BACKGROUND
botryllus schlosseri is a colonial ascidian found in shallow subtidal marine habitats around the world.
ascidians are urochordates; the sister group of vertebrates .
ascidian embryogenesis results in a chordate tadpole larva  that hatches and undergoes a short free-swimming phase.
larvae disperse, find a suitable substrate, then settle and metamorphose  into the adult form  that is now sessile.
the oozooid has a complex body plan, including a gastrointestinal tract , central and peripheral nervous system, an endocrine system, as well as a complex hematopoietic system and extracorporeal vasculature.figure  <dig>
botryllus schlosseri
sexual and asexual development  and anatomy.
a) sexual cycle showing the larva stage and metamorphosis into oozoid.
b) asexual cycle – blastogenesis: dorsal views of zooids , primary buds  and secondary buds .
a secondary bud appears as a thickening of the epidermis and the peribranchial chamber leaflet of the primary bud , which evaginates into a closed vesicle , followed by organogenesis .
gonadogenesis occurs in the secondary bud from mobile precursors .
during takeover , the secondary bud becomes the primary bud and a new blastogenic cycle begins for the next secondary bud.
after the second takeover event, the primary bud opens its siphons and becomes a functional adult .
in fertile colonies , the hermaphrodite gonad fully matures on both sides of the zooid.
c) schematic showing the anatomy of an adult fertile zooid with two primary buds.
d and e) ventral live image of both infertile and a fertile colonies .
arrows point to testes and arrowheads point to eggs.
panels a and b are modified from brown et al.
<dig>  laird et al.
<dig> and laird and de tomaso  <dig>
b. schlosseri belongs to a subset of ascidians that are colonial, and grow not by increasing in size, but by a lifelong, recurring asexual budding process during which entire bodies are regenerated de novo every week, resulting in an expanding colony of genetically identical individuals, called blastozooids .
zooids have the same body plan as the oozooid, and arrange themselves into star-shaped structures called systems .
a single tadpole larva can give rise to a colony consisting of thousands of systems with tens of thousands of zooids.
the zooids are connected by a common, extracorporeal vasculature that ramifies throughout the colony.
however, while linked by a common vasculature, the zooids and buds are independent, and zooids or systems can be separated from the colony and continue to grow .
the ability to collect pieces of the same genotype at different time points is a powerful characteristic of b. schlosseri as an experimental model for these studies  <cit> .
while the entire colony can have a lifespan ranging from nine months to several years, the zooids themselves are transient.
after completing a two-week developmental program, zooids have a defined lifespan of one week as an adult.
during that week, new zooids are regenerating in a process called blastogenesis.
this process is coordinated throughout the colony and arranged spatially such that a colony consists of three co-existing asexual generations within each system  .
the center of the colony is occupied by the zooids, which are actively feeding and, when fertile, sexually reproducing.
they are joined peripherally by primary buds, which are completing development.
in turn, these are connected to secondary buds, which are in the initial stages of development.
neither primary nor secondary buds feed or sexually reproduce.
the zooid has a lifespan of one week, then dies in a process called takeover.
during takeover, all zooids undergo simultaneous apoptosis and are removed by phagocytic cells in the blood.
the primary bud then migrates into the newly vacated region of the colony, opens its siphons and becomes a zooid, the secondary bud becomes the primary bud, and a new secondary bud begins developing from a region of the primary bud called the peribranchial epithelium .
somatic regeneration
blastogenesis is synchronized throughout a colony and takes 14 days under laboratory conditions.
this process can be divided into distinct visual stages, each lasting one day and demarcated by major morphogenetic changes .
as outlined in figure  <dig> , a new generation starts as a secondary bud, first visible as a thickening of the peribranchial epithelium of a primary bud , which evaginates and forms a closed vesicle .
next, a series of epithelial invaginations and protrusions appear as development proceeds .
after 7 days , a takeover event occurs, the secondary bud transitions to a primary bud, and over the next 7 days completes organogenesis, including initiating new secondary bud.
at day  <dig>  the zooid of the preceding generation undergoes takeover and is removed, the bud migrates to the new zooid position, and opens its siphons.
germline development
ascidians are hermaphrodites, and adult animals can develop both testes and eggs.
following metamorphosis, colonies undergo an average of eight to twelve developmental cycles prior to the development of gametes   <cit> .
in addition, natural populations show seasonal fertility, and in the lab cycle in and out of reproductive  and non-reproductive  states .
in turn, sexuality is also plastic, and colonies can be either hermaphroditic, or male only  .
when a colony is fertile, development of the gametes is synchronized with somatic development .
germline stem cells  seed the secondary bud  and begin to proliferate.
however, development of oocytes and testis are not linked.
testes develop in situ, and are visible in the secondary bud at stage b <dig>  and completing development approximately 10 days later, when that secondary bud becomes a zooid .
in contrast, oocytes appear to take several cycles to mature , and oocytes at different stages of development  will seed the secondary bud along with the gscs.
egg development
b. schlosseri is ovoviviparous and mature eggs are about 300 μm in diameter, and are surrounded by a series of cell layers.
these include a layer of outer follicle cells  a layer of inner follicle cells  and test cells   <cit> .
each egg resembles a small ovary consisting of an oocyte, the envelopes  and its own oviduct.
oocyte precursors first appear in secondary buds, ripen in primary buds, and ovulate when the primary buds are transitioning to mature zooids during takeover .
oocyte development takes place in the primary bud from stages a <dig> to c <dig> and can be described as  <dig> stages: at stage  <dig>  oocyte precursors are 10 μm in diameter, with a high nuclear to cytoplasmic ratio.
at stage  <dig>  the oocyte diameter increases up to 50 μm.
at stage  <dig> oocyte diameter is approximately 80 μ. at stage  <dig>  the oocyte diameter is 120 μm, and vitellogenesis, the process of secretion of nutrients into the oocyte by follicle cells, is initiated.
the diameter of stage  <dig> oocytes is 220 μm, vitellogenesis is almost completed, with yolk granules becoming larger.
at the end of the blastogenic cycle one to four oocytes mature and are ovulated into the peribranchial space during takeover .
usually a number  of oocytes remain arrested at stage  <dig>  which can move from the zooid into the blood stream and reach a new primary bud where they can potentially mature in successive blastogenic generations  <cit> .
to better understand the mechanisms of gonad differentiation and fertility, we used mrna-seq to identify genes that are differentially expressed between fertile and infertile colonies.
using differential expression analysis combined with in situ hybridization, we have identified a number of genes associated with fertility in botryllus, particularly genes expressed by follicle cells - surrounding both testis and oocyte precursors - that may drive maturation.
this study is the first step to comprehensively characterize the mechanisms of gonad maturation and fertility in botryllus schlosseri.
RESULTS
the recent publication of a draft genome sequence for botryllus schlosseri has identified homologs of vertebrate genes known for their function in lymphoid-mediated immunity as well as eye, ear and heart function  <cit> .
although this is an important step towards establishing botryllus as a model organism, an analysis of gene expression dynamics throughout the blastogenic cycle is an important tool to further strengthen this non-conventional model system.
one of the limitations of a newly established model organism is that we can infer the function of most proteins in botryllus only based on comparison to homologs in other species or analysis of conserved protein domains.
to study spatial and temporal gene expression in botryllus, we combined transcriptomics, quantitative rt-pcr and in situ hybridization.
to explore the genes involved in gonad formation and fertility, mrna seq analysis was performed at each stage of the blastogenic cycle  on a total of  <dig> fertile genotypes and  <dig> infertile genotypes .
after quality control analysis , the sequences were mapped to our publicly available botryllus schlosseri est database bot_asmb assembly   .
in order to identify putative homologs of the ests in our database, we performed a translated blast  analysis using the non-redundant human protein database  as well as non-redundant protein databases for mus musculus, drosophila melanogaster, ciona intestinalis and danio rerio .
contigs that are differentially expressed  between infertile and fertile animals were detected by deseq with a false discovery rate  of 10%   <cit> .
table  <dig> shows the number of de contigs at each stage of the blastogenic cycle at a 10% fdr .table  <dig>
number of differentially expressed est’s  at each stage of the blastogenic cycle and number of those est’s with human homologs
many of the homologs of these contigs have been previously demonstrated to play a role in germline development, fertility and/or fertilization in humans and other organisms .
in botryllus, conserved genes required for embryonic germ cell specification, such as vasa, piwi and nanos, are expressed in primitive germ cells that are present in juveniles as well as in both fertile and infertile adult animals .
therefore, genes that are upregulated in fertile animals in our analysis are likely to be involved in gonad formation/maturation and germ cell differentiation.table  <dig>
differentially expressed genes upregulated in fertile versus infertile colonies and their human and/or ascidian homologs
a <dig> and b1*
a <dig> and b1*
c2*
b <dig>  b <dig> and c2*
a <dig> and b1*
a <dig> and b1*
a <dig>  b1*
a <dig> and b1*
a2*
a <dig> and b1*
a1*
table  <dig> includes human and/or ascidian homologs that are differentially expressed  throughout the blastogenic cycle.
basemean infertile = mean normalized counts from infertile, basemean fertile = mean normalized counts from fertile.
foldchange = fold change from infertile to fertile, pval = p value for statistical significance; padj = p value adjusted for multiple testing at 10% false discovery rate; differentially expressed  genes at stage of the blastogenic cycle bold and  show the reported basemean infertile and fertile pval, padj for that particular contig at that particular stage.
the number of differentially expressed genes varied greatly between stages of the blastogenic cycle, from a minimum of  <dig> at stage c <dig> to a maximum of  <dig> at stage b <dig> .
in botryllus spermatogenesis begins at stage a <dig> in the testis of the zooid and continues until stage b <dig> when mature sperm is released through the oral siphon out into the water  <cit> .
consistent with this timing, we found that based on homology, a significant portion of the genes upregulated in stages a <dig> and a <dig> are functionally related to sperm formation .
since most of these homologs are involved in spermatogenesis in vertebrates , these data suggest some degree of conservation of the mechanisms governing spermatogenesis between ascidians and vertebrates.
to investigate the spatial and temporal expression pattern of de genes, we performed quantitative rt-pcr  and fluorescent in situ hybridization  for genes that were selected from the most highly de genes with human homologs: p-selectin , otoancorin , tetraspanin- <dig> , low-density lipoprotein receptor , testis-specific serine/threonine kinase  <dig>  and  <dig>  and genes with known roles in fertility in other organisms: estradiol  <dig> β dehydrogenase- <dig> , zona pellucida sperm binding protein  <dig> , vitellogenin- <dig> , tolloid-like- <dig> .
all of these genes were analyzed in fertile colonies at each stage of the blastogenic cycle by qpcr .
we found expression of these genes in: testes or developing testes ; localized to maturing eggs ; oocytes at stage  <dig> ; and other tissues  .figure  <dig>
fluorescent
in situ
hybridization  for selected differentially expressed genes on fertile colonies.
fish for selected genes with homologs in humans and other organisms was performed on whole mounts of adult fertile colonies.
nuclei were stained with dapi  and riboprobes are shown in green.
based on their expression patterns in developing gonads, these genes fall into four distinct categories: 1)  localized to testes or developing testes ; 2)  localized to maturing eggs ; 3)  oocytes at stage  <dig> ; and 4)  other tissues .
otoa = otoancorin, tspan8 = tetraspanin- <dig>  tsk1 = testis-specific serine/threonine-protein kinase <dig>  tsk2 = testis-specific serine/threonine-protein kinase <dig>  vtg1 = vitellogenin, ldlr = low-density lipoprotein receptor  <dig>  zp1 = zona-pellucida sperm binding protein  <dig>  tll1 = tolloid-like protein <dig>  hsd17β8 = estradiol 17β dehydrogenase- <dig>  selp = p-selectin.
scale bar 50 μm.
testis-specific serine/threonine protein kinases phosphorylate myelin basic protein and histones in vitro <cit> .
the testicular germ cell-specific expression of the mouse homologs suggests that these genes play an important role at and after the meiotic phase of spermatogenesis  <cit> .
in botryllus, testis-specific serine/threonine protein kinases, tsk- <dig>  tsk- <dig> and tsk- <dig> are upregulated in fertile animals based on our de analysis .
qpcr analysis showed that both tsk- <dig> and tsk- <dig> are highly upregulated between stages a <dig> and b <dig>  and in situ hybridization indicates that the mrna is expressed on developing testes  consistent with a crucial role of these genes in spermatogenesis.
in mammals, otoancorin is specific to sensory epithelia of the inner ear but in humans it has also been classified as a testis-selective cancer/testis gene  <cit> .
based on our de analysis, the botryllus homolog of otoancorin is upregulated 54-fold in fertile animals at stage a <dig>  by in situ hybridization, otoancorin shows strong expression from stage a <dig> through b <dig> in the testes of the primary bud  , indicating that in botryllus, otoancorin might be involved in spermatogenesis.
by qpcr, its expression level is highest in a <dig>  and decreases as the blastogenic cycle continues.
interestingly, testes of the zooid also show signal for otoancorin mrna, but expression is restricted to the peripheral cells of the testes .
tetraspanin/cd9-like is an oocyte factor required for sperm-oocyte fusion  <cit> .
consistent with these findings in mammals, in situ hybridization shows expression of the botryllus homolog of tetraspanin- <dig> on stage  <dig> oocytes.
the adam-integrin-tetraspanin complex, known to constitute a network of membrane microdomains called the tetraspanin web, is potentially involved in the migration of prespermatogonia from the center to the periphery of the testicular cords and in the reinitiation of mitotic activity during the initial wave of spermatogenesis  <cit> .
by qpcr, expression of tetraspanin- <dig> in botryllus is highest at stage a <dig>  coinciding with the initiation of spermatogenesis.
furthermore, our de analysis revealed a 125-fold upregulation of tetraspanin- <dig> in fertile animals at stage a <dig>  in situ hybridization showed cells expressing tetraspanin- <dig> mrna in the testes of the primary bud, and suggests that the role of tetraspanin proteins in spermatogenesis is likely to be conserved between botryllus and vertebrates.
vitellogenins are lipophosphoglycoproteins that are produced under female hormonal control in large central organs  and are transported in the circulation to the female gonads  <cit> .
in botryllus, a vitellogenin homolog is 927-fold upregulated in fertile animals at stage b <dig>  two novel isoforms of vitellogenin, both of which possess vwf-d and ct domains but not a lipovitellin or phosvitin domain, are expressed in the gonad of the ascidian halocynthia roretzi <cit> .
in situ hybridization revealed that mrnas of these proteins are specifically expressed in oocytes and test cells, accessory cells in the perivitelline space of ascidian eggs.
immunohistochemistry showed that these proteins are localized around the surface of test cells in immature oocytes  <cit> .
in botryllus, vitellogenin expression is highest at stage b <dig> , and, consistent with the findings in halocynthia, in situ hybridization shows expression on maturing eggs of the primary bud .
interestingly, vitellogenin mrna is also expressed by follicle cells on the testes of both the primary bud and the zooid .
these cells are restricted to the periphery of the testes and seem to surround them, in contrast to the mrna’s of otoancorin, testis-specific serine/threonine-protein kinase  <dig> and tetraspanin- <dig> , which are expressed by cells in the inner compartment of the testes.
to date, all molecularly characterized vitellogenin receptors belong to the low-density lipoprotein receptor  supergene family.
ldlr receptors localized in coated pits on the surface of growth-competent oocytes are able to accumulate in the yolk high concentrations of vitellogenin and other ligands they recognize  <cit> .
a botryllus schlosseri homolog of ldlr is upregulated 199-fold in fertile animals at stage b <dig>  and qpcr analysis shows that expression of ldlr in botryllus is highest at stage a <dig> .
by in situ hybridization, we found that ldlr is expressed in follicle cells of maturing eggs in the primary bud , suggesting that it might function as a vitellogenin-receptor during oocyte maturation.
in halocynthia roretzi, a 120-kda transmembrane protein with a zona pellucida domain and a  <dig> egf-like repeats , has been shown to be the receptor in the vitelline coat that binds to sperm  <cit> .
ciona intestinalis has several genes related to hrvc <dig> that are exclusively expressed in developing oocytes but not in eggs  <cit> .
we found a gene containing a zona pellucida domain, zp- <dig>  that is highly upregulated in fertile animals  and highly expressed at stage a <dig> .
by in situ hybridization, we found that zp- <dig> is expressed by the follicle cells that surround the egg in the primary bud.
to our knowledge this is the first report of a gene with a zona pellucida domain in a colonial ascidian.
this finding reflects the close relationship of these animals with vertebrates and the fact that this domain has been maintained in deuterostomes suggests that it is indispensable for sperm-egg interaction  <cit> .
mammalian tolloid  is a proteinase involved in ovulation.
during ovarian tissue remodeling, it contributes to the maturation of procollagen molecules and the deposition of collagen fibrils  <cit> .
in botryllus, a tolloid-like protein  <dig> homolog is expressed on follicle cells of maturing oocytes of the primary bud and is upregulated 261-fold in fertile animals at stage a <dig> .
a botryllus schlosseri homolog of estradiol  <dig> β dehydrogenase- <dig>  is highly upregulated in fertile animals , and as expected, given its involvement in estrogen metabolism  <cit> , its mrna localizes to oocytes at stage  <dig> .
a botryllus schlosseri homolog of p-selectin  is upregulated 191-fold in fertile animals at stage b <dig>  by in situ hybridization, this gene is expressed on several stages of oocytes .
the function of this protein in germ cells is poorly understood, but it is expressed in the oolema of oocytes in hamsters and humans, as well as in sperm following the acrosomal reaction, and is hypothesized to be involved in sperm-oocyte adhesion .
we measured the diameter of the oocytes expressing p-selectin, and found that their size increased as the blastogenic cycle advanced .
cells positive for p-selectin mrna range from 16–21 μm in diameter at stage a <dig>  suggesting they are stage  <dig> oocytes as classified by manni .
stage  <dig> oocytes are defined as being greater than 10 μm in diameter  but less than 50 μm in diameter .
by the middle of the blastogenic cycle, p-selectin-positive cells can reach up to 39 μm .
by the end of blastogenesis, one or two p-selectin-positive cells per primary bud will be about 42 μm in diameter, and could indicate that these cells are committed to become mature oocytes .
this supports the concept that there is a persisting reservoir of egg-precursors, and only one or two of those cells will increase in size to move on to stage  <dig> and develop into the egg of the primary bud for the following blastogenic cycle .table  <dig>
measurements of
p-selectin
positive cells, mean and standard deviation  in the primary bud throughout the blastogenic cycle
unexpectedly, we found that stage  <dig> oocytes were positive for the mrna of testis-specific serine/threonine-protein kinase  <dig> gene at stage b <dig> and b <dig> of the blastogenic cycle.
moreover, a strong nucleolus like signal was observed in about 30-40% of positive cells.
this finding could indicate a previously unknown function of this gene in oogenesis.
finally, we observed that some of the genes upregulated in fertile animals are expressed in tissues outside of the germ line.
particularly p-selectin mrna is expressed on the oral and excurrent siphons of both the zooid and primary bud .
interestingly, at stage d of the blastogenic cycle, p-selectin positive cells are arranged in a calyx shape  of the primary bud as it transitions to a zooid during takeover.
a botryllus schlosseri homolog of zona pellucida binding protein- <dig> showed mrna expression on cells that run along the endostyle of both zooid and primary bud, while ldlr showed positive mrna expression on secondary bud tissue at stage b <dig> figure  <dig>
fluorescent
in situ
hybridization  for selected differentially expressed genes on infertile colonies.
fish for selected genes was performed on whole mounts of adult infertile colonies .
nuclei were stained with dapi  and riboprobes are shown in green.
expression of genes detected in fertile colonies is absent in juveniles except for p-selectin  as indicated by arrows  and zona-pellucida sperm binding protein <dig>  as indicated by the arrowhead .
otoa = otoancorin, tspan8 = tetraspanin- <dig>  tsk1 = testis-specific serine/threonine-protein kinase <dig>  tsk2 = testis-specific serine/threonine-protein kinase <dig>  vtg1 = vitellogenin, ldlr = low-density lipoprotein receptor  <dig>  zp1 = zona-pellucida sperm binding protein  <dig>  tll1 = tolloid-like protein <dig>  hsd17b8 = estradiol 17β dehydrogenase- <dig>  selp = p-selectin.
scale bar 200 μm.
the set of fertility-related genes we have identified represents the first reported molecular markers for a variety of botryllus schlosseri anatomical structures.
specifically, we found transcripts expressed by the testes , by follicle cells surrounding both maturing oocytes and testes, by oocytes at stage  <dig> and by the siphons .
these markers will undoubtedly serve as a valuable resource for other researchers examining fertility in botryllus.
to obtain a broader perspective and explore the biological roles of the genes identified in our de analysis, we performed a gene ontology  analysis on the human homologs of differentially expressed genes  at each stage of the blastogenic cycle  .
specifically, annotation based on biological processes provided significant insight into functional changes related to fertility throughout the blastogenic cycle, and are discussed here.
other go annotations  are included in additional file 14: table s <dig>  additional file 15: table s <dig>  additional file 16: table s <dig>  additional file 17: table s <dig>  additional file 18: table s <dig>  additional file 19: table s <dig>  additional file 20: table s <dig>  additional file 21: table s <dig>  additional file 23: table s <dig>  additional file 24: table s <dig>  additional file 25: table s <dig>  additional file 26: table s <dig>  additional file 27: table s <dig>  additional file 28: table s <dig>  additional file 29: table s <dig>  additional file 30: table s <dig>  additional file 31: table s <dig>  additional file 32: table s <dig>  additional file 33: table s <dig>  additional file 34: table s <dig>  additional file 35: table s <dig>  additional file 36: table s <dig>  additional file 37: table s <dig>  additional file 38: table s <dig>  additional file 39: table s <dig>  additional file 40: table s <dig> and additional file 41: table s <dig>  furthermore, we conducted inter pro analysis to find conserved protein domains .
at stages a <dig> and a <dig>   <dig>  and  <dig>  genes are upregulated in fertile animals, respectively.
go analysis revealed that genes related to male meiosis, spermatogenesis, cilium, proteolysis, microtubule based movement, meiosis, spermatid development, cilliary or flagellar motility, female gamete generation and male meiosis are upregulated , correlating with the fact that spermatogenesis begins in this timeframe.
inter pro analysis revealed that these genes contain domains such as egf, ldlr, bmp, selectin and meiotic recombination spo <dig>  among others .
these results are congruent with previous non-molecular observations indicating that gametogenesis starts during these stages.
at stages b <dig> and b <dig>   <dig> and  <dig> genes are de with  <dig> and  <dig> human homologs respectively.
genes related to male meiosis, spermatogenesis, spermatid development, microtubule-based movement, cell differentiation, motility, filipodium assembly, flagellar motility, cell adhesion, female gamete generation and apoliprotein binding are upregulated in fertile animals .
inter pro analysis revealed that these genes contain domains such as egf, kringle, cub, speract/scavenger kelch, calcium binding, adam, and peptidase s1/s6/m12a, bmp1/tolloid, zona pellucida sperm-binding protein and ldlr among others .
these results reflect the complex biological processes occurring during these stages, which also have the highest number of de genes, and are consistent with the timing of sperm maturation and release.
at stage c <dig> and c <dig>   <dig> and  <dig> genes are de with  <dig> and  <dig> human homologs respectively.
the predominant go terms at these stages included oocyte maturation, oocyte growth, lipoprotein metabolic process, regulation of cholesterol transport, positive regulation of estrogen receptor, extracellular matrix disassembly, and tissue remodeling, among others .
the inter pro analysis for this stage revealed domains such as: apple-like, ldlr, kringe, aquaporin, tetraspanin, egf, zona pellucida sperm-binding protein, peptidase s1/s6/m12a and fy rich, among others .
at these stages maturing oocytes rapidly increase in size and are preparing to move into the branchial basket of the zooid.
finally at stage d  <dig> genes are de .
go analysis revealed the presence of annotations including cell adhesion, carbohydrate metabolism, regulation of developmental process and response to corticosteroids stimulus, proteolysis, lipoprotein transport, cholesterol transport, estrogen receptor signaling pathway, oocyte growth  and the inter pro analysis of the genes upregulated at this stage revealed the following protein domains: egf, notch, cub, ldlr, selectin, and peptidases s1/s1a/s <dig>  among others .
at this stage mature oocytes begin to transition from the peribranchial cavity into the branchial basket of the new zooid.
in summary, our go analysis has revealed a number of genes with potential involvement in spermatogenesis at stages a1-b <dig>  correlating with what has been described for botryllus schlosseri <cit> .
the botryllus homologs of these genes are promising candidates for future studies on testes maturation and sperm production.
homologs of genes with reported roles in lipid and steroid metabolism are upregulated at stages b1-d, representing key processes involved in oocyte maturation in other species that may be conserved in botryllus.
CONCLUSIONS
this manuscript represents the first step in the comprehensive characterization of gonad differentiation and fertility in botryllus schlosseri.
we have analyzed the transcriptomes of both infertile and fertile colonies and characterized the spatial and temporal expression of highly expressed genes involved in fertility.
our findings suggest a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.
some of the genes characterized in this study were found to have unexpected spatial localization, such as testis serine/threonine kinase  <dig>  which is expressed by egg precursors.
another example is vitellogenin, which localizes to both follicle cells surrounding the egg and equivalent cells surrounding the testes.
interestingly, known germline markers such as vasa, nanos and piwi were not significantly differentially expressed between fertile and infertile colonies, most likely because both juveniles and infertile colonies have immature germ cells expressing these genes.
we have identified specific markers for both developing gonads  throughout the blastogenic cycle in zooids, primary buds and secondary buds.
some of these markers are now being used to investigate the phenomenon of induced juvenile fertility in botryllus schlosseri .
in future studies, we will investigate differential gene expression between male and hermaphroditic colonies, which will allow us to characterize genes used exclusively in male or female gamete development and identify genes governing the process by which botryllus schlosseri assumes a given reproductive state.

 botryllus schlosseri is a colonial ascidian found in shallow subtidal marine habitats around the world. ascidians are urochordates; the sister group of vertebrates. ascidian embryogenesis results in a chordate tadpole larva that hatches and undergoes a short free-swimming phase. larvae disperse, find a suitable substrate, then settle and metamorphose into the adult form that is now sessile. the oozooid has a complex body plan, including a gastrointestinal tract, central and peripheral nervous system, an endocrine system, as well as a complex hematopoietic system and extracorporeal vasculature.figure botryllus schlosseri sexual and asexual development and anatomy. a) sexual cycle showing the larva stage and metamorphosis into oozoid. b) asexual cycle – blastogenesis: dorsal views of zooids, primary buds and secondary buds. a secondary bud appears as a thickening of the epidermis and the peribranchial chamber leaflet of the primary bud, which evaginates into a closed vesicle, followed by organogenesis. gonadogenesis occurs in the secondary bud from mobile precursors. during takeover, the secondary bud becomes the primary bud and a new blastogenic cycle begins for the next secondary bud. after the second takeover event, the primary bud opens its siphons and becomes a functional adult. in fertile colonies, the hermaphrodite gonad fully matures on both sides of the zooid. c) schematic showing the anatomy of an adult fertile zooid with two primary buds. d and e) ventral live image of both infertile and a fertile colonies. arrows point to testes and arrowheads point to eggs. panels a and b are modified from brown et al. laird et al. and laird and de tomaso b. schlosseri belongs to a subset of ascidians that are colonial, and grow not by increasing in size, but by a lifelong, recurring asexual budding process during which entire bodies are regenerated de novo every week, resulting in an expanding colony of genetically identical individuals, called blastozooids. zooids have the same body plan as the oozooid, and arrange themselves into star-shaped structures called systems. a single tadpole larva can give rise to a colony consisting of thousands of systems with tens of thousands of zooids. the zooids are connected by a common, extracorporeal vasculature that ramifies throughout the colony. however, while linked by a common vasculature, the zooids and buds are independent, and zooids or systems can be separated from the colony and continue to grow. the ability to collect pieces of the same genotype at different time points is a powerful characteristic of b. schlosseri as an experimental model for these studies. while the entire colony can have a lifespan ranging from nine months to several years, the zooids themselves are transient. after completing a two-week developmental program, zooids have a defined lifespan of one week as an adult. during that week, new zooids are regenerating in a process called blastogenesis. this process is coordinated throughout the colony and arranged spatially such that a colony consists of three co-existing asexual generations within each system. the center of the colony is occupied by the zooids, which are actively feeding and, when fertile, sexually reproducing. they are joined peripherally by primary buds, which are completing development. in turn, these are connected to secondary buds, which are in the initial stages of development. neither primary nor secondary buds feed or sexually reproduce. the zooid has a lifespan of one week, then dies in a process called takeover. during takeover, all zooids undergo simultaneous apoptosis and are removed by phagocytic cells in the blood. the primary bud then migrates into the newly vacated region of the colony, opens its siphons and becomes a zooid, the secondary bud becomes the primary bud, and a new secondary bud begins developing from a region of the primary bud called the peribranchial epithelium. somatic regeneration blastogenesis is synchronized throughout a colony and takes 14 days under laboratory conditions. this process can be divided into distinct visual stages, each lasting one day and demarcated by major morphogenetic changes. as outlined in figure , a new generation starts as a secondary bud, first visible as a thickening of the peribranchial epithelium of a primary bud, which evaginates and forms a closed vesicle. next, a series of epithelial invaginations and protrusions appear as development proceeds. after 7 days, a takeover event occurs, the secondary bud transitions to a primary bud, and over the next 7 days completes organogenesis, including initiating new secondary bud. at day the zooid of the preceding generation undergoes takeover and is removed, the bud migrates to the new zooid position, and opens its siphons. germline development ascidians are hermaphrodites, and adult animals can develop both testes and eggs. following metamorphosis, colonies undergo an average of eight to twelve developmental cycles prior to the development of gametes. in addition, natural populations show seasonal fertility, and in the lab cycle in and out of reproductive and non-reproductive states. in turn, sexuality is also plastic, and colonies can be either hermaphroditic, or male only. when a colony is fertile, development of the gametes is synchronized with somatic development. germline stem cells seed the secondary bud and begin to proliferate. however, development of oocytes and testis are not linked. testes develop in situ, and are visible in the secondary bud at stage b and completing development approximately 10 days later, when that secondary bud becomes a zooid. in contrast, oocytes appear to take several cycles to mature, and oocytes at different stages of development will seed the secondary bud along with the gscs. egg development b. schlosseri is ovoviviparous and mature eggs are about 300 μm in diameter, and are surrounded by a series of cell layers. these include a layer of outer follicle cells a layer of inner follicle cells and test cells. each egg resembles a small ovary consisting of an oocyte, the envelopes and its own oviduct. oocyte precursors first appear in secondary buds, ripen in primary buds, and ovulate when the primary buds are transitioning to mature zooids during takeover. oocyte development takes place in the primary bud from stages a to c and can be described as stages: at stage oocyte precursors are 10 μm in diameter, with a high nuclear to cytoplasmic ratio. at stage the oocyte diameter increases up to 50 μm. at stage oocyte diameter is approximately 80 μ. at stage the oocyte diameter is 120 μm, and vitellogenesis, the process of secretion of nutrients into the oocyte by follicle cells, is initiated. the diameter of stage oocytes is 220 μm, vitellogenesis is almost completed, with yolk granules becoming larger. at the end of the blastogenic cycle one to four oocytes mature and are ovulated into the peribranchial space during takeover. usually a number of oocytes remain arrested at stage which can move from the zooid into the blood stream and reach a new primary bud where they can potentially mature in successive blastogenic generations. to better understand the mechanisms of gonad differentiation and fertility, we used mrna-seq to identify genes that are differentially expressed between fertile and infertile colonies. using differential expression analysis combined with in situ hybridization, we have identified a number of genes associated with fertility in botryllus, particularly genes expressed by follicle cells - surrounding both testis and oocyte precursors - that may drive maturation. this study is the first step to comprehensively characterize the mechanisms of gonad maturation and fertility in botryllus schlosseri.  the recent publication of a draft genome sequence for botryllus schlosseri has identified homologs of vertebrate genes known for their function in lymphoid-mediated immunity as well as eye, ear and heart function. although this is an important step towards establishing botryllus as a model organism, an analysis of gene expression dynamics throughout the blastogenic cycle is an important tool to further strengthen this non-conventional model system. one of the limitations of a newly established model organism is that we can infer the function of most proteins in botryllus only based on comparison to homologs in other species or analysis of conserved protein domains. to study spatial and temporal gene expression in botryllus, we combined transcriptomics, quantitative rt-pcr and in situ hybridization. to explore the genes involved in gonad formation and fertility, mrna seq analysis was performed at each stage of the blastogenic cycle on a total of fertile genotypes and infertile genotypes. after quality control analysis, the sequences were mapped to our publicly available botryllus schlosseri est database bot_asmb assembly. in order to identify putative homologs of the ests in our database, we performed a translated blast analysis using the non-redundant human protein database as well as non-redundant protein databases for mus musculus, drosophila melanogaster, ciona intestinalis and danio rerio. contigs that are differentially expressed between infertile and fertile animals were detected by deseq with a false discovery rate of 10%. table  shows the number of de contigs at each stage of the blastogenic cycle at a 10% fdr.table number of differentially expressed est’s at each stage of the blastogenic cycle and number of those est’s with human homologs many of the homologs of these contigs have been previously demonstrated to play a role in germline development, fertility and/or fertilization in humans and other organisms. in botryllus, conserved genes required for embryonic germ cell specification, such as vasa, piwi and nanos, are expressed in primitive germ cells that are present in juveniles as well as in both fertile and infertile adult animals. therefore, genes that are upregulated in fertile animals in our analysis are likely to be involved in gonad formation/maturation and germ cell differentiation.table differentially expressed genes upregulated in fertile versus infertile colonies and their human and/or ascidian homologs a and b1* a and b1* c2* b b and c2* a and b1* a and b1* a b1* a and b1* a2* a and b1* a1* table  includes human and/or ascidian homologs that are differentially expressed throughout the blastogenic cycle. basemean infertile = mean normalized counts from infertile, basemean fertile = mean normalized counts from fertile. foldchange = fold change from infertile to fertile, pval = p value for statistical significance; padj = p value adjusted for multiple testing at 10% false discovery rate; differentially expressed genes at stage of the blastogenic cycle bold and show the reported basemean infertile and fertile pval, padj for that particular contig at that particular stage. the number of differentially expressed genes varied greatly between stages of the blastogenic cycle, from a minimum of at stage c to a maximum of at stage b. in botryllus spermatogenesis begins at stage a in the testis of the zooid and continues until stage b when mature sperm is released through the oral siphon out into the water. consistent with this timing, we found that based on homology, a significant portion of the genes upregulated in stages a and a are functionally related to sperm formation. since most of these homologs are involved in spermatogenesis in vertebrates, these data suggest some degree of conservation of the mechanisms governing spermatogenesis between ascidians and vertebrates. to investigate the spatial and temporal expression pattern of de genes, we performed quantitative rt-pcr and fluorescent in situ hybridization for genes that were selected from the most highly de genes with human homologs: p-selectin, otoancorin, tetraspanin-, low-density lipoprotein receptor, testis-specific serine/threonine kinase and and genes with known roles in fertility in other organisms: estradiol β dehydrogenase-, zona pellucida sperm binding protein, vitellogenin-, tolloid-like-. all of these genes were analyzed in fertile colonies at each stage of the blastogenic cycle by qpcr. we found expression of these genes in: testes or developing testes; localized to maturing eggs; oocytes at stage; and other tissues.figure fluorescent in situ hybridization for selected differentially expressed genes on fertile colonies. fish for selected genes with homologs in humans and other organisms was performed on whole mounts of adult fertile colonies. nuclei were stained with dapi and riboprobes are shown in green. based on their expression patterns in developing gonads, these genes fall into four distinct categories: 1) localized to testes or developing testes; 2) localized to maturing eggs; 3) oocytes at stage; and 4) other tissues. otoa = otoancorin, tspan8 = tetraspanin- tsk1 = testis-specific serine/threonine-protein kinase tsk2 = testis-specific serine/threonine-protein kinase vtg1 = vitellogenin, ldlr = low-density lipoprotein receptor zp1 = zona-pellucida sperm binding protein tll1 = tolloid-like protein hsd17β8 = estradiol 17β dehydrogenase- selp = p-selectin. scale bar 50 μm. testis-specific serine/threonine protein kinases phosphorylate myelin basic protein and histones in vitro. the testicular germ cell-specific expression of the mouse homologs suggests that these genes play an important role at and after the meiotic phase of spermatogenesis. in botryllus, testis-specific serine/threonine protein kinases, tsk- tsk- and tsk- are upregulated in fertile animals based on our de analysis. qpcr analysis showed that both tsk- and tsk- are highly upregulated between stages a and b and in situ hybridization indicates that the mrna is expressed on developing testes consistent with a crucial role of these genes in spermatogenesis. in mammals, otoancorin is specific to sensory epithelia of the inner ear but in humans it has also been classified as a testis-selective cancer/testis gene. based on our de analysis, the botryllus homolog of otoancorin is upregulated 54-fold in fertile animals at stage a by in situ hybridization, otoancorin shows strong expression from stage a through b in the testes of the primary bud, indicating that in botryllus, otoancorin might be involved in spermatogenesis. by qpcr, its expression level is highest in a and decreases as the blastogenic cycle continues. interestingly, testes of the zooid also show signal for otoancorin mrna, but expression is restricted to the peripheral cells of the testes. tetraspanin/cd9-like is an oocyte factor required for sperm-oocyte fusion. consistent with these findings in mammals, in situ hybridization shows expression of the botryllus homolog of tetraspanin- on stage oocytes. the adam-integrin-tetraspanin complex, known to constitute a network of membrane microdomains called the tetraspanin web, is potentially involved in the migration of prespermatogonia from the center to the periphery of the testicular cords and in the reinitiation of mitotic activity during the initial wave of spermatogenesis. by qpcr, expression of tetraspanin- in botryllus is highest at stage a coinciding with the initiation of spermatogenesis. furthermore, our de analysis revealed a 125-fold upregulation of tetraspanin- in fertile animals at stage a in situ hybridization showed cells expressing tetraspanin- mrna in the testes of the primary bud, and suggests that the role of tetraspanin proteins in spermatogenesis is likely to be conserved between botryllus and vertebrates. vitellogenins are lipophosphoglycoproteins that are produced under female hormonal control in large central organs and are transported in the circulation to the female gonads. in botryllus, a vitellogenin homolog is 927-fold upregulated in fertile animals at stage b two novel isoforms of vitellogenin, both of which possess vwf-d and ct domains but not a lipovitellin or phosvitin domain, are expressed in the gonad of the ascidian halocynthia roretzi. in situ hybridization revealed that mrnas of these proteins are specifically expressed in oocytes and test cells, accessory cells in the perivitelline space of ascidian eggs. immunohistochemistry showed that these proteins are localized around the surface of test cells in immature oocytes. in botryllus, vitellogenin expression is highest at stage b, and, consistent with the findings in halocynthia, in situ hybridization shows expression on maturing eggs of the primary bud. interestingly, vitellogenin mrna is also expressed by follicle cells on the testes of both the primary bud and the zooid. these cells are restricted to the periphery of the testes and seem to surround them, in contrast to the mrna’s of otoancorin, testis-specific serine/threonine-protein kinase and tetraspanin-, which are expressed by cells in the inner compartment of the testes. to date, all molecularly characterized vitellogenin receptors belong to the low-density lipoprotein receptor supergene family. ldlr receptors localized in coated pits on the surface of growth-competent oocytes are able to accumulate in the yolk high concentrations of vitellogenin and other ligands they recognize. a botryllus schlosseri homolog of ldlr is upregulated 199-fold in fertile animals at stage b and qpcr analysis shows that expression of ldlr in botryllus is highest at stage a. by in situ hybridization, we found that ldlr is expressed in follicle cells of maturing eggs in the primary bud, suggesting that it might function as a vitellogenin-receptor during oocyte maturation. in halocynthia roretzi, a 120-kda transmembrane protein with a zona pellucida domain and a egf-like repeats, has been shown to be the receptor in the vitelline coat that binds to sperm. ciona intestinalis has several genes related to hrvc that are exclusively expressed in developing oocytes but not in eggs. we found a gene containing a zona pellucida domain, zp- that is highly upregulated in fertile animals and highly expressed at stage a. by in situ hybridization, we found that zp- is expressed by the follicle cells that surround the egg in the primary bud. to our knowledge this is the first report of a gene with a zona pellucida domain in a colonial ascidian. this finding reflects the close relationship of these animals with vertebrates and the fact that this domain has been maintained in deuterostomes suggests that it is indispensable for sperm-egg interaction. mammalian tolloid is a proteinase involved in ovulation. during ovarian tissue remodeling, it contributes to the maturation of procollagen molecules and the deposition of collagen fibrils. in botryllus, a tolloid-like protein homolog is expressed on follicle cells of maturing oocytes of the primary bud and is upregulated 261-fold in fertile animals at stage a. a botryllus schlosseri homolog of estradiol β dehydrogenase- is highly upregulated in fertile animals, and as expected, given its involvement in estrogen metabolism, its mrna localizes to oocytes at stage. a botryllus schlosseri homolog of p-selectin is upregulated 191-fold in fertile animals at stage b by in situ hybridization, this gene is expressed on several stages of oocytes. the function of this protein in germ cells is poorly understood, but it is expressed in the oolema of oocytes in hamsters and humans, as well as in sperm following the acrosomal reaction, and is hypothesized to be involved in sperm-oocyte adhesion. we measured the diameter of the oocytes expressing p-selectin, and found that their size increased as the blastogenic cycle advanced. cells positive for p-selectin mrna range from 16–21 μm in diameter at stage a suggesting they are stage oocytes as classified by manni. stage oocytes are defined as being greater than 10 μm in diameter but less than 50 μm in diameter. by the middle of the blastogenic cycle, p-selectin-positive cells can reach up to 39 μm. by the end of blastogenesis, one or two p-selectin-positive cells per primary bud will be about 42 μm in diameter, and could indicate that these cells are committed to become mature oocytes. this supports the concept that there is a persisting reservoir of egg-precursors, and only one or two of those cells will increase in size to move on to stage and develop into the egg of the primary bud for the following blastogenic cycle.table measurements of p-selectin positive cells, mean and standard deviation in the primary bud throughout the blastogenic cycle unexpectedly, we found that stage oocytes were positive for the mrna of testis-specific serine/threonine-protein kinase gene at stage b and b of the blastogenic cycle. moreover, a strong nucleolus like signal was observed in about 30-40% of positive cells. this finding could indicate a previously unknown function of this gene in oogenesis. finally, we observed that some of the genes upregulated in fertile animals are expressed in tissues outside of the germ line. particularly p-selectin mrna is expressed on the oral and excurrent siphons of both the zooid and primary bud. interestingly, at stage d of the blastogenic cycle, p-selectin positive cells are arranged in a calyx shape of the primary bud as it transitions to a zooid during takeover. a botryllus schlosseri homolog of zona pellucida binding protein- showed mrna expression on cells that run along the endostyle of both zooid and primary bud, while ldlr showed positive mrna expression on secondary bud tissue at stage b figure fluorescent in situ hybridization for selected differentially expressed genes on infertile colonies. fish for selected genes was performed on whole mounts of adult infertile colonies. nuclei were stained with dapi and riboprobes are shown in green. expression of genes detected in fertile colonies is absent in juveniles except for p-selectin as indicated by arrows and zona-pellucida sperm binding protein as indicated by the arrowhead. otoa = otoancorin, tspan8 = tetraspanin- tsk1 = testis-specific serine/threonine-protein kinase tsk2 = testis-specific serine/threonine-protein kinase vtg1 = vitellogenin, ldlr = low-density lipoprotein receptor zp1 = zona-pellucida sperm binding protein tll1 = tolloid-like protein hsd17b8 = estradiol 17β dehydrogenase- selp = p-selectin. scale bar 200 μm. the set of fertility-related genes we have identified represents the first reported molecular markers for a variety of botryllus schlosseri anatomical structures. specifically, we found transcripts expressed by the testes, by follicle cells surrounding both maturing oocytes and testes, by oocytes at stage and by the siphons. these markers will undoubtedly serve as a valuable resource for other researchers examining fertility in botryllus. to obtain a broader perspective and explore the biological roles of the genes identified in our de analysis, we performed a gene ontology analysis on the human homologs of differentially expressed genes at each stage of the blastogenic cycle. specifically, annotation based on biological processes provided significant insight into functional changes related to fertility throughout the blastogenic cycle, and are discussed here. other go annotations are included in additional file 14: table s additional file 15: table s additional file 16: table s additional file 17: table s additional file 18: table s additional file 19: table s additional file 20: table s additional file 21: table s additional file 23: table s additional file 24: table s additional file 25: table s additional file 26: table s additional file 27: table s additional file 28: table s additional file 29: table s additional file 30: table s additional file 31: table s additional file 32: table s additional file 33: table s additional file 34: table s additional file 35: table s additional file 36: table s additional file 37: table s additional file 38: table s additional file 39: table s additional file 40: table s and additional file 41: table s furthermore, we conducted inter pro analysis to find conserved protein domains. at stages a and a and genes are upregulated in fertile animals, respectively. go analysis revealed that genes related to male meiosis, spermatogenesis, cilium, proteolysis, microtubule based movement, meiosis, spermatid development, cilliary or flagellar motility, female gamete generation and male meiosis are upregulated, correlating with the fact that spermatogenesis begins in this timeframe. inter pro analysis revealed that these genes contain domains such as egf, ldlr, bmp, selectin and meiotic recombination spo among others. these results are congruent with previous non-molecular observations indicating that gametogenesis starts during these stages. at stages b and b and genes are de with and human homologs respectively. genes related to male meiosis, spermatogenesis, spermatid development, microtubule-based movement, cell differentiation, motility, filipodium assembly, flagellar motility, cell adhesion, female gamete generation and apoliprotein binding are upregulated in fertile animals. inter pro analysis revealed that these genes contain domains such as egf, kringle, cub, speract/scavenger kelch, calcium binding, adam, and peptidase s1/s6/m12a, bmp1/tolloid, zona pellucida sperm-binding protein and ldlr among others. these results reflect the complex biological processes occurring during these stages, which also have the highest number of de genes, and are consistent with the timing of sperm maturation and release. at stage c and c and genes are de with and human homologs respectively. the predominant go terms at these stages included oocyte maturation, oocyte growth, lipoprotein metabolic process, regulation of cholesterol transport, positive regulation of estrogen receptor, extracellular matrix disassembly, and tissue remodeling, among others. the inter pro analysis for this stage revealed domains such as: apple-like, ldlr, kringe, aquaporin, tetraspanin, egf, zona pellucida sperm-binding protein, peptidase s1/s6/m12a and fy rich, among others. at these stages maturing oocytes rapidly increase in size and are preparing to move into the branchial basket of the zooid. finally at stage d genes are de. go analysis revealed the presence of annotations including cell adhesion, carbohydrate metabolism, regulation of developmental process and response to corticosteroids stimulus, proteolysis, lipoprotein transport, cholesterol transport, estrogen receptor signaling pathway, oocyte growth and the inter pro analysis of the genes upregulated at this stage revealed the following protein domains: egf, notch, cub, ldlr, selectin, and peptidases s1/s1a/s among others. at this stage mature oocytes begin to transition from the peribranchial cavity into the branchial basket of the new zooid. in summary, our go analysis has revealed a number of genes with potential involvement in spermatogenesis at stages a1-b correlating with what has been described for botryllus schlosseri. the botryllus homologs of these genes are promising candidates for future studies on testes maturation and sperm production. homologs of genes with reported roles in lipid and steroid metabolism are upregulated at stages b1-d, representing key processes involved in oocyte maturation in other species that may be conserved in botryllus.  this manuscript represents the first step in the comprehensive characterization of gonad differentiation and fertility in botryllus schlosseri. we have analyzed the transcriptomes of both infertile and fertile colonies and characterized the spatial and temporal expression of highly expressed genes involved in fertility. our findings suggest a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates. some of the genes characterized in this study were found to have unexpected spatial localization, such as testis serine/threonine kinase which is expressed by egg precursors. another example is vitellogenin, which localizes to both follicle cells surrounding the egg and equivalent cells surrounding the testes. interestingly, known germline markers such as vasa, nanos and piwi were not significantly differentially expressed between fertile and infertile colonies, most likely because both juveniles and infertile colonies have immature germ cells expressing these genes. we have identified specific markers for both developing gonads throughout the blastogenic cycle in zooids, primary buds and secondary buds. some of these markers are now being used to investigate the phenomenon of induced juvenile fertility in botryllus schlosseri. in future studies, we will investigate differential gene expression between male and hermaphroditic colonies, which will allow us to characterize genes used exclusively in male or female gamete development and identify genes governing the process by which botryllus schlosseri assumes a given reproductive state. 
analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates. here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis using differential expression analysis. 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. spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads. several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. we have identified several genes expressed in developing and mature gonads in b. schlosseri.
 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.  here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis using differential expression analysis. we identified genes 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ascidiantunicatefertilityinfertilitygonad formationgermlineissue-copyright-statement© the author 2014 