Concave-to-convex curve conversion of fiber cells correlates with Y-shaped suture formation at the poles of the rodent lens.

The eye lens contains convexly curved fiber cells that align in concentric layers around the lens anterior-posterior pole axis. For lens fiber differentiation at the equator, cells elongate with their apical and basal tips migrating towards the anterior and posterior poles, respectively. At each pole, the fiber tips meet opposing tips of other fiber cells, to form a suture. Although umbilical or point sutures are observed in fish and birds, line, Y- or star-shaped sutures are detected in other vertebrate lenses. Sutures that do not converge at the point are thought to result from intricate movements of the fiber tips, rather than a straightforward migration along a meridional path. The triggers that give rise to these variations are currently not understood. Our findings revealed that in the mouse embryo, the early-stage lens contains only concave curved fibers, and later, a zone of concave-to-convex curve conversion develops. At this point, a nascent suture in a linear shape appears at the posterior pole and subsequently progresses into a V-shape. This V-shape appears to further develop into a Y-shape as a branch extends from the apex of the V-shape. In lens of zebrafish and Xenopus larvae that form point sutures, this curve-conversion zone is not observed. In lens of adult birds (e.g. zebra finch) that form a point suture, these too also lack a curve-conversion zone. In our previous studies, we demonstrated that murine lens fibers undergoing curve conversion extend membrane protrusions, or lamellipodia, at their basal membranes. In line with this, we did not observe protrusions at the basal tips of fibers in the non-mammalian lenses of zebrafish, Xenopus, and zebra finch in which curve conversion does not occur. We propose that the concave-to-convex conversion in rodent lenses introduces defined paths for fiber cell tips, leading to a more elaborate and complex suture formation, compared to the simple point suture of lower vertebrates.

Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.

Fibroblast growth factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac.

The spheroidal shape of the eye lens is crucial for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that, in the mouse lens, membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating that lamellipodium formation is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controlling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin and actin reduced the height of both early and later fibers, indicating that elongation of these fibers relies on actomyosin contractility. Consistent with this, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose that it does so through regulation of Rho activity.

Fibroblast Growth Factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac.

The spheroidal shape of the eye lens is critical for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating lamellipodium is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin, and actin reduced the height of both early and later fibers, indicating elongation of these fibers relies on actomyosin contractility. Consistently, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose it to do so through regulation of Rho activity.

Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina.

Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated region (MAR)-binding protein and it functions as a tumor suppressor. At the molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here, we report that Banp is indispensable for the DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic cell accumulation and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation, chromosome segregation was not smoothly processed from prometaphase to anaphase in banp morphants, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, a DNA replication fork regulator, wrnip1, and two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of wrnip1 for recovery from DNA replication stress, and cenpt and ncapg for chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.

In order for a cell to divide, it must progress through a series of carefully controlled steps known as the cell cycle. First, the cell replicates its DNA and both copies get segregated to opposite ends. The cell then splits into two and each new cell receives a copy of the duplicated genetic material. If any of the stages in the cell cycle become disrupted or mis-regulated this can lead to uncontrolled divisions that may result in cancer. Researchers have often used a structure within the eye known as the retina to study the cell cycle in zebrafish and other animals as cells in the retina rapidly divide in a highly controlled manner. A protein called Banp is known to help stop tumors from growing in humans and mice, but its normal role in the body, particularly the cell cycle, has remained unclear. To investigate, Babu et al. studied the retina of mutant zebrafish that were unable to make the Banp protein. The experiments revealed that two stress responses indicating DNA damage or defects in copying DNA were active in the retinal cells of the mutant zebrafish. This suggested that Banp allows cell to progress through the cell cycle by repairing any DNA damage that may arise during replication. Banp does this by activating the gene for another protein called Wrnip1. Babu et al. also found that Banp helps segregate the two copies of DNA during cell division by promoting the activation of two other proteins called Cenpt and Ncapg. Further experiments identified 31 genes that were directly regulated by Banp. These findings demonstrate that Banp is required for zebrafish cells to be able to accurately copy their DNA and divide in to two new cells. In the future, the work of Babu et al. will provide a useful resource to investigate how tumors grow and spread around the body, and may contribute to the development of new treatments for cancer.

Strip1 regulates retinal ganglion cell survival by suppressing Jun-mediated apoptosis to promote retinal neural circuit formation.

In the vertebrate retina, an interplay between retinal ganglion cells (RGCs), amacrine (AC), and bipolar (BP) cells establishes a synaptic layer called the inner plexiform layer (IPL). This circuit conveys signals from photoreceptors to visual centers in the brain. However, the molecular mechanisms involved in its development remain poorly understood. Striatin-interacting protein 1 (Strip1) is a core component of the striatin-interacting phosphatases and kinases (STRIPAK) complex, and it has shown emerging roles in embryonic morphogenesis. Here, we uncover the importance of Strip1 in inner retina development. Using zebrafish, we show that loss of Strip1 causes defects in IPL formation. In strip1 mutants, RGCs undergo dramatic cell death shortly after birth. AC and BP cells subsequently invade the degenerating RGC layer, leading to a disorganized IPL. Mechanistically, zebrafish Strip1 interacts with its STRIPAK partner, Striatin 3 (Strn3), and both show overlapping functions in RGC survival. Furthermore, loss of Strip1 or Strn3 leads to activation of the proapoptotic marker, Jun, within RGCs, and Jun knockdown rescues RGC survival in strip1 mutants. In addition to its function in RGC maintenance, Strip1 is required for RGC dendritic patterning, which likely contributes to proper IPL formation. Taken together, we propose that a series of Strip1-mediated regulatory events coordinates inner retinal circuit formation by maintaining RGCs during development, which ensures proper positioning and neurite patterning of inner retinal neurons.

The back of the eye is lined with an intricate tissue known as the retina, which consists of carefully stacked neurons connecting to each other in well-defined 'synaptic' layers. Near the surface, photoreceptors cells detect changes in light levels, before passing this information through the inner plexiform layer to retinal ganglion cells (or RGCs) below. These neurons will then relay the visual signals to the brain. Despite the importance of this inner retinal circuit, little is known about how it is created as an organism develops. As a response, Ahmed et al. sought to identify which genes are essential to establish the inner retinal circuit, and how their absence affects retinal structure. To do this, they introduced random errors in the genetic code of zebrafish and visualised the resulting retinal circuits in these fast-growing, translucent fish. Initial screening studies found fish with mutations in a gene encoding a protein called Strip1 had irregular layering of the inner retina. Further imaging experiments to pinpoint the individual neurons affected showed that in zebrafish without Strip1, RGCs died in the first few days of development. Consequently, other neurons moved into the RGC layer to replace the lost cells, leading to layering defects. Ahmed et al. concluded that Strip1 promotes RGC survival and thereby coordinates proper positioning of neurons in the inner retina. In summary, these findings help to understand how the inner retina is wired; they could also shed light on the way other layered structures are established in the nervous system. Moreover, this study paves the way for future research investigating Strip1 as a potential therapeutic target to slow down the death of RGCs in conditions such as glaucoma.

Mechanisms underlying microglial colonization of developing neural retina in zebrafish.

Microglia are brain-resident macrophages that function as the first line of defense in brain. Embryonic microglial precursors originate in peripheral mesoderm and migrate into the brain during development. However, the mechanism by which they colonize the brain is incompletely understood. The retina is one of the first brain regions to accommodate microglia. In zebrafish, embryonic microglial precursors use intraocular hyaloid blood vessels as a pathway to migrate into the optic cup via the choroid fissure. Once retinal progenitor cells exit the cell cycle, microglial precursors associated with hyaloid blood vessels start to infiltrate the retina preferentially through neurogenic regions, suggesting that colonization of retinal tissue depends upon the neurogenic state. Along with blood vessels and retinal neurogenesis, IL34 also participates in microglial precursor colonization of the retina. Altogether, CSF receptor signaling, blood vessels, and neuronal differentiation function as cues to create an essential path for microglial migration into developing retina.

The immune system is comprised of many different cells which protect our bodies from infection and other illnesses. The brain contains its own population of immune cells called microglia. Unlike neurons, these cells form outside the brain during development. They then travel to the brain and colonize specific regions like the retina, the light-sensing part of the eye in vertebrates. It is poorly understood how newly formed microglia migrate to the retina and whether their entry depends on the developmental state of nerve cells (also known as neurons) in this region. To help answer these questions, Ranawat and Masai attached fluorescent labels that can be seen under a microscope to microglia in the embryos of zebrafish. Developing zebrafish are transparent, making it easy to trace the fluorescent microglia as they travel to the retina and insert themselves among its neurons. Ranawat and Masai found that blood vessels around the retina act as a pathway that microglia move along. Once they reach the retina, the microglia remain attached and only enter the retina at sites where brain cells are starting to mature in to adult neurons. Further experiments showed that microglia fail to infiltrate and colonize the retina when blood vessels are damaged or neuron maturation is blocked. These findings reveal some of the key elements that guide microglia to the retina during development. However, further work is needed to establish the molecular and biochemical processes that allow microglia to attach to blood vessels and detect when cells in the retina are starting to mature.

ß-SNAP activity in the outer segment growth period is critical for preventing BNip1-dependent apoptosis in zebrafish photoreceptors.

BNip1, which functions as a t-SNARE component of the syntaxin18 complex, is localized on the ER membrane and regulates retrograde transport from Golgi to the ER. BNip1 also has a BH3 domain, which generally releases pro-apoptotic proteins from Bcl2-mediated inhibition. Previously we reported that retinal photoreceptors undergo BNip1-dependent apoptosis in zebrafish ß-snap1 mutants. Here, we investigated physiological roles of BNip1-dependent photoreceptor apoptosis. First, we examined the spatio-temporal profile of photoreceptor apoptosis in ß-snap1 mutants, and found that apoptosis occurs only during a small developmental window, 2-4 days-post-fertilization (dpf), in which an apical photoreceptive membrane structure, called the outer segment (OS), grows rapidly. Transient expression of ß-SNAP1 during this OS growing period prevents photoreceptor apoptosis in ß-snap1 mutants, enabling cone to survive until at least 21 dpf. These observations suggest that BNip1-mediated apoptosis is linked to excessive activation of vesicular transport associated with rapid growth of the OS. Consistently, knockdown of Ift88 and Kif3b, which inhibits protein transport to the OS, rescued photoreceptor apoptosis in ß-snap1 mutants. Treatment with rapamycin, which inhibits protein synthesis via the mTOR pathway, also rescued photoreceptor apoptosis in ß-snap1 mutants. These data suggest that BNip1 performs risk assessment to detect excessive vesicular transport in photoreceptors.

Genetically Encoded Fluorescent Indicator GRAPHIC Delineates Intercellular Connections.

Intercellular contacts are essential for precise organ morphogenesis, function, and maintenance; however, spatiotemporal information of cell-cell contacts or adhesions remains elusive in many systems. We developed a genetically encoded fluorescent indicator for intercellular contacts with optimized intercellular GFP reconstitution using glycosylphosphatidylinositol (GPI) anchor, GRAPHIC (GPI anchored reconstitution-activated proteins highlight intercellular connections), which can be used for an expanded number of cell types. We observed a robust GFP signal specifically at the interface between cultured cells, without disrupting natural cell contact. Application of GRAPHIC to the fish retina specifically delineated cone-bipolar connection sites. Moreover, we showed that GRAPHIC can be used in the mouse central nervous system to delineate synaptic sites in the thalamocortical circuit. Finally, we generated GRAPHIC color variants, enabling detection of multiple convergent contacts simultaneously in cell culture system. We demonstrated that GRAPHIC has high sensitivity and versatility, which will facilitate the analysis of the complex multicellular connections without previous limitations.

A crucial role for Arf6 in the response of commissural axons to Slit.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

TNFα Induces Müller Glia to Transition From Non-proliferative Gliosis to a Regenerative Response in Mutant Zebrafish Presenting Chronic Photoreceptor Degeneration.

Unlike mammals, zebrafish have the capacity to regenerate neurons in response to damage. Most zebrafish retinal injury models employ acute damage, which is unlike the chronic, gradual damage that occurs in human retinal diseases. Here, we studied the regenerative response in the zebrafish aipl1b mutant, gold rush (gosh). In gosh mutants, both cones and rods degenerate by 3 weeks post-fertilization (wpf). Müller glia do not exhibit a regenerative response by 3 wpf; however, they do present non-proliferative gliosis. Only at 5 wpf, is proliferation of Müller cells and rod precursor cells activated. Rods start to recover at 5 wpf and by 12 wpf they reach a level of recovery comparable to wild type, but cones remain absent in the adult stage. TNFα was detected in degenerating cones at 5-7 wpf and in Müller glia at 7 wpf in gosh mutants. At 5 wpf, proliferating Müller glia express Sox2, followed by Pax6 expression in neuronal progenitor cells (NPCs), confirming that the neuronal regeneration program is activated in gosh mutants after 5 wpf. Although acute light-induced damage did not activate proliferation of Müller glia, TNFα injection caused Müller glia to commence a proliferative response at 3 wpf in gosh mutants. These results suggest that Müller glia transition from non-proliferative gliosis to a regenerative state in gosh mutants, and that ectopic introduction of TNFα promotes this Müller cell transition even at 3 wpf. Thus, zebrafish gosh mutants provide a useful model to investigate mechanisms underlying retinal regeneration in a chronic photoreceptor degeneration model.

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation.

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

Endocytic trafficking factor VPS45 is essential for spatial regulation of lens fiber differentiation in zebrafish.

In vertebrate lens, lens epithelial cells cover the anterior half of the lens fiber core. Lens epithelial cells proliferate, move posteriorly and start to differentiate into lens fiber cells at the lens equator. Although FGF signaling promotes this equatorial commencement of lens fiber differentiation, the underlying mechanism is not fully understood. Here, we show that lens epithelial cells abnormally enter lens fiber differentiation without passing through the equator in zebrafish vps45 mutants. VPS45 belongs to the Sec1/Munc18-like protein family and promotes endosome trafficking, which differentially modulates signal transduction. Ectopic lens fiber differentiation in vps45 mutants does not depend on FGF, but is mediated through activation of TGFß signaling and inhibition of canonical Wnt signaling. Thus, VPS45 normally suppresses lens fiber differentiation in the anterior region of lens epithelium by modulating TGFß and canonical Wnt signaling pathways. These data indicate a novel role of endosome trafficking to ensure equator-dependent commencement of lens fiber differentiation.

Do cGMP Levels Drive the Speed of Photoreceptor Degeneration?

Humans with mutations in the phototransduction pathway develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or Leber congenital amaurosis. Similarly, numerous phototransduction mutant animal models resemble retinal degeneration. In our lab, using a zebrafish model, we study cone-specific phototransduction mutants. cGMP is the second messenger in the phototransduction pathway, and abnormal cGMP levels are associated with photoreceptor death. Rd1, a rod-specific phosphodiesterase 6 (Pde6) subunit mutant in mice, is one of the most widely used animal models for retinal degeneration. Rd1 mutant mice accumulate cGMP, causing rapid photoreceptor degeneration. However, much less is known about photoreceptor mutants producing abnormally low levels of cGMP. Here, focusing on Pde6 mutants in zebrafish and mice, we propose a correlation between cGMP levels and speed of photoreceptor degeneration.

Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches.

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Aipl1 is required for cone photoreceptor function and survival through the stability of Pde6c and Gc3 in zebrafish.

Genetic mutations in aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) cause photoreceptor degeneration associated with Leber congenital amaurosis 4 (LCA4) in human patients. Here we report retinal phenotypes of a zebrafish aipl1 mutant, gold rush (gosh). In zebrafish, there are two aipl1 genes, aipl1a and aipl1b, which are expressed mainly in rods and cones, respectively. The gosh mutant gene encodes cone-specific aipl1, aipl1b. Cone photoreceptors undergo progressive degeneration in the gosh mutant, indicating that aipl1b is required for cone survival. Furthermore, the cone-specific subunit of cGMP phosphodiesterase 6 (Pde6c) is markedly decreased in the gosh mutant, and the gosh mutation genetically interacts with zebrafish pde6c mutation eclipse (els). These data suggest that Aipl1 is required for Pde6c stability and function. In addition to Pde6c, we found that zebrafish cone-specific guanylate cyclase, zGc3, is also decreased in the gosh and els mutants. Furthermore, zGc3 knockdown embryos showed a marked reduction in Pde6c. These observations illustrate the interdependence of cGMP metabolism regulators between Aipl1, Pde6c, and Gc3 in photoreceptors.

Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish.

In vertebrates, lens epithelial cells cover the anterior half of the lens fiber core. During development, lens epithelial cells proliferate, move posteriorly and differentiate into lens fiber cells after passing through the equator. To elucidate the mechanisms underlying lens epithelial cell movement, we conducted time-lapse imaging of zebrafish lens epithelium. Lens epithelial cells do not intermingle but maintain their relative positions during development. Cell division induces epithelial rearrangement, which subsequently promotes cell movement towards the equator. These data suggest that cell division is the major driving force for cell movement. In zebrafish, E-cadherin is expressed in lens epithelium, whereas N-cadherin is required for lens fiber growth. E-cadherin reduced lens epithelial cell movement, whereas N-cadherin enhanced it. Laser ablation experiments revealed that lens epithelium is governed by pulling tension, which is modulated by these cadherins. Thus, cell division and cadherin-mediated adhesion regulate lens epithelial cell movement via modulation of epithelial tension.

Spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium.

Cell proliferation is a key regulator of tissue morphogenesis. We examined cell proliferation and cell division in zebrafish lens epithelium by visualizing cell-cycle phases and nuclear positions, using fluorescent-labeled geminin and histone proteins. Proliferation was low in the anterior region of lens epithelium and higher in the marginal zone anterior to the equator, suggesting that the proliferation zone, called the germinative zone, is formed in zebrafish lens. Interestingly, cell-division orientation was biased longitudinally in the anterior region, shifted from longitudinal to circumferential along the anterior-posterior axis of lens sphere, and was biased circumferentially in the peripheral region. These data suggest that cell-division orientation is spatially regulated in zebrafish lens epithelium. The Hertwig rule indicates that cells tend to divide along their long axes. Orientation of long axes and cell division were biased similarly in zebrafish lens epithelium, suggesting that cell geometry correlates with cell-division orientation. A cell adhesion molecule, E-cadherin, is expressed in lens epithelium. In a zebrafish e-cadherin mutant, the long axes and cell-division orientation were shifted more longitudinally. These data suggest that E-cadherin is required for the spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium.

Stem-loop binding protein is required for retinal cell proliferation, neurogenesis, and intraretinal axon pathfinding in zebrafish.

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.