Single cell transcriptome analyses of the developing zebrafish eye- perspectives and applications.

Within a relatively short period of time, single cell transcriptome analyses (SCT) have become increasingly ubiquitous with transcriptomic research, uncovering plentiful details that boost our molecular understanding of various biological processes. Stemming from SCT analyses, the ever-growing number of newly assigned genetic markers increases our understanding of general function and development, while providing opportunities for identifying genes associated with disease. SCT analyses have been carried out using tissue from numerous organisms. However, despite the great potential of zebrafish as a model organism, other models are still preferably used. In this mini review, we focus on eye research as an example of the advantages in using zebrafish, particularly its usefulness for single cell transcriptome analyses of developmental processes. As studies have already shown, the unique opportunities offered by zebrafish, including similarities to the human eye, in combination with the possibility to analyze and extract specific cells at distinct developmental time points makes the model a uniquely powerful one. Particularly the practicality of collecting large numbers of embryos and therefore isolation of sufficient numbers of developing cells is a distinct advantage compared to other model organisms. Lastly, the advent of highly efficient genetic knockouts methods offers opportunities to characterize target gene function in a more cost-efficient way. In conclusion, we argue that the use of zebrafish for SCT approaches has great potential to further deepen our molecular understanding of not only eye development, but also many other organ systems.

A temporal single cell transcriptome atlas of zebrafish anterior segment development.

Anterior segment dysgenesis (ASD), resulting in vision impairment, stems from maldevelopment of anterior segment (AS) tissues. Incidence of ASD has been linked to malfunction of periocular mesenchyme cells (POM). POM cells specify into anterior segment mesenchyme (ASM) cells which colonize and produce AS tissues. In this study we uncover ASM developmental trajectories associated with formation of the AS. Using a transgenic line of zebrafish that fluorescently labels the ASM throughout development, Tg[foxc1b:GFP], we isolated GFP+ ASM cells at several developmental timepoints (48-144 hpf) and performed single cell RNA sequencing. Clustering analysis indicates subdifferentiation of ASM as early as 48 hpf and subsequent diversification into corneal epithelium/endothelium/stroma, or annular ligament (AL) lineages. Tracking individual clusters reveals common developmental pathways, up to 72 hpf, for the AL and corneal endothelium/stroma and distinct pathways for corneal epithelium starting at 48 hpf. Spatiotemporal validation of over 80 genes found associated with AS development demonstrates a high degree of conservation with mammalian trabecular meshwork and corneal tissues. In addition, we characterize thirteen novel genes associated with annular ligament and seven with corneal development. Overall, the data provide a molecular verification of the long-standing hypothesis that POM derived ASM give rise to AS tissues and highlight the high degree of conservation between zebrafish and mammals.

Zebrafish anterior segment mesenchyme progenitors are defined by function of tfap2a but not sox10.

The anterior segment is a critical component of the visual system. Developing independent of the retina, the AS relies partially on cranial neural crest cells (cNCC) as its earliest progenitors. The cNCCs are thought to first adopt a periocular mesenchyme (POM) fate and subsequently target to the AS upon formation of the rudimentary retina. AS targeted POM is termed anterior segment mesenchyme (ASM). However, it remains unknown when and how the switch from cNCC to POM or POM to ASM takes place. As such, we sought to visualize the timing of these transitions and identify the regulators of this process using the zebrafish embryo model. Using two color fluorescence in situ hybridization, we tracked cNCC and ASM target gene expression from 12 to 24hpf. In doing so, we identified a tfap2a and foxC1a co-expression at 16hpf, identifying the earliest ASM to arrive at the AS. Interestingly, expression of two other key regulators of NCC, foxD3 and sox10 was not associated with early ASM. Functional analysis of tfap2a, foxD3 and sox10 revealed that tfap2a and foxD3 are both critical regulators of ASM specification and AS formation while sox10 was dispensable for either specification or development of the AS. Using genetic knockout lines, we show that in the absence of tfap2a or foxD3 function ASM cells are not specified, and subsequently the AS is malformed. Conversely, sox10 genetic mutants or CRISPR Cas9 injected embryos displayed no defects in ASM specification, migration or the AS. Lastly, using transcriptomic analysis, we show that GFP + cNCCs derived from Tg [foxD3:GFP] and Tg [foxC1b:GFP] share expression profiles consistent with ASM development whereas cNCCs isolated from Tg [sox10:GFP] exhibit expression profiles associated with vasculogenesis, muscle function and pigmentation. Taken together, we propose that the earliest stage of anterior segment mesenchyme (ASM) specification in zebrafish is approximately 16hpf and involves tfap2a/foxC1a positive cNCCs.

Deep Diversity: Extensive Variation in the Components of Complex Visual Systems across Animals.

Understanding the molecular underpinnings of the evolution of complex (multi-part) systems is a fundamental topic in biology. One unanswered question is to what the extent do similar or different genes and regulatory interactions underlie similar complex systems across species? Animal eyes and phototransduction (light detection) are outstanding systems to investigate this question because some of the genetics underlying these traits are well characterized in model organisms. However, comparative studies using non-model organisms are also necessary to understand the diversity and evolution of these traits. Here, we compare the characteristics of photoreceptor cells, opsins, and phototransduction cascades in diverse taxa, with a particular focus on cnidarians. In contrast to the common theme of deep homology, whereby similar traits develop mainly using homologous genes, comparisons of visual systems, especially in non-model organisms, are beginning to highlight a "deep diversity" of underlying components, illustrating how variation can underlie similar complex systems across taxa. Although using candidate genes from model organisms across diversity was a good starting point to understand the evolution of complex systems, unbiased genome-wide comparisons and subsequent functional validation will be necessary to uncover unique genes that comprise the complex systems of non-model groups to better understand biodiversity and its evolution.

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes.

BACKGROUND: The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. RESULTS: Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. CONCLUSIONS: We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Spatiotemporal Characterization of Anterior Segment Mesenchyme Heterogeneity During Zebrafish Ocular Anterior Segment Development.

Assembly of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to anterior segment dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and transcriptional identities during early establishment of the zebrafish AS. Two-color fluorescent in situ hybridization suggested that early AS associated POM comprise of a heterogenous population. In vivo and time-course imaging analysis of POM distribution and migratory dynamics analyzed using transgenic zebrafish embryos (Tg[foxc1b:GFP], Tg[foxd3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP]) revealed unique AS distribution and migratory behavior among the reporter lines. Based on fixed timepoint and real-time analysis of POM cell behavior a comprehensive model for colonization of the zebrafish AS was assembled. Furthermore, we generated single cell transcriptomic profiles (scRNA) from our POM reporter lines and characterized unique subpopulation expression patterns. Based on scRNA clustering analysis we observed cluster overlap between neural crest associated (sox10/foxd3), POM (pitx2) and finally AS specified cells (lmx1b, and foxc1b). scRNA clustering also revealed several novel markers potentially associated with AS development and/or function including lum, fmoda, adcyap1b, tgfbi, and hmng2. Taken together, our data indicates that AS-associated POM, or Anterior Segment Mesenchyme (ASM), is not homogeneous but rather comprised of several subpopulations with differing colonization patterns, migration behavior, and transcriptomic profiles.

Temporal characterization of optic fissure basement membrane composition suggests nidogen may be an initial target of remodeling.

Fusion of the optic fissure is necessary to complete retinal morphogenesis and ensure proper function of the optic stalk. Failure of this event leads to congenital coloboma, one of the leading causes of pediatric blindness. Mechanistically it is widely accepted that the basement membrane (BM) surrounding the maturing retina needs to be remodeled within the fissure in order to facilitate subsequent epithelial sheet fusion. However, the mechanism driving BM remodeling has yet to be elucidated. As a first step to understanding this critical molecular event we comprehensively characterized the core composition of optic fissure BMs in the zebrafish embryos. Zebrafish optic fissure BMs were found to express laminin a1, a4, b1a, c1 and c3, nidogen 1a, 1b and 2a, collagen IV a1 and a2 as well as perlecan. Furthermore, we observed that laminin, perlecan and collagen IV expression persists in the fissure during fusion, up to 56 hpf, while nidogen expression is downregulated upon initiation of fusion, at 36 hpf. Using immunohistochemistry we also show that nidogen is removed from the BM prior to that of laminin, indicating that remodeling of the BM is an ordered event. Lastly, we characterized retinal morphogenesis in the absence of nidogen function and documented retinal malformation similar to what is observed in laminin mutants. Taken together, we propose a model of BM remodeling where nidogen acts as a linchpin during initiation of optic fissure fusion.

Co-expression of xenopsin and rhabdomeric opsin in photoreceptors bearing microvilli and cilia.

Ciliary and rhabdomeric opsins are employed by different kinds of photoreceptor cells, such as ciliary vertebrate rods and cones or protostome microvillar eye photoreceptors, that have specialized structures and molecular physiologies. We report unprecedented cellular co-expression of rhabdomeric opsin and a visual pigment of the recently described xenopsins in larval eyes of a mollusk. The photoreceptors bear both microvilli and cilia and express proteins that are orthologous to transporters in microvillar and ciliary opsin trafficking. Highly conserved but distinct gene structures suggest that xenopsins and ciliary opsins are of independent origin, irrespective of their mutually exclusive distribution in animals. Furthermore, we propose that frequent opsin gene loss had a large influence on the evolution, organization and function of brain and eye photoreceptor cells in bilaterian animals. The presence of xenopsin in eyes of even different design might be due to a common origin and initial employment of this protein in a highly plastic photoreceptor cell type of mixed microvillar/ciliary organization.

Owenia fusiformis - a basally branching annelid suitable for studying ancestral features of annelid neural development.

BACKGROUND: Comparative investigations on bilaterian neurogenesis shed light on conserved developmental mechanisms across taxa. With respect to annelids, most studies focus on taxa deeply nested within the annelid tree, while investigations on early branching groups are almost lacking. According to recent phylogenomic data on annelid evolution Oweniidae represent one of the basally branching annelid clades. Oweniids are thought to exhibit several plesiomorphic characters, but are scarcely studied - a fact that might be caused by the unique morphology and unusual metamorphosis of the mitraria larva, which seems to be hardly comparable to other annelid larva. In our study, we compare the development of oweniid neuroarchitecture with that of other annelids aimed to figure out whether oweniids may represent suitable study subjects to unravel ancestral patterns of annelid neural development. Our study provides the first data on nervous system development in basally branching annelids. RESULTS: Based on histology, electron microscopy and immunohistochemical investigations we show that development and metamorphosis of the mitraria larva has many parallels to other annelids irrespective of the drastic changes in body shape during metamorphosis. Such significant changes ensuing metamorphosis are mainly from diminution of a huge larval blastocoel and not from major restructuring of body organization. The larval nervous system features a prominent apical organ formed by flask-shaped perikarya and circumesophageal connectives that interconnect the apical and trunk nervous systems, in addition to serially arranged clusters of perikarya showing 5-HT-LIR in the ventral nerve cord, and lateral nerves. Both 5-HT-LIR and FMRFamide-LIR are present in a distinct nerve ring underlying the equatorial ciliary band. The connections arising from these cells innervate the circumesophageal connectives as well as the larval brain via dorsal and ventral neurites. Notably, no distinct somata with 5-HT -LIR in the apical organ are detectable in the larval stages of Owenia. Most of the larval neural elements including parts of the apical organ are preserved during metamorphosis and contribute to the juvenile nervous system. CONCLUSIONS: Our studies in Owenia fusiformis strongly support that early branching annelids are comparable to other annelids with regard to larval neuroanatomy and formation of the juvenile nervous system. Therefore, Owenia fusiformis turns out to be a valuable study subject for comparative investigations and unravelling ancestral processes in neural development in Annelida and Bilateria in general.

Posterior eyespots in larval chitons have a molecular identity similar to anterior cerebral eyes in other bilaterians.

BACKGROUND: Development of cerebral eyes is generally based on fine-tuned networks and closely intertwined with the formation of brain and head. Consistently and best studied in insects and vertebrates, many signaling pathways relaying the activity of eye developmental factors to positional information in the head region are characterized. Though known from several organisms, photoreceptors developing outside the head region are much less studied and the course of their development, relation to cerebral eyes and evolutionary origin is in most cases unknown. To explore how position influences development of otherwise similar photoreceptors, we analyzed the molecular characteristics of photoreceptors we discovered at the very anterior, the posttrochal mid-body and posterior body region of larval Leptochiton asellus, a representative of the chiton subgroup of mollusks. RESULTS: Irrespective of their position, all found photoreceptors exhibit a molecular signature highly similar to cerebral eye photoreceptors of related animals. All photoreceptors employ the same subtype of visual pigments (r-opsin), and the same key elements for phototransduction such as GNAq, trpC and arrestin and intracellular r-opsin transport such as rip11 and myosinV as described from other protostome cerebral eyes. Several transcription factors commonly involved in cerebral eye and brain development such as six1/2, eya, dachshund, lhx2/9 and prox are also expressed by all found photoreceptor cells, only pax6 being restricted to the anterior most cells. Coexpression of pax6 and MITF in photoreceptor-associated shielding pigment cells present at the mid-body position matches the common situation in cerebral eye retinal pigment epithelium specification and differentiation. Notably, all photoreceptors, even the posterior ones, further express clear anterior markers such as foxq2, irx, otx, and six3/6 (only the latter absent in the most posterior photoreceptors), which play important roles in the early patterning of the anterior neurogenic area throughout the animal kingdom. CONCLUSIONS: Our data suggest that anterior eyes with brain-associated development can indeed be subject to heterotopic replication to developmentally distinct and even posterior body regions. Retention of the transcriptional activity of a broad set of eye developmental factors and common anterior markers suggests a mode of eye development induction, which is largely independent of body regionalization.

Sperm dynamics in spiders (Araneae): ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772).

Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.