Biology subjects

Psenicka, M.

Publications and source records attributed to Psenicka, M..

13 recordsLinked to original sources

bioRxiv2025

Molecular insights into electroreceptor ribbon synapses from differential gene expression in sturgeon lateral line organs

In fishes and aquatic-stage amphibians, mechanosensory neuromasts are arranged in characteristic lines in the skin of the head and trunk, with afferent innervation from anterior or posterior lateral line nerves. In electroreceptive non-teleost jawed fishes and amphibians, fields of electrosensory ampullary organs flank some or all of the cranial neuromast lines, innervated by the anterior lateral line nerve. Like the mechanosensory hair cells found in neuromasts and the inner ear, electroreceptor cells in ampullary organs form specialised ribbon synapses with afferent nerve terminals. Ribbon synapses in hair cells are distinct from other glutamatergic synapses, including the ribbon synapses in photoreceptors: otoferlin is the Ca2+ sensor for synaptic vesicle exocytosis and synaptic vesicles are loaded with glutamate by vGlut3. We previously showed that the genes encoding otoferlin and vGlut3 are expressed by ampullary organs as well as neuromasts in a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula), suggesting that electroreceptor ribbon synapses are very similar to those in hair cells. In this study, we selected seven additional synapse-related candidate genes from our previously published dataset of putatively lateral line organ-enriched genes from late-larval paddlefish, and examined their expression in developing lateral line organs in a related chondrostean, the sterlet sturgeon (Acipenser ruthenus). We found that genes encoding the presynaptic cell adhesion molecule Nrxn3, the calcium-independent synaptotagmin Syt14, the high-affinity glutamate re-uptake transporter EAAT1 (GLAST), calmodulin regulator protein PCP4 (PEP-19) and cell adhesion molecule DSCAML1 were expressed in both neuromasts and ampullary organs. In contrast, Cbln18, encoding a secreted trans-synaptic scaffolding protein, was only expressed in neuromasts and Tulp1, encoding tubby-related protein 1 (required for the development and function of photoreceptor ribbon synapses), was only expressed in ampullary organs. Our results support electroreceptor ribbon synapses being glutamatergic and suggest further commonalities, but also some differences, with hair cell ribbon synapses.

developmental biology↗

bioRxiv2025

Mitochondria as Indispensable Yet Replaceable Components of Germ Plasm: Insights into Primordial Germ Cell Specification in Non-Teleost Sturgeons

While it is widely recognized that mitochondria are components of germ plasm, their specific role in the formation and specification of primordial germ cells (PGCs) remains poorly understood. Furthermore, it has not been established whether mitochondria in germ plasm possess unique characteristics essential for their function. In this study, we demonstrate that mitochondria are indispensable for PGCs development in non-teleost fishes and that their role is not dependent on their origin from germ plasm. Using sturgeon embryos, we showed that UV radiation applied to the vegetal pole effectively eliminates germ plasm, including mitochondria, and prevents PGCs formation. Remarkably, we restored germ plasm function and PGCs development by injecting mitochondria derived from donor eggs, even when these mitochondria were not originally part of the germ plasm. Transplanted mitochondria were successfully identified in larval PGCs using a fluorescent PKH26 tracer, and in interspecies transplantation experiments, their presence was confirmed using species-specific mtDNA and mtRNA primers in larvae and individual PGCs. Our findings reveal that mitochondria are critical but not germ plasm-specific determinants of PGCs formation. This study provides novel insights into the developmental pathways of germ cells and establishes a previously unrecognized flexibility in mitochondrial functionality within the germline. These findings also offer a potential method for conserving matrilineal genetics in critically endangered species like sturgeons, while simultaneously opening new avenues for studying germlines with high interspecies mitochondrial heteroplasmy and contributing to broader evolutionary and conservation biology.

developmental biology↗

bioRxiv2024

Two opposing roles for Bmp signalling in the development of electrosensory lateral line organs

The lateral line system enables all fishes and aquatic-stage amphibians to detect local water movement via mechanosensory hair cells in neuromasts, and many species to detect weak electric fields via electroreceptors (modified hair cells) in ampullary organs. Both neuromasts and ampullary organs develop from lateral line placodes. However, the molecular mechanisms underpinning ampullary organ formation are understudied relative to neuromasts, as the ancestral lineages of zebrafish (teleosts) and Xenopus (frogs) independently lost electroreception. We identified Bmp5 as a promising candidate via differential RNA-seq in an electroreceptive ray-finned fish, the Mississippi paddlefish (Polyodon spathula; Modrell et al., 2017, eLife 6: e24197). In an experimentally tractable relative, the sterlet sturgeon (Acipenser ruthenus), we found that Bmp5 and four other Bmp pathway genes are expressed in the developing lateral line, and that Bmp signalling is active. Furthermore, CRISPR/Cas9-mediated mutagenesis targeting Bmp5 in G0-injected sterlet embryos resulted in fewer ampullary organs. Conversely, when Bmp signalling was inhibited by DMH1 treatment shortly before the formation of ampullary organ primordia, supernumerary ampullary organs developed. These data suggest that Bmp5 promotes ampullary organ development, whereas Bmp signalling via another ligand(s) prevents their overproduction. Taken together, this demonstrates two opposing roles for Bmp signalling during ampullary organ formation.

developmental biology↗

bioRxiv2023

Atoh1 is required for the formation of lateral line electroreceptors and hair cells, whereas Foxg1 represses an electrosensory fate.

In electroreceptive jawed fishes and amphibians, individual lateral line placodes form lines of neuromasts on the head containing mechanosensory hair cells, flanked by fields of ampullary organs containing electroreceptors - modified hair cells that respond to weak electric fields. Extensively shared gene expression between neuromasts and ampullary organs suggests that conserved molecular mechanisms are involved in their development, but a few transcription factor genes are restricted either to the developing electrosensory or mechanosensory lateral line. Here, we used CRISPR/Cas9-mediated mutagenesis in F0-injected sterlet embryos (Acipenser ruthenus, a sturgeon) to test the function of three such genes. We found that the hair cell transcription factor gene Atoh1 is required for both hair cell and electroreceptor differentiation in sterlet, and for Pou4f3 and Gfi1 expression in both neuromasts and ampullary organs. These data support the conservation of developmental mechanisms between hair cells and electroreceptors. Targeting ampullary organ-restricted Neurod4 did not yield any phenotype, potentially owing to redundancy with other Neurod genes that we found to be expressed in sterlet ampullary organs. After targeting mechanosensory-restricted Foxg1, ampullary organs formed within neuromast lines, suggesting that Foxg1 normally represses their development. We speculate that electrosensory organs may be the default fate of lateral line primordia in electroreceptive vertebrates.

developmental biology↗

bioRxiv2023

Identification of multiple transcription factor genes potentially involved in the development of electrosensory versus mechanosensory lateral line organs

In electroreceptive jawed vertebrates, embryonic lateral line placodes give rise to electrosensory ampullary organs as well as mechanosensory neuromasts. Previous reports of shared gene expression suggest that conserved mechanisms underlie electroreceptor and mechanosensory hair cell development and that electroreceptors evolved as a transcriptionally related sister cell type to hair cells. We previously identified only one transcription factor gene, Neurod4, as ampullary organ-restricted in the developing lateral line system of a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula). The other 16 transcription factor genes we previously validated in paddlefish were expressed in both ampullary organs and neuromasts. Here, we used our published lateral line organ-enriched gene-set (arising from differential bulk RNA-seq in late-larval paddlefish), together with a candidate gene approach, to identify 23 transcription factor genes expressed in the developing lateral line system of a more experimentally tractable chondrostean, the sterlet (Acipenser ruthenus, a small sturgeon), and/or that of paddlefish. Twelve are expressed in both ampullary organs and neuromasts, consistent with conservation of molecular mechanisms. Six are electrosensory-restricted on the head (Irx5, Insm1, Sp5, Satb2, MafA and Rorc), and five are the first-reported mechanosensory-restricted transcription factor genes (Foxg1, Sox8, Isl1, Hmx2 and Rorb). However, as previously reported, Sox8 is expressed in ampullary organs as well as neuromasts in a shark (Scyliorhinus canicula), suggesting the existence of lineage-specific differences between cartilaginous and ray-finned fishes. Overall, our results support the hypothesis that ampullary organs and neuromasts develop via largely conserved transcriptional mechanisms, and identify multiple transcription factors potentially involved in the formation of electrosensory versus mechanosensory lateral line organs.

developmental biology↗

bioRxiv2023

Characterization of regeneration initiating cells during Xenopus laevis tail regeneration

Embryos are regeneration and wound healing masters. They not only rapidly close their wounds, remodel injured tissue without a scar, but also regenerate body parts. Many animal models with variable regenerative capabilities have already been studied. Additionally, with the introduction of high throughput techniques, novel regeneration mechanisms including genes and signaling pathways, and specialized cell types required for regeneration control in spatial and temporal aspects have been identified. Until now our knowledge has been limited to primarily the late phases of regeneration (> 1 day post injury). In this paper, we reveal the critical steps for regeneration initiation. We have discovered Regeneration Initiating Cells (RICs) using single cell and spatial transcriptomic analyses during tail regeneration in Xenopus laevis. RICs are formed transiently from the basal epidermal cells and are critical for the modification of the surrounding extracellular matrix to allow for migration of other cell types such as regeneration organizing cells that further promote regeneration. Absence or deregulation of RICs leads to excessive extracellular matrix deposition and regeneration defects.

developmental biology↗

bioRxiv2021

Clonal gametogenesis is triggered by intrinsic stimuli in the hybrids germ cells but is dependent on sex differentiation

Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrids reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreduplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrids gonial cells or whether it is regulated by the surrounding gonadal tissue. We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrids SSCs lead to sterility when transplanted into sexual males, but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females. This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals. Significance StatementAlthough sexual reproduction is a dominant trait among all eukaryotes, many taxa have evolved the ability to reproduce asexually. While asexuality appears to be linked to interspecific hybridization, it remains unknown how the coexistence of diverged genomes may initiate such a swap in reproduction. In our study, we transplanted germ cells between asexual hybrids and their parents. On one hand, the ability of clonal gametogenesis occurred exclusively in hybrid germ cells, suggesting that asexual development is directly triggered by the hybrid genomic constitution of the cell. On the other hand, clonality was observed only in cells transplanted into females, suggesting that the execution of clonal development is influenced by signals from the gonadal environment and regulated by somatic factors.

developmental biology↗

bioRxiv2021

Who is the best surrogate for germ stem cell transplantation in fish?

Surrogate reproduction technology in fish has potential for aquaculture as well as endangered species preservation and propagation. Species with some unfavourable biological characteristics for culturing such as a late maturation or a large body size are ideal candidates for surrogate reproduction using smaller and faster-maturing host. One of the general prerequisites for the successful surrogacy and the pure donor-derived gamete production is the sterility of the host. Various sterilization methods have been developed and used in fish surrogacy; however, a direct comparison of available methods is missing. Such a knowledge gap hinders choice for the surrogate in various fish species, including those in high commercial demand such as tuna or sturgeons, where is a particular limitation from the point of the live material availability and difficulty to perform a high throughput assessment of different surrogates. Yet, large sturgeons or tuna species are one of the most prominent candidates for surrogacy. Zebrafish was utilized in this study as a model species to answer whether and to which extent different sterilization strategies can affect the surrogacy. Germ cell-depleted recipients (produced using knockdown of dead end gene), triploid recipients, and zebrafish x pearl danio hybrid recipients were tested as they represent the most frequently used types of surrogates. Spermatogonia isolated from vas::EGFP transgenic strain were intraperitoneally transplanted into swim-up 5-day old zebrafish. Transplantation success, survival, gonadal development, and reproductive output of the fish was analyzed. Germ cell-depleted recipients with empty gonads were identified as the most convenient among tested sterilization methods considering surrogacy induction success and reproductive output. The present study stands as significant aid for selecting suitable surrogates in various fish species.

cell biology↗

bioRxiv2021

Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized mRNAs is still limited to a few candidates. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four models, Xenopus laevis, Danio rerio, Ambystoma mexicanum and Acipenser ruthenus using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important genes such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. There was also a poor correlation between vegetally localized genes but a relatively good correlation between animally localized genes. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

developmental biology↗

bioRxiv2020

Production of common carp donor-derived offspring from goldfish surrogate broodstock

BackgroundCommon carp is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining carp gene bank in situ can be considered as demanding due to its big body size. Recent progress in reproductive biotechnologies in fish allows improving some unfavourable characteristics of a target species using surrogate reproduction. Germ stem cells (gamete precursors) from one species are transplanted into different surrogate species with small body size. After maturation, surrogates are producing donor-derived progeny. Efficient protocols for cryopreservation of carp male and female germ stem cells have been developed lately. Thus, the next logical goal was to assess the potential of goldfish surrogate to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells. ResultsHigh transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp. More importantly, both viable eggs and sperm giving rise to pure common carp progeny were produced, witnessing sustainability of the presented method. Donor-derived identity of the offspring was confirmed by genotyping and typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control carp and goldfish eggs. Interestingly, flagellum length in donor-derived spermatozoa was comparable to common carp flagellum and significantly shorter than goldfish flagellum. ConclusionsIn this study, we succeeded in the production of pure common carp progeny from surrogate goldfish recipients transplanted intraperitoneally by testicular germ cells. Here we reported production of viable eggs between most distant species up to date. Good reproductive performance of goldfish germline chimeras gives a promising prospect for further analysis about the long-term reproductive performance of surrogates, recovery of cryopreserved germ cells or production of monosex stocks. Presented technology is ready to ease needs for carp breeds preservation and their recovery using many times smaller goldfish surrogates.

cell biology↗

bioRxiv2019

Parthenogenesis as a solution to hybrid sterility: the mechanistic basis of meiotic distortions in clonal and sterile hybrids

Formation of species generally occurs in a continuum from potentially intermixing populations to independent entities isolated from other species by pre- and postzygotic barriers. Especially the establishment of hybrid sterility (HS) is a hallmark of speciation, which usually emerges at different rates between hybrid sexes. However, although HS is frequently observed, the underlying molecular mechanisms remain poorly understood. Here we report that speciation proceeds through a previously unnoticed stage at which gene flow is completely interrupted on side of both hybrids sexes, although only male hybrids are sterile, while female fertility is rescued due to a particular gametogenetic deviation leading to the formation of clonal gametes. Specifically, analysis of gametogenetic pathways in hybrids between fish species Cobitis elongatoides and C. taenia revealed that male HS resulted from extensive asynapses and crossover reduction among elongatoides-taenia chromosomal pairs followed by apoptosis. By contrast, hybrid females exhibited premeiotic genome endoreplication which ensured proper formation of bivalents between identical chromosomal copies. This deviation ultimately restored fertility in females but since it simultaneously leads to the production of unreduced clonal gametes, it restricts interspecific gene flow thereby directly contributing to speciation. In conclusion, our data demonstrate that the emergence of asexuality may remedy HS in a sex-specific manner and is intermingled with the speciation process. Although gametogenetic mechanisms employed by asexual animals and plants have rarely been scrutinized, available evidence suggests that premeiotic endoreplication is relatively widespread. This suggests that observed link between HS and clonality may have general validity in taxa able of asexual reproduction.\n\nAuthors summarySpecies are fundamental evolutionary units that presumably evolve in a continuum from potentially intermixing populations to independent entities isolated from other species by pre- and postzygotic barriers. Especially the establishment of hybrid sterility (HS) is a hallmark of speciation, which usually emerges at different rates between hybrid sexes. However, although HS is frequently observed, the underlying molecular mechanisms remain poorly understood. Here we report the existence of a previously unnoticed stage of speciation at which gene flow is completely interrupted, although only male hybrids are sterile, while female fertility is rescued due to a particular gametogenetic deviation leading to formation of clonal gametes. Specifically, HS resulted from extensive asynapses in male gonads, but in females the hybridization provoked premeiotic endoreplication which rescued chromosome pairing and fertility. Simultaneously, this meiotic deviation caused clonal transmission of maternal genome, thereby effectively restricting the interspeficic gene flow. Our results emphasize that emergence of clonality is a type of hybrid incompatibility that is intermingled with the formation of biological species and may remedy hybrid sterility in a sex-specific manner.

evolutionary biology↗

bioRxiv2019

Production and use of triploid zebrafish for surrogate reproduction

We report for first time comparison of two approaches for zebrafish triploid production using cold shock and heat shock treatment. Subsequently, produced triploid zebrafish were used as a recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Heat shock treatment was far more effective evaluated as success rate of triploid production and viability in comparison to cold shock. Triploids were produced with up to 100% efficiency in particular females. As expected, all triploids were males. Subsequently, germ cells transplantation revealed that triploids are suitable surrogate hosts. Production of donor-derived sperm was achieved in 23% and 16% of triploids transplanted by testicular and ovarian cells, respectively. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups.

zoology↗

bioRxiv2018

Cryopreservation and transplantation of common carp spermatogonia

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation, however, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10 {degrees}C/min) and different concentrations of Me2SO were tested resulting in the highest survival using 2 M Me2SO and cooling rate of -1 Q59 A When testing different tissue sizes and incubation times in the cryomedium, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the gonads in goldfish recipients was confirmed by molecular markers and incorporation rate was over 40% in both groups at 3 months post transplantation. Results of this study can serve as an alternative way for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

bioengineering↗