Genotype-specific expression of uncle fester suggests a role in allorecognition education in a basal chordate.

Histocompatibility is the ability to discriminate between self and non-self tissues, and has been described in species throughout the metazoa. Despite its universal presence, histocompatibility genes utilized by different phyla are unique- those found in sponges, cnidarians, ascidians and vertebrates are not orthologous. Thus, the origins of these sophisticated recognition systems, and any potential functional commonalities between them are not understood. We are studying histocompatibility in the botryllid ascidians, members of the chordate subphylum, Tunicata, which provide a powerful model to understand both the origins and functional aspects of this process. Histocompatibility in the botryllids occurs at the tips of an extracorporeal vasculature that come into contact when two individuals grow into proximity. If compatible, the vessels will fuse, forming a parabiosis between the two individuals. If incompatible, the two vessels will reject- an inflammatory reaction that results in melanin scar formation at the point of contact, blocking anastomosis. Compatibility is determined by a single, highly polymorphic locus called the fuhc with the following rules: individuals that share one or both fuhc alleles will fuse, while those who share neither will reject. The fuhc locus encodes at least six proteins with known roles in allorecognition. One of these genes, called uncle fester, is necessary and sufficient to initiate the rejection response. Here we report the existence of genotype-specific expression levels of uncle fester, differing by up to 8-fold at the mRNA-level, and that these expression levels are constant and maintained for the lifetime of an individual. We also found that these differences had functional consequences: the expression level of uncle fester correlated with the speed and severity of the rejection response. These findings support previous conclusions that uncle fester levels modulate the rejection response, and may be responsible for controlling the variation observed in the timing and intensity of the reaction. The maintenance of genotype specific expression of uncle fester is also evidence of an education process reminiscent of that which occurs in mammalian Natural Killer (NK) cells. In turn, this suggests that while histocompatibility receptors and ligands evolve via convergent evolution, they may utilize conserved intracellular machinery to interpret binding events at the cell surface.

Genotype-specific expression of uncle fester suggests a role in allorecognition education in a basal chordate.

Histocompatibility is the ability to discriminate between self and non-self tissues, and has been described in species throughout the metazoa. Despite its universal presence, histocompatibility genes utilized by different phyla are unique- those found in sponges, cnidarians, ascidians and vertebrates are not orthologous. Thus, the origins of these sophisticated recognition systems, and any potential functional commonalities between them are not understood. A well-studied histocompatibility system exists in the botryllid ascidians, members of the chordate subphylum, Tunicata, and provides an opportunity to do so. Histocompatibility in the botryllids occurs at the tips of an extracorporeal vasculature that come into contact when two individuals grow into proximity. If compatible, the vessels will fuse, forming a parabiosis between the two individuals. If incompatible, the two vessels will reject- an inflammatory reaction that results in melanin scar formation at the point of contact, blocking anastomosis. Compatibility is determined by a single, highly polymorphic locus called the fuhc with the following rules: individuals that share one or both fuhc alleles will fuse, while those who share neither will reject. The fuhc locus encodes multiple proteins with roles in allorecognition, including one called uncle fester, which is necessary and sufficient to initiate the rejection response. Here we report the existence of genotype-specific expression levels of uncle fester, differing by up to 8-fold at the mRNA-level, and that these expression levels are constant and maintained for the lifetime of an individual. We also found that these differences had functional consequences: the expression level of uncle fester correlated with the speed and severity of the rejection response. These findings support previous conclusions that uncle fester levels modulate the rejection response, and may be responsible for controlling the variation observed in the timing and intensity of the reaction. The maintenance of genotype specific expression of uncle fester is also evidence of an education process reminiscent of that which occurs in mammalian Natural Killer (NK) cells. In turn, this suggests that while histocompatibility receptors and ligands evolve via convergent evolution, they may utilize conserved intracellular machinery to interpret binding events at the cell surface.

Rtf1-dependent transcriptional pausing regulates cardiogenesis.

During heart development, a well-characterized network of transcription factors initiates cardiac gene expression and defines the precise timing and location of cardiac progenitor specification. However, our understanding of the post-initiation transcriptional events that regulate cardiac gene expression is still incomplete. The PAF1C component Rtf1 is a transcription regulatory protein that modulates pausing and elongation of RNA Pol II, as well as cotranscriptional histone modifications. Here we report that Rtf1 is essential for cardiogenesis in fish and mammals, and that in the absence of Rtf1 activity, cardiac progenitors arrest in an immature state. We found that Rtf1's Plus3 domain, which confers interaction with the transcriptional pausing and elongation regulator Spt5, was necessary for cardiac progenitor formation. ChIP-seq analysis further revealed changes in the occupancy of RNA Pol II around the transcription start site (TSS) of cardiac genes in rtf1 morphants reflecting a reduction in transcriptional pausing. Intriguingly, inhibition of pause release in rtf1 morphants and mutants restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes. Our findings highlight the crucial role that transcriptional pausing plays in promoting normal gene expression levels in a cardiac developmental context.

Rtf1 Transcriptionally Regulates Neonatal and Adult Cardiomyocyte Biology.

The PAF1 complex component Rtf1 is an RNA Polymerase II-interacting transcription regulatory protein that promotes transcription elongation and the co-transcriptional monoubiquitination of histone 2B. Rtf1 plays an essential role in the specification of cardiac progenitors from the lateral plate mesoderm during early embryogenesis, but its requirement in mature cardiac cells is unknown. Here, we investigate the importance of Rtf1 in neonatal and adult cardiomyocytes using knockdown and knockout approaches. We demonstrate that loss of Rtf1 activity in neonatal cardiomyocytes disrupts cell morphology and results in a breakdown of sarcomeres. Similarly, Rtf1 ablation in mature cardiomyocytes of the adult mouse heart leads to myofibril disorganization, disrupted cell-cell junctions, fibrosis, and systolic dysfunction. Rtf1 knockout hearts eventually fail and exhibit structural and gene expression defects resembling dilated cardiomyopathy. Intriguingly, we observed that loss of Rtf1 activity causes a rapid change in the expression of key cardiac structural and functional genes in both neonatal and adult cardiomyocytes, suggesting that Rtf1 is continuously required to support expression of the cardiac gene program.

Glutamate 73 Promotes Anti-arrhythmic Effects of Voltage-Dependent Anion Channel Through Regulation of Mitochondrial Ca2+ Uptake.

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.

A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

Integrin-alpha-6+ Candidate stem cells are responsible for whole body regeneration in the invertebrate chordate Botrylloides diegensis.

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. WBR cannot proceed when proliferating IA6+ cells are ablated with Mitomycin C, and injection of a single IA6+ Candidate stem cell can rescue WBR after ablation. Lineage tracing using EdU-labeling demonstrates that donor-derived IA6+ Candidate stem cells directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of IA6+ Candidate stem cells, indicating that these two pathways regulate their activation. In conclusion, we show that IA6+ Candidate stem cells are responsible for whole body regeneration and give rise to regenerating tissues.

Mitochondrial Calcium Uniporter Deficiency in Zebrafish Causes Cardiomyopathy With Arrhythmia.

Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.

Catalytic Enantioselective Synthesis of Guvacine Derivatives through [4 + 2] Annulations of Imines with α-Methylallenoates.

P-Chiral [2.2.1] bicyclic phosphines (HypPhos catalysts) have been applied to reactions between α-alkylallenoates and imines, producing guvacine derivatives. These HypPhos catalysts were assembled from trans-4-hydroxyproline, with the modular nature of the synthesis allowing variations of the exocyclic P and N substituents. Among them, exo-( p-anisyl)-HypPhos was most efficacious for [4 + 2] annulations between ethyl α-methylallenoate and imines. Through this method, ( R)-aplexone was identified as being responsible for the decrease in the cellular levels of cholesterol.

Multiscale light-sheet for rapid imaging of cardiopulmonary system.

The ability to image tissue morphogenesis in real-time and in 3-dimensions (3-D) remains an optical challenge. The advent of light-sheet fluorescence microscopy (LSFM) has advanced developmental biology and tissue regeneration research. In this review, we introduce a LSFM system in which the illumination lens reshapes a thin light-sheet to rapidly scan across a sample of interest while the detection lens orthogonally collects the imaging data. This multiscale strategy provides deep-tissue penetration, high-spatiotemporal resolution, and minimal photobleaching and phototoxicity, allowing in vivo visualization of a variety of tissues and processes, ranging from developing hearts in live zebrafish embryos to ex vivo interrogation of the microarchitecture of optically cleared neonatal hearts. Here, we highlight multiple applications of LSFM and discuss several studies that have allowed better characterization of developmental and pathological processes in multiple models and tissues. These findings demonstrate the capacity of multiscale light-sheet imaging to uncover cardiovascular developmental and regenerative phenomena.

The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes.

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.

In vivo manipulation of the extracellular matrix induces vascular regression in a basal chordate.

We investigated the physical role of the extracellular matrix (ECM) in vascular homeostasis in the basal chordate Botryllus schlosseri, which has a large, transparent, extracorporeal vascular network encompassing an area >100 cm2 We found that the collagen cross-linking enzyme lysyl oxidase is expressed in all vascular cells and that in vivo inhibition using ß-aminopropionitrile (BAPN) caused a rapid, global regression of the entire network, with some vessels regressing >10 mm within 16 h. BAPN treatment changed the ultrastructure of collagen fibers in the vessel basement membrane, and the kinetics of regression were dose dependent. Pharmacological inhibition of both focal adhesion kinase (FAK) and Raf also induced regression, and levels of phosphorylated FAK in vascular cells decreased during BAPN treatment and FAK inhibition but not Raf inhibition, suggesting that physical changes in the vessel ECM are detected via canonical integrin signaling pathways. Regression is driven by apoptosis and extrusion of cells through the basal lamina, which are then engulfed by blood-borne phagocytes. Extrusion and regression occurred in a coordinated manner that maintained vessel integrity, with no loss of barrier function. This suggests the presence of regulatory mechanisms linking physical changes to a homeostatic, tissue-level response.

Tbx20 drives cardiac progenitor formation and cardiomyocyte proliferation in zebrafish.

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis.

Transcriptional Regulation of Heart Development in Zebrafish.

Cardiac transcription factors orchestrate the complex cellular and molecular events required to produce a functioning heart. Misregulation of the cardiac transcription program leads to embryonic developmental defects and is associated with human congenital heart diseases. Recent studies have expanded our understanding of the regulation of cardiac gene expression at an additional layer, involving the coordination of epigenetic and transcriptional regulators. In this review, we highlight and discuss discoveries made possible by the genetic and embryological tools available in the zebrafish model organism, with a focus on the novel functions of cardiac transcription factors and epigenetic and transcriptional regulatory proteins during cardiogenesis.

Temporally and spatially dynamic germ cell niches in Botryllus schlosseri revealed by expression of a TGF-beta family ligand and vasa.

BACKGROUND: Germ cells are specified during early development and are responsible for generating gametes in the adult. After germ cells are specified, they typically migrate to a particular niche in the organism where they reside for the remainder of its lifetime. For some model organisms, the specification and migration of germ cells have been extensively studied, but how these events occur in animals that reproduce both sexually and asexually is not well understood. RESULTS: We have identified a novel TGF-ß family member in Botryllus schlosseri, tgfß-f, and found that it is expressed by follicle cell progenitors and the differentiated follicle and support cells surrounding the maturing gametes. Using the expression of tgfß-f and the germ cell marker vasa, we have found that nearly all germ cells in Botryllus are associated with tgfß-f-expressing follicle progenitors in clusters consisting solely of those two cell types. These clusters were mostly small, consisting of ten or fewer cells, and generally contained between a 2:1 and 1:1 ratio of follicle progenitors to germ cells. Clusters of germ and follicle progenitor cells were primarily localized to niches in the primary and secondary buds, but could also be found in other locations including the vasculature. We analyzed the location of germ cell clusters throughout the asexual life cycle of Botryllus and found that at the stage when germ cells are first detected in the secondary bud niche, a dramatic change in the size and location of germ/follicle cell clusters also occurred. CONCLUSIONS: Our findings suggest that germ/follicle cell clusters have predictable migratory patterns during the weekly asexual developmental cycle in Botryllus. An increased number of small clusters and the presence of clusters in the vasculature coinciding with the appearance of clusters in the secondary bud suggest that fragmentation of clusters and the migration of smaller clusters through the vasculature may be an important aspect of Botryllus reproductive biology, ensuring the transmission of the germline to subsequent asexual generations.

Migration of germline progenitor cells is directed by sphingosine-1-phosphate signalling in a basal chordate.

The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. The germ line of the newly developing bodies is derived from migrating germ cell precursors, but the signals governing this homing process are unknown. Here we show that germ cell precursors can be prospectively isolated based on expression of aldehyde dehydrogenase and integrin alpha-6, and that these cells express germ cell markers such as vasa, pumilio and piwi, as well as sphingosine-1-phosphate receptor. In vitro, sphingosine-1-phosphate (S1P) stimulates migration of germ cells, which depends on integrin alpha-6 activity. In vivo, S1P signalling is essential for homing of germ cells to newly developing bodies. S1P is generated by sphingosine kinase in the developing germ cell niche and degraded by lipid phosphate phosphatase in somatic tissues. These results demonstrate a previously unknown role of the S1P signalling pathway in germ cell migration in the ascidian Botryllus schlosseri.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor.

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Aging in the colonial chordate, Botryllus schlosseri.

What mechanisms underlie aging? One theory, the wear-and-tear model, attributes aging to progressive deterioration in the molecular and cellular machinery which eventually lead to death through the disruption of physiological homeostasis. The second suggests that life span is genetically programmed, and aging may be derived from intrinsic processes which enforce a non-random, terminal time interval for the survivability of the organism. We are studying an organism that demonstrates both properties: the colonial ascidian, Botryllus schlosseri. Botryllus is a member of the Tunicata, the sister group to the vertebrates, and has a number of life history traits which make it an excellent model for studies on aging. First, Botryllus has a colonial life history, and grows by a process of asexual reproduction during which entire bodies, including all somatic and germline lineages, regenerate every week, resulting in a colony of genetically identical individuals. Second, previous studies of lifespan in genetically distinct Botryllus lineages suggest that a direct, heritable basis underlying mortality exists that is unlinked to reproductive effort and other life history traits. Here we will review recent efforts to take advantage of the unique life history traits of B. schlosseri and develop it into a robust model for aging research.

Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments.

Analysis of the basal chordate Botryllus schlosseri reveals a set of genes associated with fertility.

BACKGROUND: Gonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively. RESULTS: Here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads. CONCLUSION: We have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.