Biology Of Spermatogonial Stem Cells Research Articles

Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†.

Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.

Primary Cultures of Spermatogonia and Testis Cells.

Spermatogonial stem cells (SSCs) maintain adult spermatogenesis in mammals by undergoing self-renewal and differentiation into spermatozoa. In order to study the biology of SSCs as related to spermatogenesis, an in vitro, long-term expansion system of SSCs constitutes an ideal tool. In this chapter, we describe a robust culture system for mouse and rat SSCs in vitro. In the presence of GDNF, GFRα1, and bFGF, SSCs maintained on STO feeder layers with serum-free medium continuously proliferate for over 6months. Complete spermatogenesis in infertile recipient mice can be attained following transplantation of the cultured mouse and rat SSCs. Using the in vitro SSC culture systems, elucidation of stem cell biology can be advanced that significantly advances our understanding of spermatogenesis and male fertility.

Perspectives: Approaches for Studying Livestock Spermatogonia.

At present, the knowledge base on characteristics and biology of spermatogonia in livestock is limited in comparison to rodents, yet the importance of studying these cells for comparative species analysis and enhancing reproductive capacity in food animals is high. Previous studies have established that although many core attributes of organ physiology and mechanisms governing essential cellular functions are conserved across eutherians, significant differences exist between mice and higher order mammals. In this chapter, we briefly discuss distinguishing aspects of testicular anatomy and the spermatogenic lineage in livestock and critical considerations for studying spermatogonial stem cell biology in these species.

Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration.

Spermatogonial stem cells (SSCs) are a group of adult stem cells in the testis that serve as the foundation of continuous spermatogenesis and male fertility. SSCs are capable of self-renewal to maintain the stability of the stem cell pool and differentiation to produce mature spermatozoa. Dysfunction of SSCs leads to male infertility. Therefore, dissection of the regulatory network of SSCs is of great significance in understanding the fundamental molecular mechanisms of spermatogonial stem cell function in spermatogenesis and the pathogenesis of male infertility. Furthermore, a better understanding of SSC biology will allow us to culture and differentiate SSCs in vitro, which may provide novel stem cell-based therapy for assisted reproduction. This review summarizes the latest research progress on the regulation of SSCs, and the potential application of SSCs for fertility restoration through in vivo and in vitro spermatogenesis. We anticipate that the knowledge gained will advance the application of SSCs to improve male fertility. Furthermore, in vitro spermatogenesis from SSCs sets the stage for the production of SSCs from induced pluripotent stem cells (iPSCs) and subsequent spermatogenesis.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, itis used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editingwill be historic achievement for the foothold of the SSCs technology. Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Recent advances in isolation, identification, and culture of mammalian spermatogonial stem cells.

Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.

Altered Biology of Testicular VSELs and SSCs by Neonatal Endocrine Disruption Results in Defective Spermatogenesis, Reduced Fertility and Tumor Initiation in Adult Mice.

Reproductive health of men has declined in recent past with reduced sperm count and increased incidence of infertility and testicular cancers mainly attributed to endocrine disruption in early life. Present study aims to evaluate whether testicular stem cells including very small embryonic-like stem cells (VSELs) and spermatogonial stem cells (SSCs) get affected by endocrine disruption and result in pathologies in adult life. Effect of treatment onmice pups with estradiol (20μg on days 5-7) and diethylstilbestrol (DES, 2μg on days 1-5) was studied on VSELs, SSCs and spermatogonial cells in adult life. Treatment affected spermatogenesis, tubules in Stage VIII & sperm count were reduced along with reduction of meiotic (4n) cells and markers (Prohibitin, Scp3, Protamine). Enumeration of VSELs by flow cytometry (2-6μm, 7AAD-, LIN-CD45-SCA-1+) and qRT-PCR using specific transcripts for VSELs (Oct-4a, Sox-2, Nanog, Stella, Fragilis), SSCs (tOct-4, Gfra-1, Gpr-125) and early germ cells (Mvh, Dazl) showed several-fold increase but transition from c-Kit negative to c-Kit positive spermatogonial cells was blocked on D100 after treatment. Transcripts specific for apoptosis (Bcl2, Bax) remained unaffected but tumor suppressor (p53) and epigenetic regulator (NP95) transcripts showed marked disruption. 9 of 10 mice exposed to DES showed tumor-like changes. To conclude, endocrine disruption resulted in a tilt towards excessive self-renewal of VSELs (leading to testicular cancer after DES treatment) and blocked differentiation (reduced numbers of c-Kit positive cells, meiosis, sperm count and fertility). Understanding the underlying basis for infertility and cancer initiation from endogenous stem cells through murine modelling will hopefully improve human therapies in future.

Contribution of Single-Cell Transcriptomics to the Characterization of Human Spermatogonial Stem Cells: Toward an Application in Male Fertility Regenerative Medicine?

Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.

Spermatogonial stem cell biology in the bull: development of isolation, culture, and transplantation methodologies and their potential impacts on cattle production

Widespread adoption of artificial insemination as a breeding practice has allowed for expanded use of desirable genetics from specific sires and greatly influenced production traits in dairy cattle populations worldwide. In fact, the average dairy cow in the US in 2009 produced 4.5 times more milk than in 1940 when commercialization of artificial insemination began. While many factors have contributed to this rapid increase in levels of milk production, genetic gain through expanded utilization of germlines from specific sires has been a major contribution. In comparison, use of artificial insemination in beef cattle populations has been limited due to challenges with implementing intensive management strategies required for success. Thus, there is need for alternative reproductive tools to expand use of desirable male genetics in the beef cattle industry. The process of sperm production, termed spermatogenesis, is supported by a tissue- specific stem cell population referred to as spermatogonial stem cells (SSCs). These unique cells have the capacity for infinite self-renewal and long-term regeneration of spermatogenesis following transplantation. In rodents, methods for isolating, culturing, and transplanting SSCs have been devised. For beef cattle, transplanting SSCs isolated from a donor male into the testes of recipient males in which donor-derived spermatogenesis occurs and offspring with donor genetics are produced from natural breeding has great potential as an alternative to artificial insemination. This potential reproductive strategy would allow for expansive use of genetics from desirable sires that overcomes the logistical challenges of artificial insemination. Translation of the methods devised for rodents to cattle is at the forefront of development. Devising means for isolating an SSC-enriched cell fraction from donor testes and identifying conditions that support long-term maintenance and proliferation of bovine SSCs in vitro are two tools that would greatly accelerate the pace at which transplantation will become a commercially viable option for cattle industries. Recent studies showed that expression of THY1 by SSCs is a conserved phenotype between rodents and cattle, and selection of the THY1+ fraction from donor testes can be used for isolating an SSC-enriched germ cell population. In addition, the conditions devised tor expanding the number of rodent SSCs in vitro continues to serve as the basis for developing conditions that support bovine SSCs. With these tools in hand major advances in developing implementable reproductive tools with SSCs for commercial cattle production will be made in the coming decade.

Brinster Spermatogonial Stem Cell Transplantation 25th Anniversary Symposium.

The Symposium, co-sponsored by the Institute of Regenerative Medicine, the University Research Foundation, the Center for Research on Reproduction and Women's Health, the Penn Center for the Study of Epigenetics in Reproduction, and Penn Vet at the University of Pennsylvania, commemorated the 25th anniversary of the first publications describing spermatogonial stem cell (SSC) transplantation in mice. This transformative approach has propelled advances in our understanding of germ cell biology, has been translated to a variety of vertebrate species, and holds translational potential for fertility restoration in patients. The symposium opened with a lecture by Dr. Brinster reflecting on the origin of the work, as well as advances over the 25 years up to present ongoing studies. Following Dr. Brinster's remarks, 10 lectures were presented by distinguished scientists, including several of Dr. Brinster's former trainees and colleagues. The symposium closed with a keynote lecture by Dr. David Page. Topics ranged from aspects of basic SSC biology to applications in large animal models and potential translation to treating human male infertility. Many of the studies presented directly resulted from SSC transplantation technology highlighting its tremendous impact in advancing the field. The Symposium program and the lectures can be found at https://spark.adobe.com/page/jS0cDLzLHvOiJ.

Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides)

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male’s life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

Postnatal testis development in the collared peccary (Tayassu tajacu), with emphasis on spermatogonial stem cells markers and niche

Collared peccaries (Tayassu tajacu) present a unique testis cytoarchitecture, where Leydig cells (LC) are mainly located in cords around the seminiferous tubules (ST) lobes. This peculiar arrangement is very useful to better investigate and understand the role of LC in spermatogonial stem cells (SSCs) biology and niche. Recent studies from our laboratory using adult peccaries have shown that the undifferentiated type A spermatogonia (Aund or SSCs) are preferentially located in ST regions adjacent to the intertubular compartment without LC. Following these studies, our aims were to investigate the collared peccary postnatal testis development, from birth to adulthood, with emphasis on the establishment of LC cytoarchitecture and the SSCs niche. Our findings demonstrated that the unique LC cytoarchitecture is already present in the neonate peccary’s testis, indicating that this arrangement is established during fetal development. Based on the most advanced germ cell type present at each time period evaluated, puberty (the first sperm release in the ST lumen) in this species was reached at around one year of age, being preceded by high levels of estradiol and testosterone and the end of Sertoli cell proliferation. Almost all gonocytes and SSCs expressed Nanos1, Nanos2 and GFRA1. The analysis of SSCs preferential location indicated that the establishment of SSCs niche is coincident with the occurrence of puberty. Taken together, our findings reinforced and extended the importance of the collared peccary as an animal model to investigate testis function in mammals, particularly the aspects related to testis organogenesis and the SSCs biology and niche.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

Culture and transplantation of spermatogonial stem cells

The spermatogonial transplantation technique was developed by Dr. Ralph Brinster in 1994. Transplanted spermatogonial stem cells (SSCs) produce germ cell colonies after microinjection into the seminiferous tubules of infertile mice. This technique provided the first functional assay for SSCs. Although it became possible to produce transgenic animals using this transplantation technique in 2001, the lack of SSC culture systems prevented efficient genetic manipulation. To overcome this problem, a long-term SSC culture technique was developed in 2003. Cultured SSCs, designated as germline stem cells, allow drug selection of transfected SSCs, and knockout mice were produced in 2006. Using these techniques, it is now possible to address basic biological questions of SSC biology. They also open up new possibilities for male germline manipulation. In this review, we will briefly summarize our findings on SSCs and discuss unresolved issues that remain to be addressed.

Comparative analysis of single‐cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells

Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells.

Long-Term Propagation of Porcine Undifferentiated Spermatogonia.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.

Diethylstilbestrol induces oxidative DNA damage, resulting in apoptosis of spermatogonial stem cells in vitro.

The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2-) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

Spermatogonial Stem Cell Self - renewal and Differentiation

Mammalian spermatogenesis is a complicated and precisely controlled process that requires spermatogonial stem cells (SSCs). SSCs maintain the stem cell pool, balance self-renewal–commitment with differentiation, and produce millions of sperm daily. Self-renewal and differentiation are controlled by intrinsic factors within SSCs and extrinsic factors from the niche. In this review, we discuss the biology of SSCs and the factors regulating their self-renewal and differentiation. Key words: Differentiation; Self-renewal; Spermatogonial Stem Cells

Can mesenchymal stem cells improve spermatogonial stem cell transplantation efficiency?

Improved treatments have led to an increased survival rate in cancer patients. However, in pre-pubertal boys, these gonadotoxic treatments can result in the depletion of the spermatogonial stem cell (SSC) pool causing lifelong infertility. SSC transplantation has been proposed as a promising technique to preserve the fertility of these patients. In mice, this technique has resulted in live-born offspring, but the efficiency of colonization remained low. This could be because of a deficient microenvironment, leading to apoptosis of the transplanted SSCs. Interestingly, mesenchymal stem cells (MSCs), being multipotent and easy to isolate and multiply invitro, are nowadays successfully and widely used in regenerative medicine. Here, we shortly review the current understanding of MSC and SSC biology, and we hypothesize that a combined MSC-SSC transplantation might improve the efficiency of SSC colonization and differentiation as paracrine factors from MSCs may contribute to the SSC niche.

Identification, Prokaryote Expression of Medaka gdnfa/b and Their Biological Activity in a Spermatogonial Cell Line.

The origin and evolution of molecular mechanisms underlying the self-renewal and differentiation of spermatogonial stem cells (SSCs) are fundamental questions in stem cell biology as well as reproduction medicine. In mammals, glial cell line-derived neurotrophic factor (GDNF) is crucial for SSC self-renewal and maintenance. However, in nonmammals, the role of Gdnf in SSCs still remains unknown. In this study, we report that the two GDNF homologs from medaka fish (Oryzias latipes), namely OlGdnfa and OlGdnfb, can promote proliferation activity and retain the spermatogonial property of SG3, a spermatogonial cell line derived from adult medaka showing the intrinsic property of SSCs by self-renewal and differentiation potential during 2 years of culture. Cloning and sequencing led to the identification of two cDNA sequences as Olgdnfa and Olgdnfb, which are 780-nt and 744-nt in length for 253 and 245 amino acid residues, respectively. Both are homologs of mammalian GDNF and share over 45% identity with the other known vertebrate homologs. Importantly, in a well-defined condition, the recombinant proteins, OlGdnfa and OlGdnfb, can significantly promote the proliferative activity of SG3 cells and retain the spermatogonial gene expression pattern and alkaline phosphatase activity. Meanwhile, both of the two recombinant proteins can upregulate the mRNA expression level of bcl6b, one of the prominent GDNF-regulated genes involved in SSC self-renewal and maintenance in mammals. Taken together, our findings suggest that just like the mammalian counterpart, the nonmammalian Gdnfs might mediate the self-renewal and maintenance of SSCs; moreover, Bcl6b might be a conserved regulator in SSC self-renewal across vertebrate taxa. This study extends our knowledge of GDNF functions in SSC biology during evolution.