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 exhibit a subset of 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 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 Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish.

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.

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.

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.

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.

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.

Posttranscriptional control over rapid development and ciliogenesis in Marsilea.

Marsilea vestita is a semiaquatic fern that produces its spores (meiotic products) as it undergoes a process of natural desiccation. During the period of desiccation, the spores mature, and produce large quantities of pre-mRNA, which is partially processed and stored in nuclear speckles and can remain stable during a period of extended quiescence in the dry spore. Rehydration of the spores initiates a highly coordinated developmental program, featuring nine successive mitotic division cycles that occur at precise times and in precise planes within the spore wall to produce 39 cells, 32 of which are spermatids. The spermatids then undergo de novo basal body formation, the assembly of a massive cytoskeleton, nuclear and cell elongation, and finally ciliogenesis, before being released from the spore wall. The entire developmental program requires only 11 h to reach completion, and is synchronous in a population of spores rehydrated at the same time. Rapid development in this endosporic gametophyte is controlled posttranscriptionally, where stored pre-mRNAs, many of which are intron-retaining transcripts, are unmasked, processed, and translated under tight spatial and temporal control. Here, we describe posttranscriptional mechanisms that exert temporal and spatial control over this developmental program, which culminates in the production of ∼140 ciliary axonemes in each spermatozoid.

Removal of retained introns regulates translation in the rapidly developing gametophyte of Marsilea vestita.

The utilization of stored RNA is a driving force in rapid development. Here, we show that retention and subsequent removal of introns from pre-mRNAs regulate temporal patterns of translation during rapid and posttranscriptionally controlled spermatogenesis of the fern Marsilea vestita. Analysis of RNAseq-derived transcriptomes revealed a large subset of intron-retaining transcripts (IRTs) that encode proteins essential for gamete development. Genomic and IRT sequence comparisons show that other introns have been previously removed from the IRT pre-mRNAs. Fully spliced isoforms appear at distinct times during development in a spliceosome-dependent and transcription-independent manner. RNA interference knockdowns of 17/17 IRTs produced anomalies after the time points when those transcripts would normally be spliced. Intron retention is a functional mechanism for forestalling precocious translation of transcripts in the male gametophyte of M. vestita. These results have broad implications for plant gene regulation, where intron retention is widespread.

Extremes in rapid cellular morphogenesis: post-transcriptional regulation of spermatogenesis in Marsilea vestita.

The endosporic male gametophyte of the water fern, Marsilea vestita, provides a unique opportunity to study the mechanisms that control cell fate determination during a burst of rapid development. In this review, we show how the spatial and temporal control of development in this simple gametophyte involves several distinct modes of RNA processing that allow the translation of specific mRNAs at distinct stages during gametogenesis. During the early part of development, nine successive cell division cycles occur in precise planes within a closed volume to produce seven sterile cells and 32 spermatids. There is no cell movement in the gametophyte; so, cell position and size within the spore wall define cell fate. After the division cycles have been completed, the spermatids become sites for the de novo formation of basal bodies, for the assembly of a complex cytoskeleton, for nuclear and cell elongation, and for ciliogenesis. In contrast, the adjacent sterile cells exhibit none of these changes. The spermatids differentiate into multiciliated, corkscrew-shaped gametes that resemble no other cells in the entire plant. Development is controlled post-transcriptionally. The transcripts stored in the microspore are released (unmasked) in the gametophyte at different times during development. At the start of these studies, we identified several key mRNAs that undergo translation at specific stages of gametophyte development. We developed RNA silencing protocols that enabled us to block the translation of these proteins and thereby establish their necessity and sufficiency for the completion of specific stages of gametogenesis. In addition, RNAi enabled us to identify additional proteins that are essential for other phases of development. Since the distributions of mRNAs and the proteins they encode are not identical in the gametophyte, transcript processing is apparently important in allowing translation to occur under strict temporal and spatial control. Transcript polyadenylation occurs in the spermatogenous cells in ways that match the translation of specific mRNAs. We have found that the exon junction complex plays key roles in transcript regulation and modifications that underlie cell specification in the gametophyte. We have recently become interested in the mechanisms that control the unmasking of the stored transcripts and have linked the synthesis and redistribution of spermidine in the gametophyte to the control of mRNA release from storage during early development and later to basal body formation, cytoskeletal assembly, and nuclear and cell elongation in the differentiating spermatids.

Spermidine is a morphogenetic determinant for cell fate specification in the male gametophyte of the water fern Marsilea vestita.

Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.

Mago nashi is essential for spermatogenesis in Marsilea.

Spermatogenesis in Marsilea vestita is a rapid process that is activated by placing dry microspores into water. Nine division cycles produce seven somatic cells and 32 spermatids, where size and position define identity. Spermatids undergo de novo formation of basal bodies in a particle known as a blepharoplast. We are interested in mechanisms responsible for spermatogenous initial formation. Mago nashi (Mv-mago) is a highly conserved gene present as stored mRNA and stored protein in the microspore. Mv-mago protein increases in abundance during development and it localizes at discrete cytoplasmic foci (Mago-dots). RNA interference experiments show that new Mv-mago protein is required for development. With Mv-mago silenced, asymmetric divisions become symmetric, cell fate is disrupted, and development stops. The alpha-tubulin protein distribution, centrin translation, and Mv-PRP19 mRNA distribution are no longer restricted to the spermatogenous cells. Centrin aggregations, resembling blepharoplasts, occur in jacket cells. Mago-dots are undetectable after the silencing of Mv-mago, Mv-Y14, or Mv-eIF4AIII, three core components of the exon junction complex (EJC), suggesting that Mago-dots are either EJCs in the cytoplasm, or Mv-mago protein aggregations dependent on EJCs. Mv-mago protein and other EJC components apparently function in cell fate determination in developing male gametophytes of M. vestita.

Differential segregation and modification of mRNA during spermiogenesis in Marsilea vestita.

We are interested in the mechanisms that underlie cell fate determination in the endosporic male gametophytes of the fern, Marsilea vestita. Synchronous development is initiated by placing dry spores into water and involves the translation of stored mRNAs, with little transcription. Nine division cycles produce 32 spermatids surrounded by 7 sterile cells, and then each spermatid differentiates into a multiciliate gamete. Here, we focus on changes in the distribution of particular proteins, mRNAs, and patterns of polyadenylation as essential prerequisites for cell fate determination and gametogenesis. Earlier, we showed that alpha- and beta-tubulin proteins become concentrated in spermatogenous initials, and that centrin mRNA is translated only in spermatogenous initials. In situ hybridizations reveal that centrin, cyclin B, and beta-tubulin mRNAs are present in both sterile and spermatogenous cells, but that transcripts encoding RNA helicase and PRP-19 (a spliceosome component) become localized in spermatogenous cells. The targeted destruction of these two transcripts by RNAi treatments does not affect the numbers of division cycles, but the gametophytes exhibit anomalous patterns of cytokinesis, and a subsequent failure of spermatid differentiation. Thus, cell fate determination in the gametophyte involves localized translation, and the localization of mRNAs for proteins involved in transcript processing. We found differences in polyadenylation levels in sterile and spermatogenous cells that match the distribution of cytoplasmic poly(A) polymerase (PAP), which, in immunolocalizations, is abundant in spermatogenous cells, but undetectable in sterile cells. The activation of translation in spermatogenous initials, but not in sterile cells, may be under the control of mRNA processing enzymes, which become localized either as proteins or mRNAs in the spermatogenous subdomains before any divisions occur.

A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth. Roughly uniform elongation in the meristem and also, after an abrupt acceleration, in the elongation zone.

A requirement for understanding morphogenesis is being able to quantify expansion at the cellular scale. Here, we present new software (RootflowRT) for measuring the expansion profile of a growing root at high spatial and temporal resolution. The software implements an image processing algorithm using a novel combination of optical flow methods for deformable motion. The algorithm operates on a stack of nine images with a given time interval between each (usually 10 s) and quantifies velocity confidently at most pixels of the image. The root does not need to be marked. The software calculates components of motion parallel and perpendicular to the local tangent of the root's midline. A variation of the software has been developed that reports the overall root growth rate versus time. Using this software, we find that the growth zone of the root can be divided into two distinct regions, an apical region where the rate of motion, i.e. velocity, rises gradually with position and a subapical region where velocity rises steeply with position. In both zones, velocity increases almost linearly with position, and the transition between zones is abrupt. We observed this pattern for roots of Arabidopsis, tomato (Lycopersicon lycopersicum), lettuce (Lactuca sativa), alyssum (Aurinia saxatilis), and timothy (Phleum pratense). These velocity profiles imply that relative elongation rate is regulated in a step-wise fashion, being low but roughly uniform within the meristem and then becoming high, but again roughly uniform, within the zone of elongation. The executable code for RootflowRT is available from the corresponding author on request.