Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.

Genetic association and characterization of FSTL5 in isolated clubfoot.

Talipes equinovarus (clubfoot, TEV) is a congenital rotational foot deformity occurring in 1 per 1000 births with increased prevalence in males compared with females. The genetic etiology of isolated clubfoot (iTEV) remains unclear. Using a genome-wide association study, we identified a locus within FSTL5, encoding follistatin-like 5, significantly associated with iTEV. FSTL5 is an uncharacterized gene whose potential role in embryonic and postnatal development was previously unstudied. Utilizing multiple model systems, we found that Fstl5 was expressed during later stages of embryonic hindlimb development, and, in mice, expression was restricted to the condensing cartilage anlage destined to form the limb skeleton. In the postnatal growth plate, Fstl5 was specifically expressed in prehypertrophic chondrocytes. As Fstl5 knockout rats displayed no gross malformations, we engineered a conditional transgenic mouse line (Fstl5LSL) to overexpress Fstl5 in skeletal osteochondroprogenitors. We observed that hindlimbs were slightly shorter and that bone mineral density was reduced in adult male, but not female, Prrx1-cre;Fstl5LSL mice compared with control. No overt clubfoot-like deformity was observed in Prrx1-cre;Fstl5LSL mice, suggesting FSTL5 may function in other cell types to contribute to iTEV pathogenesis. Interrogating published mouse embryonic single-cell expression data showed that Fstl5 was expressed in cell lineage subclusters whose transcriptomes were associated with neural system development. Moreover, our results suggest that lineage-specific expression of the Fstl genes correlates with their divergent roles as modulators of transforming growth factor beta and bone morphogenetic protein signaling. Results from this study associate FSTL5 with iTEV and suggest a potential sexually dimorphic role for Fstl5 in vivo.

Loss of Piccolo Function in Rats Induces Cerebellar Network Dysfunction and Pontocerebellar Hypoplasia Type 3-like Phenotypes.

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO (Pclogt/gt ) rats. Analysis of rats of both sexes revealed a dramatic reduction in brain size compared with WT (Pclowt/wt ) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar, and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber afferents from pontine neurons and the expression of the α6 GABAA receptor subunit at the mossy fiber-granule cell synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers. Ultrastructural and functional studies revealed a reduced size of mossy fiber boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation, and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adult Pclogt/gt rats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together, these data suggest that loss of Piccolo function in patients with PCH3 could be involved in many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.SIGNIFICANCE STATEMENT Pontocerebellar Hypoplasia Type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures, and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the PCLO gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo KO rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with Pontocerebellar Hypoplasia Type 3 are also exhibited by these Piccolo deficient animals.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

Long Evans rat spermatogonial lines are effective germline vectors for transgenic rat production.

Long Evans rat strains are applied as research models in a broad spectrum of biomedical fields (>15,800 citations, NCBI PubMed). Here, we report an approach to genetically modify the Long Evans rat germline in donor spermatogonial stem cells. Long Evans rat spermatogonial lines were derived from freshly isolated laminin-binding spermatogonia. Laminin-binding spermatogonia were cultured over multiple passages on fibroblast feeder layers in serum-free culture medium containing GDNF and FGF2. Long Evans rat spermatogonial lines were genetically modified by transposon transduction to express a germline, tdTomato reporter gene. Donor rat spermatogonial lines robustly regenerated spermatogenesis after transplantation into testes of busulfan-treated, allogenic, Long Evans rats. Donor-derived spermatogenesis largely restored testis size in the chemically sterilized, recipient Long Evans rats. Recipient Long Evans rats stably transmitted the tdTomato germline marker to subsequent generations. Overall, Long Evans rat spermatogonial lines provided effective donor germline vectors for genetically modifying Long Evans rats.

Linking spermatid ribonucleic acid (RNA) binding protein and retrogene diversity to reproductive success.

Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness.

A-single spermatogonia heterogeneity and cell cycles synchronize with rat seminiferous epithelium stages VIII-IX.

In mammalian testes, "A-single" spermatogonia function as stem cells that sustain sperm production for fertilizing eggs. Yet, it is not understood how cellular niches regulate the developmental fate of A-single spermatogonia. Here, immunolabeling studies in rat testes define a novel population of ERBB3(+) germ cells as approximately 5% of total SNAP91(+) A-single spermatogonia along a spermatogenic wave. As a function of time, ERBB3(+) A-single spermatogonia are detected during a 1- to 2-day period each 12.9-day sperm cycle, representing 35%-40% of SNAP91(+) A-single spermatogonia in stages VIII-IX of the seminiferous epithelium. Local concentrations of ERBB3(+) A-single spermatogonia are maintained under the mean density measured for neighboring SNAP91(+) A-single spermatogonia, potentially indicative of niche saturation. ERBB3(+) spermatogonia also synchronize their cell cycles with epithelium stages VIII-IX, where they form physical associations with preleptotene spermatocytes transiting the blood-testis barrier and Sertoli cells undergoing sperm release. Thus, A-single spermatogonia heterogeneity within this short-lived and reoccurring microenvironment invokes novel theories on how cellular niches integrate with testicular physiology to orchestrate sperm development in mammals.

Generating knockout rats by transposon mutagenesis in spermatogonial stem cells.

Disrupting genes in the rat on a genome-wide scale will allow the investigation of many biological processes linked to human health. Here we used transposon-mediated mutagenesis to knock out genes in rat spermatogonial stem cells. Given the capacity of the testis to support spermatogenesis from thousands of transplanted, genetically manipulated spermatogonia, this approach paves a way for high-throughput functional genomic studies in the laboratory rat.

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats.

OBJECTIVE: Male rats transgenic for HLA-B27 and human ß(2) -microglobulin (hß(2) m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/hß(2) m-transgenic rat cross-strain (21-3 × 382-2)F(1) , only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 × 382-2)F(1) rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. METHODS: Male B27/hß(2) m-transgenic (21-3 × 382-2)F(1) rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. RESULTS: In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and anti-testis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-γ, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. CONCLUSION: Autoimmune EO develops spontaneously in HLA-B27/hß(2) m-transgenic (21-3 × 283-2)F(1) rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of SpA.

MOV10L1 is necessary for protection of spermatocytes against retrotransposons by Piwi-interacting RNAs.

Piwi-interacting RNAs (piRNAs) comprise a broad class of small noncoding RNAs that function as an endogenous defense system against transposable elements. Here we show that the putative DExD-box helicase MOV10-like-1 (MOV10L1) is essential for silencing retrotransposons in the mouse male germline. Mov10l1 is specifically expressed in germ cells with increasing expression from gonocytes/type A spermatogonia to pachytene spermatocytes. Primary spermatocytes of Mov10l1(-/-) mice show activation of LTR and LINE-1 retrotransposons, followed by cell death, causing male infertility and a complete block of spermatogenesis at early prophase of meiosis I. Despite the early expression of Mov10l1, germline stem cell maintenance appears unaffected in Mov10l1(-/-) mice. MOV10L1 interacts with the Piwi proteins MILI and MIWI. MOV10L1 also interacts with heat shock 70-kDa protein 2 (HSPA2), a testis-enriched chaperone expressed in pachytene spermatocytes and also essential for male fertility. These studies reveal a crucial role of MOV10L1 in male fertility and piRNA-directed retrotransposon silencing in male germ cells and suggest that MOV10L1 functions as a key component of a safeguard mechanism for the genetic information in male germ cells of mammals.

Rattus norvegicus Spermatogenesis Colony-Forming Assays.

Knowledge gaps persist on signaling pathways and metabolic states in germ cells sufficient to support spermatogenesis independent of a somatic environment. Consequently, methods to culture mammalian stem cells through spermatogenesis in defined systems have not been established. Lack of success at culturing mammalian stem cells through spermatogenesis in defined systems reflects an inability to experimentally recapitulate biochemical events that develop in germ cells within the testis-specific seminiferous epithelium. Complex germ and somatic cell associations that develop each seminiferous epithelial cycle support such a hypothesis, conceivably explaining why highly pure mammalian spermatogonia do not effectively develop into and through meiosis without somatic cells. Here, we outline an in vitro spermatogenesis colony-forming assay to study how differentiating spermatogonial syncytia develop from rat spermatogonial stem cell lines. Robust spermatogonial differentiation under defined culture conditions, once established, is anticipated to facilitate molecular biology studies on pre-meiotic steps in gametogenesis by providing soma-free bioassays to systematically identify spermatogenic factors that promote meiotic progression in vitro.

Sleeping Beauty transposon mutagenesis of the rat genome in spermatogonial stem cells.

Since several aspects of physiology in rats have evolved to be more similar to humans than that of mice, it is highly desirable to link the rat into the process of annotating the human genome with function. However, the lack of technology for generating defined mutants in the rat genome has hindered the identification of causative relationships between genes and disease phenotypes. As an important step towards this goal, an approach of establishing transposon-mediated insertional mutagenesis in rat spermatogonial stem cells was recently developed. Transposons can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest such as a mutagenic gene trap cassette cloned between the inverted repeat sequences of a transposon-based vector can be utilized for stable genomic insertion in a regulated and highly efficient manner. Gene-trap transposons integrate into the genome in a random fashion, and those mutagenic insertions that occurred in expressed genes can be selected in vitro based on activation of a reporter. Selected monoclonal as well as polyclonal libraries of gene trap clones are transplanted into the testes of recipient/founder male rats allowing passage of the mutation through the germline to F1 progeny after only a single cross with wild-type females. This paradigm enables a powerful methodological pipeline for forward genetic screens for functional gene annotation in the rat, as well as other vertebrate models. This article provides a detailed description on how to culture rat spermatogonial stem cell lines, their transfection with transposon plasmids, selection of gene-trap insertions with antibiotics, transplantation of genetically modified stem cells and genotyping of knockout animals.

Spermatogonial Gene Networks Selectively Couple to Glutathione and Pentose Phosphate Metabolism but Not Cysteine Biosynthesis.

In adult males, spermatogonia maintain lifelong spermatozoa production for oocyte fertilization. To understand spermatogonial metabolism we compared gene profiles in rat spermatogonia to publicly available mouse, monkey, and human spermatogonial gene profiles. Interestingly, rat spermatogonia expressed metabolic control factors Foxa1, Foxa2, and Foxa3. Germline Foxa2 was enriched in Gfra1Hi and Gfra1Low undifferentiated A-single spermatogonia. Foxa2-bound loci in spermatogonial chromatin were overrepresented by conserved stemness genes (Dusp6, Gfra1, Etv5, Rest, Nanos2, Foxp1) that intersect bioinformatically with conserved glutathione/pentose phosphate metabolism genes (Tkt, Gss, Gc l c , Gc l m, Gpx1, Gpx4, Fth), marking elevated spermatogonial GSH:GSSG. Cystine-uptake and intracellular conversion to cysteine typically couple glutathione biosynthesis to pentose phosphate metabolism. Rat spermatogonia, curiously, displayed poor germline stem cell viability in cystine-containing media, and, like primate spermatogonia, exhibited reduced transsulfuration pathway markers. Exogenous cysteine, cysteine-like mercaptans, somatic testis cells, and ferroptosis inhibitors counteracted the cysteine-starvation-induced spermatogonial death and stimulated spermatogonial growth factor activity in vitro.

Characterization of a Dazl-GFP germ cell-specific reporter.

In this study, we characterized the promoter activity of a 1.7 kb sequence in the 5' flanking region of the mouse Deleted in Azoospermia-Like (Dazl) gene. We found the 1.7 kb sequence sufficient to drive robust germ cell-specific expression of green fluorescent protein (GFP) in adult mouse testis and lower levels of expression in adult ovary and in fetal and newborn gonads of both sexes. This expression pattern was confirmed in two independently-derived transgenic mouse lines. In adult testis, Dazl-GFP exhibited a developmentally-regulated, stage-specific expression pattern during spermatogenesis. GFP was highly expressed in spermatocyte stages, with strongest expression in pachytene spermatocytes. Weaker expression was observed in round and elongating spermatids, as well as spermatogonial cells. In the fetal gonad, GFP transcript was detected by e12.5 in both sexes; however, GFP fluorescence was only detected during later embryonic stages. In addition, we produced mouse embryonic stem cell (ESC) lines harboring the Dazl-GFP reporter and used this reporter to isolate putative germ cell populations derived from mouse ESCs following embryoid body differentiation and fluorescence activated cell sorting.

Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.

beta-Glucosidase 2 (GBA2) is a resident enzyme of the endoplasmic reticulum thought to play a role in the metabolism of bile acid-glucose conjugates. To gain insight into the biological function of this enzyme and its substrates, we generated mice deficient in GBA2 and found that these animals had normal bile acid metabolism. Knockout males exhibited impaired fertility. Microscopic examination of sperm revealed large round heads (globozoospermia), abnormal acrosomes, and defective mobility. Glycolipids, identified as glucosylceramides by mass spectrometry, accumulated in the testes, brains, and livers of the knockout mice but did not cause obvious neurological symptoms, organomegaly, or a reduction in lifespan. Recombinant GBA2 hydrolyzed glucosylceramide to glucose and ceramide; the same reaction catalyzed by the beta-glucosidase acid 1 (GBA1) defective in subjects with the Gaucher's form of lysosomal storage disease. We conclude that GBA2 is a glucosylceramidase whose loss causes accumulation of glycolipids and an endoplasmic reticulum storage disease.

Sohlh2 knockout mice are male-sterile because of degeneration of differentiating type A spermatogonia.

The spermatogenesis and oogenesis-specific transcription factor Sohlh2 is normally expressed only in premeiotic germ cells. In this study, Sohlh2 and several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that overexpresses bone morphogenetic protein 4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile because of a block in spermatogenesis. Although normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type B spermatogonia by postnatal day 7. By day 10, development to the preleptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type A spermatogonia (DAZL+/PLZF+) to differentiating type A spermatogonia (DAZL+/PLZF-) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice proliferated normally but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase cKit. Thus, Sohlh2 is required for progression of differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Critical role for Piccolo in synaptic vesicle retrieval.

Loss of function of the active zone protein Piccolo has recently been linked to a disease, Pontocerebellar Hypoplasia type 3, which causes brain atrophy. Here, we address how Piccolo inactivation in rat neurons adversely affects synaptic function and thus may contribute to neuronal loss. Our analysis shows that Piccolo is critical for the recycling and maintenance of synaptic vesicles. We find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by reduced synaptic recruitment of Pra1, known to interact selectively with the zinc finger domains of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to developmental and psychiatric disorders.

Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.