Ichiro Masai

Microglia, brain‐resident immune cells, maintain brain homeostasis. However, homeostatic microglia dynamically change into disease‐associated microglia in human neurodegenerative diseases. Furthermore, microglia‐mediated inflammation is required to initiate neuronal regeneration in zebrafish brain. To understand how functional states of microglia are regulated in response to neuronal degeneration and regeneration, we focused on pde6c mutants, a chronic photoreceptor degeneration zebrafish model. We conducted scRNA‐seq analysis on microglia in wild‐type sibling and pde6c mutant retinas at the onset of photoreceptor degeneration (5 dpf) and Müller glia‐mediated neuronal regeneration (4 wpf). At 5 dpf, retinal microglia consist of three clusters, which correspond to homeostatic, degeneration‐response, and stress‐response microglia, respectively. The degeneration‐response cluster expands in pde6c mutants and expresses genes for neuroprotection and tissue repair. At 4 wpf, retinal microglia comprise four clusters, two of which are specifically produced in pde6c mutants and approach the photoreceptor layer. Furthermore, another cluster is prominently localized in the retinal stem cell niche and shows a transcriptomic profile similar to that of neurogenic‐associated microglia (NAM). Comparison of transcriptomic similarity between 4 wpf and 5 dpf microglial clusters revealed that each 4 wpf microglial cluster inherits characteristics of homeostatic, degeneration‐response, and stress‐response state of 5 dpf microglia in different combinations. Thus, there is a unique heterogeneity of microglia in the initial stage of Müller glia‐mediated regeneration. Taken together, our findings reveal a dynamic change of retinal microglia during the transition from photoreceptor degeneration to Müller glia‐mediated neuronal regeneration in zebrafish.

Social behaviors include some of the most interesting interactions in living systems yet their principled characterization remains unsolved. Here we suggest that at the core of social interactions is the notion of mutual prediction, which we analyze in the context of two male zebrafish engaged in a dominance contest. Using 3D velocity trajectories, we construct the mutual information between a two-animal past and one-animal future, and we quantify the redundant, unique, and synergistic components using partial information decomposition across time windows. We find that predictive information decomposition naturally aligns with important social concepts, such as mirroring and dominance. At contest end, we find asymmetries in self-unique and redundant information that reflect the emergent dominance relationship. Applied to mecp2 zebrafish mutants, an autism model, we find that predictive information is reduced overall, but especially for synergistic flows, which is indicative of difficulties in more complex social dynamics. Significance Statement: Social interactions are rich and diverse, ranging from mirroring to complementary actions. A unifying framework for defining and analyzing such interaction types has long been needed. Here, based on modern information theory, we formulate how the past state of interacting organisms encodes the future state of an individual. This framework provides a natural decomposition of pairwise social dynamics into mirroring, independent action, directed influence, and joint action. Applied to dominance contests in zebrafish, these modes of interaction capture distinct phases of conflict, their assessment strategies, and the resulting dominance relationships. Moreover, our analysis reveals a specific disruption in the social behavior of mutant zebrafish linked to autism, shedding new light on impairments in communication and social learning.

Cone photoreceptors assemble to form a regular mosaic pattern in vertebrate retinas. In zebrafish, four distinct spectral cone types (red, green, blue, and ultraviolet), form a lattice-like pattern. However, the mechanism of cone mosaic formation has been unknown. Here we show that Down Syndrome Cell Adhesion Molecule b (Dscamb) regulates the cone mosaic pattern in zebrafish, especially via red-cone spacing. During photoreceptor differentiation, newly formed cones extend filopodium-like processes laterally to apical surfaces of neighboring cones. Interestingly, red cones extend filopodia, but promptly retract them when they meet their own cone type, suggesting filopodium-mediated, homotypic recognition and self-avoidance. This self-avoidance is compromised in zebrafish dscamb mutants, leading to abnormal clustering of red cones and subsequent disruption of regular cone spacing. Thus, apical filopodium-mediated spacing of the same cone type depends on Dscamb and is essential for cone mosaic formation in zebrafish.

Zebrafish genetic mutants have emerged as a valuable model system for studying various aspects of disease and developmental biology. Mutant zebrafish embryos are generally identified based on phenotypic defects at later developmental stages, making it difficult to investigate underlying molecular mechanisms at earlier stages. This protocol presents a PCR-based genotyping method that enables the identification of wild-type, heterozygous, and homozygous zebrafish genetic mutants at any developmental stage, even when they are phenotypically indistinguishable. The approach involves the amplification of specific genomic regions using carefully designed primers, followed by gel electrophoresis. This genotyping method facilitates the investigation of the molecular mechanisms driving phenotypic defects that are observed at later timepoints. This protocol allows researchers to perform analyses such as immunofluorescence, RT-PCR, RNA sequencing, and other molecular experiments on early developmental stages of mutants. The availability of this protocol expands the utility of zebrafish genetic mutants for elucidating the molecular underpinnings of various biological processes throughout development.

While two-body fighting behavior occurs throughout the animal kingdom to settle dominance disputes, important questions such as how the dynamics ultimately lead to a winner and loser are unresolved. Here we examine fighting behavior at high resolution in male zebrafish. We combine multiple cameras, a large volume containing a transparent interior cage to avoid reflection artifacts, with computer vision to track multiple body points across multiple organisms while maintaining individual identity in three dimensions. In the body point trajectories we find a spectrum of timescales which we use to build informative joint coordinates consisting of relative orientation and distance. We use the distribution of these coordinates to automatically identify fight epochs, and we demonstrate the postfight emergence of an abrupt asymmetry in relative orientations—a clear and quantitative signal of hierarchy formation. We identify short-time, multi-animal behaviors as clustered transitions between joint configurations, and show that fight epochs are spanned by a subset of these clusters, which we denote as maneuvers. The resulting space of maneuvers is rich but interpretable, including motifs such as “attacks” and “circling.” In the longer-time dynamics of maneuver frequencies we find differential and changing strategies, including that the eventual loser attacks more often towards the end of the contest. Our results suggest a reevaluation of relevant assessment models in zebrafish, while our approach is generally applicable to other animal systems. Published by the American Physical Society 2024

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.

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.

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. Summary statement: FGF differentially regulates Rho- and Rac-activity at the lens equator to promote lens fiber cell elongation and migration.

Two-body fighting behavior occurs throughout the animal kingdom to settle dominance disputes. While some universal features have been identified, important questions such as how the dynamics ultimately lead to a winner and loser remain unresolved. Here we analyze fighting behavior in pairs of adult male zebrafish, Danio rerio. We combine a custom imaging apparatus consisting of multiple cameras, a large volume, and a transparent interior cage to avoid reflection artifacts, with computer vision to track multiple body points across multiple organisms while maintaining individual identity in 3D. In the body point dynamics we find a hierarchy of timescales which we use to construct interpretable joint coordinates consisting of relative orientation and distance. We use the distribution of these coordinates to automatically identify fight epochs and we demonstrate the post-fight emergence of an abrupt asymmetry in relative orientations. To quantify short-time behaviors we cluster transitions between joint configurations, and show that fight epochs are spanned by a small number of dynamical clusters, which we denote as maneuvers. The maneuver clusters include motifs such as “attacks” and “circling” and we quantify longer-time dynamics through maneuver frequencies. We find that the eventual loser attacks more often towards the end of the contest, suggesting a loser assessment component of the dominance decision.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments (OS), 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 lack OSs and undergo apoptosis. Interestingly, zebrafish mak mutants fail to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies are 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 is critical in ciliary axoneme development and photoreceptor survival. Thus, MAK is required for ciliogenesis and OS formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival. Summary statement: Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase that promotes axoneme development during ciliogenesis in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.