, where definitive erythrocytes begin to differentiate (Paik and Zon, 2010; Gore et al., 2018). Eventually, definitive hematopoiesis moves to the thymus and head kidneys in adults. The primitive and definitive waves of hematopoiesis are potential targets for adaptive changes induced by hypoxia.

Hematopoiesis is directed by a series of transcription factors producing different types of blood cells (Davidson and Zon, 2004; Carroll and North, 2014). The growth factor independence 1 (gfi1aa) gene, which encodes a transcriptional repressor of many different genes (Möröy et al., 2015), is important for primitive hematopoiesis at several developmental stages (Wei et al., 2008; Cooney et al., 2013; Moore et al., 2018). In zebrafish, gfi1aa expression occurs throughout the period of embryonic segmentation, first in the PLM, then in the intermediate cell mass, and finally in the AGM (Wei et al., 2008; Cooney et al., 2013), where it directs the development of hemogenic endothelia into hematopoietic stem and progenitor cells, which ultimately differentiate into red blood cells (Moore et al., 2018). The gfi1aa gene is also expressed in the ALM, albeit weakly and only for a short time (Wei et al., 2008). Its expression at different stages of hematopoiesis makes gfi1aa an excellent marker for investigating differences between surface fish and cavefish hematopoiesis. The LIM domain only 2 gene encodes another transcription factor essential for determination of hematopoietic domains early in primitive hematopoiesis (Patterson et al., 2007) and is also an excellent marker for differences during the initial stages of hematopoiesis.

Oxygen is required in aerobic organisms to produce energy, and insufficient oxygen leads to the activation of an evolutionarily conserved transcriptional response (Majmundar et al., 2010). The responses to oxygen deprivation are coordinated by activation of the hypoxia-inducible factors (HIF1, HIF2, and HIF3) (Semenza and Wang, 1992), a family of transcription factors controlling a large number of downstream target genes involved in promoting different responses to hypoxia (Pelster and Egg, 2018; Rashid et al., 2017). Increased expression of the α subunit of HIF1 and the HIF regulated genes are indicators of hypoxia (Mandic et al., 2021). Notable responses include the improvement of oxygen supply through increasing red blood cell production and function (Haase, 2013; Kulkarni et al., 2010; Cai et al., 2020), metabolic homeostasis balancing ATP production with oxygen consumption, management of Reactive Oxygen Species (ROS) via glycolysis, modulating anaerobic respiration and the pentose phosphate pathway (PPP) (Singh et al., 2017; Wheaton and Chandel, 2011; Stincone et al., 2015), and conservation of energy by growth inhibition (Kamei, 2020). Astyanax cavefish (Hüppop, 1986; Moran et al., 2014; Aspiras et al., 2015) and other cave-adapted animals (Bishop et al., 2004) have evolved reduced metabolic rates to conserve energy in the cave environment. However, the strategies used to conserve energy in hypoxic cave environments are poorly understood.

The present investigation shows that cavefish embryos develop significantly more erythrocytes than surface fish. The results also revealed that cavefish larvae have constitutively high expression of some of the HIF1 pathway genes and show no effects of oxygen depletion on growth, traits that could promote larval survival in hypoxic environments. These studies reveal that changes in development of the circulatory system may underlie adaptation of cavefish to hypo-oxygenated environments.

We show that cavefish embryos develop more red blood cells than surface fish embryos, a classic response to hypoxia (Martinez et al., 2004; Timmerman and Chapman, 2004; Rutjes et al., 2007; Haase, 2013), when they are raised under normoxic laboratory conditions. This conclusion is based on direct counts of blood cells, including erythrocytes, increased expression of multiple erythropoietic marker genes, and hyper-expression of the gfi1aa gene, a crucial regulator of hematopoiesis. In addition, we report that the increase in erythrocytes may be caused by expansion of embryonic hematopoietic domains in cavefish compared to surface fish. Finally, we provide evidence that cavefish have evolved a permanent response to hypoxia involving the constitutive overexpression of the some of the hif1 genes, which persists under normoxic laboratory conditions.

Hematopoiesis occurs in the primitive and definitive waves in vertebrates (Davidson and Zon, 2004; Paik and Zon, 2010). Increased hbb2, hbbe2, and gfi1aa expression was observed during embryogenesis and after larval hatching in cavefish. In zebrafish, transfusion experiments indicate that blood cells circulating during the first four days of development are primitive erythrocytes (Weinstein et al., 1996). Accordingly, our findings suggest that Astyanax larval blood cells are primitive erythrocytes derived from the first wave of hematopoiesis. Nevertheless, we also found stronger expression of erythropoietic marker genes in the AGM and caudal hematopoietic tissue, which are involved in definitive hematopoiesis, implying that the second wave may also be expanded in cavefish. Further investigation will be required to determine whether erythrocytes are also increased in adults, although this possibility seems likely because adult cavefish have increased levels of hemoglobin transcripts (Sears et al., 2020) and more erythrocytes in their head kidneys compared to surface fish (Peuß et al., 2020). We conclude that cavefish have evolved a permanent enhancement in red blood cells as a potential adaptation for survival in hypoxic cave waters.

The results showed that surface fish and cavefish primitive erythrocytes are derived from two different hematopoietic domains, one located anteriorly in the ALM and the other posteriorly in the PLM. Importantly, both the anterior and posterior embryonic domains show expanded marker gene expression in cavefish compared to surface fish. It is noteworthy that expansion of the posterior hematopoietic domain can also be induced in zebrafish by overexpressing LMO2 together with its partner transcription factor SCL/TAL1 (Gering et al., 2003), suggesting that teleosts have the capacity to modulate blood cell numbers by changing the size of primitive erythropoietic domains during development. The existence of erythrocyte forming capacity in the ALM of surface fish, albeit smaller than in cavefish, suggests that the expansion of primitive erythropoiesis may pre-adapt Astyanax for colonizing hypoxic cave environments.

The expansion of erythropoiesis into the ALM during primitive hematopoiesis in Astyanax contrasts strikingly to zebrafish, medaka, and other teleosts that have been investigated, in which primitive erythropoiesis is confined to the PLM, and the ALM is devoted only to myelopoiesis (Detrich et al., 1995; Govoni et al., 2005; Moriyama et al., 2010). Expression of the macrophage marker lcp1 (Herbomel et al., 2001) indicated that myelopoietic precursors had already differentiated into macrophages as erythrocyte progenitors were being formed in the ALM during early Astyanax development. Cell-tracing studies have shown that blood precursor cells can produce either erythrocytes or macrophages, but not both cell types (Warga et al., 2009), indicating an opposing relationship between the erythroid and myeloid cell populations. However, this relationship can be reversed by knockdown of the myeloid-controlling transcription factor PU.1, which induces blood precursors of the ALM to form erythrocytes instead of macrophages (Rhodes et al., 2005). Apparently, teleosts are potentially able to undergo erythropoiesis in the ALM and PLM, but in most species this capacity is normally confined to the PLM. In Astyanax, the balanced relationship between myeloid and erythroid cell specification appears to have tilted toward the production of erythrocytes. However, the development of erythrocytes in the ALM may not be at the expense of fewer myeloid cells because there is also an overall expansion of the ALM (and PLM), which may promote the specification of more blood precursor cells. In future studies, it will be interesting to determine how the expansion of erythropoietic potential is controlled in cavefish.

Specification of cells in the primitive blood lineage can be traced to their positions during gastrulation in zebrafish (Kimmel et al., 1990): ALM macrophages are originally derived from progenitors on the dorsal side of the embryo, and PLM erythrocytes originate from the ventral side of the embryo (Davidson and Zon, 2004; Warga et al., 2009). Erythrocyte progenitors corresponding to the ALM and PLM are also likely to be positioned on the dorsal or ventral sides respectively in gastrulating Astyanax embryos. Therefore, increased erythropoiesis in the ALM suggests that a relative ventralization of hematopoietic development has evolved in cavefish compared to surface fish. This possibility is supported by expanded erythroid development induced by overexpression of the ventralizing factors bmp2b and bmp7 in zebrafish gastrulae (Lengerke et al., 2008) and is in line with evidence implicating the importance of maternal factors in dorsal-ventral axis determination of cavefish embryos (Ren et al., 2018; Ma et al., 2018; Torres-Paz et al., 2019). Maternal control of primitive erythropoiesis may promote maximal oxygen distribution at the early stages of development in hypoxic environments. This finding extends our understanding of evolutionary differences in early hematopoiesis to events beginning during oogenesis.

In the present investigation, we have examined primitive hematopoiesis and red blood cells in the Pachón cavefish population. About 30 different Astyanax cavefish populations have been described in northeastern Mexico, and some of these cavefish are thought to have been derived independently from surface fish ancestors (Gross, 2012; Herman et al., 2018). It is conceivable that diverse cavefish lineages inhabit cave pools with different levels of oxygenation. Therefore, in the future it would be interesting to determine the status of erythrocytes in additional cavefish populations. It will also be important to determine if other aquatic cave animals modify their oxygen delivery systems to cope with hypoxia.

Teleosts show considerable diversity in utilizing hemoglobin and red blood cells for oxygen transport during development: some species begin hemoglobin synthesis during embryogenesis, whereas others postpone hemoglobin and erythrocyte production until much later in larval development (Wells, 2009). Our studies on the induction of hemolytic anemia with PHZ confirm the functional significance of red blood cells produced during early Astyanax development. At the same PHZ concentrations, surface fish, but not cavefish with residual red blood cells, show negative effects on growth, and hypoxia treatment inhibited growth in surface fish but not in cavefish. These results suggest that red blood cells are necessary for oxygen transport at early stages of A. mexicanus development and that increased erythrocytes may be advantageous in cavefish. This is contrary to zebrafish, where PHZ induced anemia showed that red blood cells are not required during the first few weeks of development under normoxic conditions (Pelster and Burggren, 1996), although under severe hypoxia zebrafish embryos also use red blood cells to increase the supply of oxygen (Rombough and Drader, 2009).

Teleosts have evolved different strategies to cope with environments depleted in oxygen, including changes in gill morphology (Sollid et al., 2003; Dhillon et al., 2013), behavioral avoidance of hypoxic waters (Congleton, 1980; Mandic et al., 2009), and altered metabolism (Torres et al., 2012). Astyanax surface fish larvae appear to deal with hypoxia by conserving energy and reducing growth. It is likely that growth is regulated similarly to zebrafish via upregulation of igfbp1a (Kajimura et al., 2005). The upregulation of igfbp1 under hypoxia is regulated by HIF1 (Kajimura et al., 2006). Astyanax cavefish, which are presumably unable to avoid hypoxic waters in their natural environment, show increased expression of some of the hif1 genes in either hypoxic or normoxic conditions. The constitutive increase in hif1-related gene expression may have conferred growth resistance to hypoxia, even under conditions in which igfbp1 is upregulated, and may be responsible for increased red blood cell number.

The increased expression of the metabolic gene hk1 in cavefish compared to surface fish indicates an increase in glycolysis, consistent with the observations of Krishnan et al., 2020. The increase in glycolysis does not seem to affect the TCA cycle since ldha and pdk1 are not differentially expressed between cavefish and surface fish (Medley et al., 2020). Wild captured cavefish also have higher expression of the genes encoding enzymes involved in glutathione metabolism compared to wild caught surface fish (Krishnan et al., 2020; Medley et al., 2020). Glutathione is an important player in the mitigation of ROS, and the Pentose Phosphate Pathway produces a large portion of the NADPH used in the production of glutathione (Stincone et al., 2015). Therefore, the constitutive expression of hif1-genes in cavefish could play a role in ROS mitigation via glutathione during hypoxia (Birnie-Gauvin et al., 2017).

In conclusion, the present investigation has revealed two potential adaptations that may have permitted Astyanax cavefish to cope with hypoxic cave waters. First, cavefish have enhanced erythrocyte development, and as a maternal effect this phenotype may have increased the capacity of cavefish to carry and distribute essential oxygen to tissues and organs early in development. Second, cavefish have adjusted to hypoxic conditions by evolving constitutively high expression levels of some of the hif1 genes. Together, these changes may be important for the successful adaptation of Astyanax to hypoxic subterranean environments.

All data generated or analyzed during this study are included in the manuscript and supporting file. Source data files have been provided for Figures 1, 2, 4, 5, 6 and 7.

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Author details

1. Corine M van der Weele

   Department of Biology, University of Maryland, College Park, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. William R Jeffery

   Department of Biology, University of Maryland, College Park, United States

   Contribution

   Conceptualization, Funding acquisition, Project administration, Supervision, Writing - original draft, Writing - review and editing

   For correspondence

   Jeffery@umd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6997-2946

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