Vertebrate Stem Research Articles

Postembryonic Maintenance of Nephron Progenitor Cells with Low Translational Activity in the Chondrichthyan Scyliorhinus canicula.

Key Points Unlike mammals, chondrichthyan species exhibit postembryonic nephrogenesis, where new nephrons are continuously added in the kidney.Nephron progenitor cells in catsharks display slow cycling property, akin to other somatic stem cells, indicating their potential for tissue renewal and regeneration.Molecular analysis suggests a potential link between protein synthesis rate and nephron progenitor cell maintenance. Background While adult mammals are unable to grow new nephrons, cartilaginous fish kidneys display nephrogenesis throughout life. In this study, we investigated the molecular properties of nephron progenitor cells (NPCs) within the kidney of the catshark (Scyliorhinus canicula). Methods We used branched DNA in situ hybridization to analyze markers expressed in catshark NPCs. Bromodesoxyuridine pulse-chase labeling was also performed to test whether NPCs are slow-cycling cells. To question the mechanisms allowing NPC maintenance in the catshark postembryonic kidney, we measured global protein synthesis rates using in vivo OP-puromycin incorporation. We also investigated the expression of two targets of the mammalian target of rapamycin pathway, an important signaling pathway for translation initiation. Results We found that NPCs express molecular markers previously identified in mice and teleost embryonic NPCs, such as the transcription factors Six2, Pax2, and Wt1. At postembryonic stages, these NPCs are integrated into a specific nephrogenic area of the kidney and contain slow-cycling cells. We also evidenced that NPCs have lower protein synthesis levels than the differentiated cells present in forming nephrons. Such transition from low to high translation rates has been previously observed in several populations of vertebrate stem cells as they undergo differentiation. Finally, we reported the phosphorylation of two targets of the mammalian target of rapamycin pathway, p4E-BP1 and pS6K1, in catshark differentiated epithelial cells but not in the NPCs. Conclusions This first molecular analysis of NPCs in a chondrichthyan species indicates that translation rate increases in NPCs as they differentiate into epithelial cells of the nephron.

Shared features of blastula and neural crest stem cells evolved at the base of vertebrates.

The neural crest is a vertebrate-specific stem cell population that helped drive the origin and evolution of vertebrates. A distinguishing feature of these cells is their multi-germ layer potential, which has parallels to another stem cell population-pluripotent stem cells of the vertebrate blastula. Here, we investigate the evolutionary origins of neural crest potential by comparing neural crest and pluripotency gene regulatory networks of a jawed vertebrate, Xenopus, and a jawless vertebrate, lamprey. We reveal an ancient evolutionary origin of shared regulatory factors in these gene regulatory networks that dates to the last common ancestor of extant vertebrates. Focusing on the key pluripotency factor pou5, we show that a lamprey pou5 orthologue is expressed in animal pole cells but is absent from neural crest. Both lamprey and Xenopus pou5 promote neural crest formation, suggesting that pou5 activity was lost from the neural crest of jawless vertebrates or acquired along the jawed vertebrate stem. Finally, we provide evidence that pou5 acquired novel, neural crest-enhancing activity after evolving from an ancestral pou3-like clade. This work provides evidence that both the neural crest and blastula pluripotency networks arose at the base of the vertebrates and that this may be linked to functional evolution of pou5.

A long-headed Cambrian soft-bodied vertebrate from the American Great Basin region.

The fossil record suggests that chordates might have been minor components of marine ecosystems during the first major diversification of animal life in the Cambrian. Vertebrates are represented by a handful of rare soft-bodied stem-lineage taxa known from Konservat-Lagerstätten, including Myllokunmingia and Yunnanozoon from the Stage 3 of South China, and Emmonsaspis and Metaspriggina from Stage 4-Drumian deposits of northeast USA and British Columbia. Here, we describe the first soft-bodied vertebrate from the American Great Basin, a region home to a dozen Cambrian Konservat-Lagerstätten. Found in the Drumian Marjum Formation of Utah, Nuucichthys rhynchocephalus gen. et sp. nov. is characterized by a finless torpedo-shaped body that includes a snout-like anterior head bearing anterolateral eyes, approximately 25 thick myomeres, a large branchial chamber with a keel and approximately seven putative dorsal bars and a spiniform caudal process. Using Bayesian inference, our analysis recovers Nuucichthys within the vertebrate stem, closer to the crown than Pikaia, Yunnanozoon and Myllokunmingia, where it forms a polytomy with its Laurentian relatives, Emmonsaspis and Metaspriggina, and a scion consisting of conodonts and crown-group vertebrates. Based on the eye orientation and absence of fins, we tentatively reconstruct Nuucichthys as a pelagic organism with limited swimming abilities (planktonektic).

The Evolution of Transglutaminases Underlies the Origin and Loss of Cornified Skin Appendages in Vertebrates.

Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.

The origins of gas exchange and ion regulation in fish gills: evidence from structure and function.

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid-base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills.

Comment on "Ultrastructure reveals ancestral vertebrate pharyngeal skeleton in yunnanozoans".

Tian et al. (Reports, 8 July 2022, p. 218) claim that Cambrian yunnanozoan animals are stem vertebrates, based partly on their observation at the nanometer scale of microfibrillar tissue located in the branchial arches. They interpret this to represent cellular cartilage with an extracellular matrix of microfibrils. Instead, we argue that the 'microfibrils' are more likely modern organic contamination.

Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchangethat is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

Metabolic Scaling in Birds and Mammals: How Taxon Divergence Time, Phylogeny, and Metabolic Rate Affect the Relationship between Scaling Exponents and Intercepts.

Simple SummaryThis study is based on a large dataset and re-evaluates data on the metabolic rate, providing new insights into the similarities and differences across different groups of birds and mammals. We compared six taxonomic groups of mammals and birds according to their energetic characteristics and the geological time of evolutionary origin. The overall metabolic rate of a taxonomic group increases with the geological time of evolutionary origin. The terrestrial mammals and flightless birds have almost equal metabolic levels. The higher the metabolic rate in a group, the less it increases within increasing body size in this group.Analysis of metabolic scaling in currently living endothermic animal species allowed us to show how the relationship between body mass and the basal metabolic rate (BMR) has evolved in the history of endothermic vertebrates. We compared six taxonomic groups according to their energetic characteristics and the time of evolutionary divergence. We transformed the slope of the regression lines to the common value and analyzed three criteria for comparing BMR of different taxa regardless of body size. Correlation between average field metabolic rate (FMR) of the group and its average BMR was shown. We evaluated the efficiency of self-maintenance in ordinary life (defined BMR/FMR) in six main groups of endotherms. Our study has shown that metabolic scaling in the main groups of endothermic animals correlates with their evolutionary age: the younger the group, the higher the metabolic rate, but the rate increases more slowly with increasing body weight. We found negative linear relationship for scaling exponents and the allometric coefficient in five groups of endotherms: in units of mL O2/h per g, in relative units of allometric coefficients, and also in level or scaling elevation. Mammals that diverged from the main vertebrate stem earlier have a higher “b” exponent than later divergent birds. A new approach using three criteria for comparing BMR of different taxa regardless of body mass will be useful for many biological size-scaling relationships that follow the power function.

Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria

BackgroundSpeciation genes contribute disproportionately to species divergence, but few examples exist, especially in vertebrates. Here we test whether Zan, which encodes the sperm acrosomal protein zonadhesin that mediates species-specific adhesion to the egg’s zona pellucida, is a speciation gene in placental mammals.ResultsGenomic ontogeny reveals that Zan arose by repurposing of a stem vertebrate gene that was lost in multiple lineages but retained in Eutheria on acquiring a function in egg recognition. A 112-species Zan sequence phylogeny, representing 17 of 19 placental Orders, resolves all species into monophyletic groups corresponding to recognized Orders and Suborders, with <5% unsupported nodes. Three other rapidly evolving germ cell genes (Adam2, Zp2, and Prm1), a paralogous somatic cell gene (TectA), and a mitochondrial gene commonly used for phylogenetic analyses (Cytb) all yield trees with poorer resolution than the Zan tree and inferior topologies relative to a widely accepted mammalian supertree. Zan divergence by intense positive selection produces dramatic species differences in the protein’s properties, with ordinal divergence rates generally reflecting species richness of placental Orders consistent with expectations for a speciation gene that acts across a wide range of taxa. Furthermore, Zan’s combined phylogenetic utility and divergence exceeds those of all other genes known to have evolved in Eutheria by positive selection, including the only other mammalian speciation gene, Prdm9.ConclusionsSpecies-specific egg recognition conferred by Zan’s functional divergence served as a mode of prezygotic reproductive isolation that promoted the extraordinary adaptive radiation and success of Eutheria.

Ultrastructure reveals ancestral vertebrate pharyngeal skeleton in yunnanozoans.

Pharyngeal arches are a key innovation that likely contributed to the evolution of the jaws and braincase of vertebrates. It has long been hypothesized that the pharyngeal (branchial) arch evolved from an unjointed cartilaginous rod in vertebrate ancestors such as that in the nonvertebrate chordate amphioxus, but whether such ancestral anatomy existed remains unknown. The pharyngeal skeleton of controversial Cambrian animals called yunnanozoans may contain the oldest fossil evidence constraining the early evolution of the arches, yet its correlation with that of vertebrates is still disputed. By examining additional specimens in previously unexplored techniques (for example, x-ray microtomography, scanning and transmission electron microscopy, and energy dispersive spectrometry element mapping), we found evidence that yunnanozoan branchial arches consist of cellular cartilage with an extracellular matrix dominated by microfibrils, a feature hitherto considered specific to vertebrates. Our phylogenetic analysis provides further support that yunnanozoans are stem vertebrates.

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives.

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them.

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives

Evolution of a histone variant involved in compartmental regulation of NAD metabolism.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

An atlas of neural crest lineages along the posterior developing zebrafish at single-cell resolution.

Neural crest cells (NCCs) are vertebrate stem cells that give rise to various cell types throughout the developing body in early life. Here, we utilized single-cell transcriptomic analyses to delineate NCC-derivatives along the posterior developing vertebrate, zebrafish, during the late embryonic to early larval stage, a period when NCCs are actively differentiating into distinct cellular lineages. We identified several major NCC/NCC-derived cell-types including mesenchyme, neural crest, neural, neuronal, glial, and pigment, from which we resolved over three dozen cellular subtypes. We dissected gene expression signatures of pigment progenitors delineating into chromatophore lineages, mesenchyme cells, and enteric NCCs transforming into enteric neurons. Global analysis of NCC derivatives revealed they were demarcated by combinatorial hox gene codes, with distinct profiles within neuronal cells. From these analyses, we present a comprehensive cell-type atlas that can be utilized as a valuable resource for further mechanistic and evolutionary investigations of NCC differentiation.

The evolutionary origins of the vertebrate olfactory system.

Vertebrates develop an olfactory system that detects odorants and pheromones through their interaction with specialized cell surface receptors on olfactory sensory neurons. During development, the olfactory system forms from the olfactory placodes, specialized areas of the anterior ectoderm that share cellular and molecular properties with placodes involved in the development of other cranial senses. The early-diverging chordate lineages amphioxus, tunicates, lampreys and hagfishes give insight into how this system evolved. Here, we review olfactory system development and cell types in these lineages alongside chemosensory receptor gene evolution, integrating these data into a description of how the vertebrate olfactory system evolved. Some olfactory system cell types predate the vertebrates, as do some of the mechanisms specifying placodes, and it is likely these two were already connected in the common ancestor of vertebrates and tunicates. In stem vertebrates, this evolved into an organ system integrating additional tissues and morphogenetic processes defining distinct olfactory and adenohypophyseal components, followed by splitting of the ancestral placode to produce the characteristic paired olfactory organs of most modern vertebrates.

Mast4 knockout shows the regulation of spermatogonial stem cell self-renewal via the FGF2/ERM pathway.

Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual's lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSCself-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.

CaMKII oxidation is a performance-disease tradeoff in vertebrate evolution

Reactive oxygen species (ROS) contribute to health and disease. CaMKII is a widely expressed enzyme whose activation by oxidation of regulatory domain methionines (ox-CaMKII) contributes to cardiovascular disease, asthma, and cancer. Here we integrate comparative genomic and experimental data to show that CaMKII activation by ROS arose more than half-a-billion years ago on the vertebrate stem lineage where it constituted a bridge between ROS and increased intracellular Ca2+ release, exercise responsive gene transcription, and improved performance in skeletal muscle. These enhancements to fight-or-flight physiology were likely key in facilitating a well-evidenced shift in the behavioural ecology of our immediate chordate ancestors, and, in turn, the evolutionary success of vertebrates. Still, the ox-CaMKII innovation for augmenting performance must be considered a critical evolutionary trade-off, as it rendered us more susceptible to common and often fatal diseases linked to excessive ROS.

RNA-binding protein PUM2 regulates mesenchymal stem cell fate via repression of JAK2 and RUNX2 mRNAs.

The differentiation of mesenchymal stem cells (MSCs) into unwanted lineages can generate potential problems in clinical trials. Thus, understanding the molecular mechanisms, involved in this process, would help prevent unexpected complications. Regulation of gene expression, at the posttranscriptional level, is a new approach in cell therapies. PUMILIO is a conserved posttranscriptional regulator. However, the underlying mechanisms of PUMILIO, in vertebrate stem cells, remain elusive. Here, we show that depletion of PUMILIO2 (PUM2) blocks MSC adipogenesis and enhances osteogenesis. We also demonstrate that PUM2 works as a negative regulator on the 3'-untranslated regions of JAK2 and RUNX2 via direct binding. CRISPR/Cas9-mediated gene silencing of Pum2 inhibited lipid accumulation and induced excessive bone formation in zebrafish larvae. Our findings reveal novel roles of PUM2 in MSCs and provide potential therapeutic targets for related diseases.

Neural stem cells induce the formation of their physical niche during organogenesis.

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches.