Hypoxia-Sonic Hedgehog Axis as a Driver of Primitive Hematopoiesis Development and Evolution in Cavefish.

The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and show constructive and regressive phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by treatment with the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and the Shh signaling system. Lastly, we demonstrate that hypoxia treatment enhances expression of Shh signaling along the midline of surface fish embryos. Thus, we conclude that a hypoxia-Shh axis may drive the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish. Highlights: Hypoxia increases hematopoiesis and erythrocytes in surface fishShh upregulation increases hematopoiesis and erythrocytes in surface fishShh inhibition decreases hematopoiesis and erythrocytes in cavefishHypoxia upregulates Shh along the embryonic midline in surface fish.

Natural reversal of cavefish heart asymmetry is controlled by Sonic Hedgehog effects on the left-right organizer.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.

Differentially expressed chaperone genes reveal a stress response required for unidirectional regeneration in the basal chordate Ciona.

BACKGROUND: Unidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments. RESULTS: We identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration. CONCLUSIONS: The chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.

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

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.

Cost Implications of Using Carbetocin Injection to Prevent Postpartum Hemorrhage in a Canadian Urban Hospital.

OBJECTIVE: Recent World Health Organization (WHO) recommendations regarding uterotonics for the prevention of postpartum hemorrhage (PPH) state that carbetocin should be considered a first-line prophylactic agent for all births where its cost is comparable to other effective uterotonics. This study evaluated whether a room temperature stable formulation of carbetocin met this recommendation in a Canadian urban hospital setting. METHODS: A decision tree model was developed to assess the financial implications of replacing oxytocin with carbetocin as a first-line prophylactic agent for PPH prevention in a Greater Toronto Area (GTA) hospital. The analysis accounted for the mode of delivery, efficacies of carbetocin and oxytocin in PPH prevention, occurrence of PPH-related health outcomes, and health care resource costs for PPH interventions. RESULTS: This study found that a GTA hospital, with 3242 deliveries per year, could save over CAD $349 000 annually by switching to room temperature stable carbetocin for PPH prevention. Carbetocin was able to lower institution costs by reducing the use of health care resources for PPH management in low-risk and high-risk PPH patients. The cost-saving potential of carbetocin relative to oxytocin was largely attributed to its greater efficacy in preventing the consequences of PPH. CONCLUSION: The use of room temperature stable carbetocin as a first-line prophylactic agent for PPH prevention meets WHO recommendations regarding uterotonics for PPH in a GTA hospital. The model from this study can be used to determine the financial impact of switching from oxytocin to carbetocin in other jurisdictions while diversifying a hospital's pool of PPH prophylactic agents.

Maternal control of visceral asymmetry evolution in Astyanax cavefish.

The direction of visceral organ asymmetry is highly conserved during vertebrate evolution with heart development biased to the left and pancreas and liver development restricted to opposing sides of the midline. Here we show that reversals in visceral organ asymmetry have evolved in Astyanax mexicanus, a teleost species with interfertile surface-dwelling (surface fish) and cave-dwelling (cavefish) forms. Visceral organ asymmetry is conventional in surface fish but some cavefish have evolved reversals in heart, liver, and pancreas development. Corresponding changes in the normally left-sided expression of the Nodal-Pitx2/Lefty signaling system are also present in the cavefish lateral plate mesoderm (LPM). The Nodal antagonists lefty1 (lft1) and lefty2 (lft2), which confine Nodal signaling to the left LPM, are expressed in most surface fish, however, lft2, but not lft1, expression is absent during somitogenesis of most cavefish. Despite this difference, multiple lines of evidence suggested that evolutionary changes in L-R patterning are controlled upstream of Nodal-Pitx2/Lefty signaling. Accordingly, reciprocal hybridization of cavefish and surface fish showed that modifications of heart asymmetry are present in hybrids derived from cavefish mothers but not from surface fish mothers. The results indicate that changes in visceral asymmetry during cavefish evolution are influenced by maternal genetic effects.

Apoptosis is a generator of Wnt-dependent regeneration and homeostatic cell renewal in the ascidian Ciona.

In the ascidian Ciona intestinalis, basal body parts regenerate distal structures but distal body parts do not replace basal structures. Regeneration involves the activity of adult stem cells in the branchial sac, which proliferate and produce migratory progenitor cells for tissue and organ replacement. Branchial sac-derived stem cells also replenish recycling cells lining the pharyngeal fissures during homeostatic growth. Apoptosis at injury sites occurs early during regeneration and continuously in the pharyngeal fissures during homeostatic growth. Caspase 1 inhibitor, caspase 3 inhibitor, or pan-caspase inhibitor Z-VAD-FMK treatment blocked apoptosis, prevented regeneration, and suppressed branchial sac growth and function. A pharmacological screen and siRNA-mediated gene knockdown indicated that regeneration requires canonical Wnt signaling. Wnt3a protein rescued both caspase-blocked regeneration and branchial sac growth. Inhibition of apoptosis did not affect branchial sac stem cell proliferation but prevented the survival of progenitor cells. After bisection across the mid-body, apoptosis occurred only in the regenerating basal fragments, although both fragments contained a part of the branchial sac, suggesting that apoptosis is unilateral at the wound site and the presence of branchial sac stem cells is insufficient for regeneration. The results suggest that apoptosis-dependent Wnt signaling mediates regeneration and homeostatic growth in Ciona.

Incremental Temperature Changes for Maximal Breeding and Spawning in Astyanax mexicanus.

The Mexican tetra, Astyanax mexicanus, is an emerging model system for studies in development and evolution. The existence of eyed surface (surface fish) and blind cave (cave fish) morphs in this species presents an opportunity to interrogate the mechanisms underlying morphological and behavioral evolution. Cave fish have evolved novel constructive and regressive traits. The constructive changes include increases in taste buds and jaws, lateral line sensory organs, and body fat. The regressive changes include loss or reduction of eyes. melanin pigmentation, schooling behavior, aggression, and sleep. To experimentally interrogate these changes, it is crucial to obtain large numbers of spawned embryos. Since the original A. mexicanus surface fish and cave fish were collected in Texas and Mexico in the 1990s, their descendants have been routinely stimulated to breed and spawn large numbers of embryos bimonthly in the Jeffery laboratory. Although breeding is controlled by food abundance and quality, light-dark cycles, and temperature, we have found that incremental temperature changes play a key role in stimulating maximal spawning. The gradual increase of temperature from 72 °F to 78 °F in the first three days of a breeding week provides two-three consecutive spawning days with maximal numbers of high-quality embryos, which is then followed by a gradual decrease of temperature from 78 °F to 72 °F during the last three days of the spawning week. The procedures shown in this video outline the workflow before and during a laboratory breeding week for incremental temperature stimulated spawning.

Author Correction: A hypomorphic cystathionine ß-synthase gene contributes to cavefish eye loss by disrupting optic vasculature.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fundamental research questions in subterranean biology.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.

Astyanax surface and cave fish morphs.

The small teleost fish Astyanax mexicanus has emerged as an outstanding model for studying many biological topics in the context of evolution. A major attribute is conspecific surface dwelling (surface fish) and blind cave dwelling (cavefish) morphs that can be raised in the laboratory and spawn large numbers of transparent and synchronously developing embryos. More than 30 cavefish populations have been discovered, mostly in northeastern Mexico, and some are thought to have evolved independently from surface fish ancestors, providing excellent models of parallel and convergent evolution. Cavefish have evolved eye and pigmentation regression, as well as modifications in brain morphology, behaviors, heart regenerative capacity, metabolic processes, and craniofacial organization. Thus, the Astyanax model provides researchers with natural "mutants" to study life in the challenging cave environment. The application of powerful genetic approaches based on hybridization between the two morphs and between the different cavefish populations are key advantages for deciphering the developmental and genetic mechanisms regulating trait evolution. QTL analysis has revealed the genetic architectures of gained and lost traits. In addition, some cavefish traits resemble human diseases, offering novel models for biomedical research. Astyanax research is supported by genome assemblies, transcriptomes, tissue and organ transplantation, gene manipulation and editing, and stable transgenesis, and benefits from a welcoming and interactive research community that conducts integrated community projects and sponsors the International Astyanax Meeting (AIM).

A hypomorphic cystathionine ß-synthase gene contributes to cavefish eye loss by disrupting optic vasculature.

Vestigial structures are key indicators of evolutionary descent, but the mechanisms underlying their development are poorly understood. This study examines vestigial eye formation in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling morph and multiple populations of blind cave morphs. Cavefish embryos initially develop eyes, but they subsequently degenerate and become vestigial structures embedded in the head. The mutated genes involved in cavefish vestigial eye formation have not been characterized. Here we identify cystathionine ß-synthase a (cbsa), which encodes the key enzyme of the transsulfuration pathway, as one of the mutated genes responsible for eye degeneration in multiple cavefish populations. The inactivation of cbsa affects eye development by increasing the transsulfuration intermediate homocysteine and inducing defects in optic vasculature, which result in aneurysms and eye hemorrhages. Our findings suggest that localized modifications in the circulatory system may have contributed to the evolution of vestigial eyes in cavefish.

Phenotypic plasticity as a mechanism of cave colonization and adaptation.

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that many cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

The Mexican tetra is a fish that has two forms: a surface-dwelling form, which has eyes and silvery grey appearance, and a cave-dwelling form, which is blind and has lost its pigmentation. Recent studies have shown that the cave-dwelling form evolved rapidly within the last 200,000 years from an ancestor that lived at the surface. The recent evolution of the cave-dwelling form of the tetra poses an interesting evolutionary question: how did the surface-dwelling ancestor of the tetra quickly adapt to the new and challenging environment found in the caves? 'Phenotypic plasticity' is a phenomenon through which a single set of genes can produce different observable traits depending on the environment. An example of phenotypic plasticity occurs in response to diet: in animals, poor diets can lead to an increase in the size of the digestive organs and to the animals eating more. To see if surface-dwelling tetras can quickly adapt to cave environments through phenotypic plasticity, Bilandzija et al. have exposed these fish to complete darkness (the major feature of the cave environment) for two years. After spending up to two years in the dark, these fish were compared to normal surface-dwelling and cave-dwelling tetras. Results revealed that surface-dwelling tetras raised in the dark exhibited traits associated with cave-dwelling tetras. These traits included changes in the activity of many genes involved in diverse processes, resistance to starvation, metabolism, and levels of hormones and molecules involved in neural signaling, which could lead to changes in behavior. However, the fish also exhibited traits, including an increase in the cells responsible for pigmentation, that would have no obvious benefit in the darkness. Even though the changes observed require no genetic mutations, they can help or hinder the fish's survival once they occur, possibly determining subsequent evolution. Thus, a trait beneficial for surviving in the dark that appears simply through phenotypic plasticity may eventually be selected for and genetic mutations that encode it more reliably may appear too. These results shed light on how species may quickly adapt to new environments without accumulating genetic mutations, which can take hundreds of thousands of years. They also may help to explain how colonizer species succeed in challenging environments. The principles described by Bilandzija et al. can be applied to different organisms adapting to new environments, and may help understand the role of phenotypic plasticity in evolution.

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration.

Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration.

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.