Medaka as a model for seasonal plasticity: Photoperiod-mediated changes in behaviour, cognition, and hormones.

Teleosts display the highest level of brain plasticity of all vertebrates. Yet we still know little about how seasonality affects fish behaviour and the underlying cognitive mechanisms since the common neurobehavioral fish models are native to tropical environments where seasonal variation is absent or reduced. The medaka, Oryzias latipes, which inhabits temperate zone habitats, represents a promising model in this context given its large phenotypic changes associated with seasonality and the possibility to induce seasonal plasticity by only manipulating photoperiod. Here, we report the first extended investigation of seasonal plasticity in medaka behaviour and cognition, as well as the potential underlying molecular mechanisms. We compared medaka exposed to summer photoperiod (16 h light:8 h dark) with medaka exposed to winter photoperiod (8 h light:16 h dark), and detected substantial differences. Medaka were more active and less social in summer photoperiod conditions, two effects that emerged in the second half of an open-field and a sociability test, respectively, and might be at least in part related to habituation to the testing apparatus. Moreover, the cognitive phenotype was significantly affected: in the early response to a social stimulus, brain functional lateralisation shifted between the two hemispheres under the two photoperiod conditions, and inhibitory and discrimination learning performance were reduced in summer conditions. Finally, the expression of genes encoding key pituitary hormones, tshß and gh, and of the tshß regulatory transcription factor tef in the brain was increased in summer photoperiod conditions. This work reveals remarkable behavioural and cognitive phenotypic plasticity in response to photoperiod in medaka, and suggests a potential regulatory role for the same hormones involved in seasonal plasticity of other vertebrates.

Identification, visualization and clonal analysis of intestinal stem cells in fish.

Recently, a stochastic model of symmetrical stem cell division followed by neutral drift has been proposed for intestinal stem cells (ISCs), which has been suggested to represent the predominant mode of stem cell progression in mammals. In contrast, stem cells in the retina of teleost fish show an asymmetric division mode. To address whether the mode of stem cell division follows phylogenetic or ontogenetic routes, we analysed the entire gastrointestinal tract of the teleost medaka (Oryzias latipes). X-ray microcomputed tomography shows a correlation of 3D topography with the functional domains. Analysis of ISCs in proliferation assays and via genetically encoded lineage tracing highlights a stem cell niche in the furrow between the long intestinal folds that is functionally equivalent to mammalian intestinal crypts. Stem cells in this compartment are characterized by the expression of homologs of mammalian ISC markers - sox9, axin2 and lgr5 - emphasizing the evolutionary conservation of the Wnt pathway components in the stem cell niche of the intestine. The stochastic, sparse initial labelling of ISCs ultimately resulted in extended labelled or unlabelled domains originating from single stem cells in the furrow niche, contributing to both homeostasis and growth. Thus, different modes of stem cell division co-evolved within one organism, and in the absence of physical isolation in crypts, ISCs contribute to homeostatic growth.

Gastrulation in an annual killifish: Molecular and cellular events during germ layer formation in Austrolebias.

BACKGROUND: Comparative studies beyond the traditional model organisms have been instrumental in enhancing our understanding of the conserved and derived features of gastrulation, a fundamental process in which the germ layers are specified and shaped to form the body axis. Here, we analyzed gastrulation in a vertebrate group with an extreme mode of early development, the annual killifish. RESULTS: Gastrulation in annual killifish of the genus Austrolebias takes place after the initially dispersed deep blastomeres congregate to form the so-called reaggregate. Cells from the early reaggregate do not appear to form part of any recognizable axial embryonic structure and are possibly extraembryonic. In contrast, later reaggregate cells become engaged in morphogenetic transformations indicative of a process of gastrulation and axis formation. The expression of brachyury and goosecoid suggests that gastrulation takes place in a compressed blastopore-like structure with an organizer region displaced to one end. No collective cell internalization proper of blastopore architecture is observed, though, and it appears that gastrulation primarily involves the reorganization of individual cells. CONCLUSIONS: The unique mode of gastrulation in annual killifish demonstrates that a process so ancient and fundamental to ontogenesis can have striking morphogenetic variations nonpredicted from the sole examination of model species. Developmental Dynamics 246:812-826, 2017. © 2017 Wiley Periodicals, Inc.

ArhGEF18 regulates RhoA-Rock2 signaling to maintain neuro-epithelial apico-basal polarity and proliferation.

The vertebrate central nervous system develops from an epithelium where cells are polarized along the apicobasal axis. Loss of this polarity results in abnormal organ architecture, morphology and proliferation. We found that mutations of the guanine nucleotide exchange factor ArhGEF18 affect apicobasal polarity of the retinal neuroepithelium in medaka fish. We show that ArhGEF18-mediated activation of the small GTPase RhoA is required to maintain apicobasal polarity at the onset of retinal differentiation and to control the ratio of neurogenic to proliferative cell divisions. RhoA signals through Rock2 to regulate apicobasal polarity, tight junction localization and the cortical actin cytoskeleton. The human ArhGEF18 homologue can rescue the mutant phenotype, suggesting a conserved function in vertebrate neuroepithelia. Our analysis identifies ArhGEF18 as a key regulator of tissue architecture and function, controlling apicobasal polarity and proliferation through RhoA activation. We thus identify the control of neuroepithelial apicobasal polarity as a novel role for RhoA signaling in vertebrate development.

Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences.

Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a "small eye" phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.

A Yap-dependent mechanoregulatory program sustains cell migration for embryo axis assembly.

The assembly of the embryo's primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development.

Comparison of anxiety-like and social behaviour in medaka and zebrafish.

The medaka, Oryzias latipes, is rapidly growing in importance as a model in behavioural research. However, our knowledge of its behaviour is still incomplete. In this study, we analysed the performance of medaka in 3 tests for anxiety-like behaviour (open-field test, scototaxis test, and diving test) and in 3 sociability tests (shoaling test with live stimuli, octagonal mirror test, and a modified shoaling test with mirror stimulus). The behavioural response of medaka was qualitatively similar to that observed in other teleosts in the open-field test (thigmotaxis), and in 2 sociability tests, the shoaling test and in the octagonal mirror test (attraction towards the social stimulus). In the remaining tests, medaka did not show typical anxiety (i.e., avoidance of light environments and preference for swimming at the bottom of the aquarium) and social responses (attraction towards the social stimulus). As a reference, we compared the behaviour of the medaka to that of a teleost species with well-studied behaviour, the zebrafish, tested under the same conditions. This interspecies comparison indicates several quantitative and qualitative differences across all tests, providing further evidence that the medaka responds differently to the experimental settings compared to other fish models.

Conserved and diverged asymmetric gene expression in the brain of teleosts.

Morphological left-right brain asymmetries are universal phenomena in animals. These features have been studied for decades, but the functional relevance is often unclear. Studies from the zebrafish dorsal diencephalon on the genetics underlying the establishment and function of brain asymmetries have uncovered genes associated with the development of functional brain asymmetries. To gain further insights, comparative studies help to investigate the emergence of asymmetries and underlying genetics in connection to functional adaptation. Evolutionarily distant isogenic medaka inbred lines, that show divergence of complex traits such as morphology, physiology and behavior, are a valuable resource to investigate intra-species variations in a given trait of interest. For a detailed study of asymmetry in the medaka diencephalon we generated molecular probes of ten medaka genes that are expressed asymmetrically in the zebrafish habenulae and pineal complex. We find expression of eight genes in the corresponding brain areas of medaka with differences in the extent of left-right asymmetry compared to zebrafish. Our marker gene analysis of the diverged medaka inbred strains revealed marked inter-strain size differences of the respective expression domains in the parapineal and the habenulae, which we hypothesize may result from strain-specific gene loss. Thus, our analysis reveals both inter-species differences but also intra-species plasticity of gene expression in the teleost dorsal diencephalon. These findings are a starting point showing the potential to identify the genetics underlying the emergence and modulations of asymmetries. They are also the prerequisite to examine whether variance in habenular gene expression may cause variation of behavioral traits.

The Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel.

BACKGROUND: Unraveling the relationship between genetic variation and phenotypic traits remains a fundamental challenge in biology. Mapping variants underlying complex traits while controlling for confounding environmental factors is often problematic. To address this, we establish a vertebrate genetic resource specifically to allow for robust genotype-to-phenotype investigations. The teleost medaka (Oryzias latipes) is an established genetic model system with a long history of genetic research and a high tolerance to inbreeding from the wild. RESULTS: Here we present the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel: the first near-isogenic panel of 80 inbred lines in a vertebrate model derived from a wild founder population. Inbred lines provide fixed genomes that are a prerequisite for the replication of studies, studies which vary both the genetics and environment in a controlled manner, and functional testing. The MIKK panel will therefore enable phenotype-to-genotype association studies of complex genetic traits while allowing for careful control of interacting factors, with numerous applications in genetic research, human health, drug development, and fundamental biology. CONCLUSIONS: Here we present a detailed characterization of the genetic variation across the MIKK panel, which provides a rich and unique genetic resource to the community by enabling large-scale experiments for mapping complex traits.

Genomic variations and epigenomic landscape of the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel.

BACKGROUND: The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains. Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals. RESULTS: We exploit this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We use Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allows us to identify a large variety of high-quality structural variants, and we present results and methods using a pan-genome graph representation of 12 individual medaka lines. This graph-based reference MIKK panel genome reveals novel differences between the MIKK panel lines and standard linear reference genomes. We find additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We are also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigate line-specific CpG methylation and performed differential DNA methylation analysis across these 12 lines. CONCLUSIONS: We present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel-specific pan genome reference dataset allowing for investigation of novel variation types that would be elusive using standard approaches.

Nlcam modulates midline convergence during anterior neural plate morphogenesis.

During development, different cell types must undergo distinct morphogenetic programs so that tissues develop the right dimensions in the appropriate place. In early eye morphogenesis, retinal progenitor cells (RPCs) move first towards the midline, before turning around to migrate out into the evaginating optic vesicles. Neighbouring forebrain cells, however, converge rapidly and then remain at the midline. These differential behaviours are regulated by the transcription factor Rx3. Here, we identify a downstream target of Rx3, the Ig-domain protein Nlcam, and characterise its role in regulating cell migration during the initial phase of optic vesicle morphogenesis. Through sophisticated live imaging and comprehensive cell tracking experiments in zebrafish, we show that ectopic expression of Nlcam in RPCs, as is observed in Rx3 mutants, causes enhanced convergence of these cells. Expression levels of Nlcam therefore regulate the migratory properties of RPCs. Our results provide evidence that the two phases of optic vesicle morphogenesis: slowed convergence and outward-directed migration, are under different genetic control. We propose that Nlcam forms part of the guidance machinery directing rapid midline migration of forebrain precursors, where it is normally expressed, and that its ectopic expression upon loss of Rx3 imparts these migratory characteristics upon RPCs.

A high-throughput chemically induced inflammation assay in zebrafish.

BACKGROUND: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. RESULTS: Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. CONCLUSIONS: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Strain-Specific Liver Metabolite Profiles in Medaka.

The relationship between genetic variation and phenotypic traits is often poorly understood since specific genotypes do not always easily translate into associated phenotypes, especially for complex disorders. The genetic background has been shown to affect metabolic pathways and thus contribute to variations in the metabolome. Here, we tested the suitability of NMR metabolomics for comparative analysis of fish lines as a first step towards phenotype-genotype association studies. The Japanese rice fish, medaka (Oryzias latipes), is a widely used genetic vertebrate model with several isogenic inbred laboratory strains. We used liver extracts of medaka iCab and HO5 strains as a paradigm to test the feasibility of distinguishing the metabolome of two different inbred strains. Fifteen metabolites could be detected in uni- and multivariate analyses that showed strain-specific levels. Differences could be assigned to specific metabolic pathways. Our results show that NMR spectroscopy is a suitable method to detect variance of the metabolome caused by subtle genetic differences. Thus, it has the potential to address genotype-phenotype associations in medaka, providing an additional level of phenotypic analysis.

epiTracker: A Framework for Highly Reliable Particle Tracking for the Quantitative Analysis of Fish Movements in Tanks.

Behavioral analysis of moving animals relies on a faithful recording and track analysis to extract relevant parameters of movement. To study group behavior and social interactions, often simultaneous analyses of individuals are required. To detect social interactions, for example to identify the leader of a group as opposed to followers, one needs an error-free segmentation of individual tracks throughout time. While automated tracking algorithms exist that are quick and easy to use, inevitable errors will occur during tracking. To solve this problem, we introduce a robust algorithm called epiTracker for segmentation and tracking of multiple animals in two-dimensional (2D) videos along with an easy-to-use correction method that allows one to obtain error-free segmentation. We have implemented two graphical user interfaces to allow user-friendly control of the functions. Using six labeled 2D datasets, the effort to obtain accurate labels is quantified and compared to alternative available software solutions. Both the labeled datasets and the software are publicly available.

Long photoperiod impairs learning in male but not female medaka.

Day length in conjunction with seasonal cycles affects many aspects of animal biology. We have studied photoperiod-dependent alterations of complex behavior in the teleost, medaka (Oryzias latipes), a photoperiodic breeder, in a learning paradigm whereby fish have to activate a sensor to obtain a food reward. Medaka were tested under a long (14:10 LD) and short (10:14 LD) photoperiod in three different groups: mixed-sex, all-males, and all-females. Under long photoperiod, medaka mixed-sex groups learned rapidly with a stable response. Unexpectedly, males-only groups showed a strong learning deficit, whereas females-only groups performed efficiently. In mixed-sex groups, female individuals drove group learning, whereas males apparently prioritized mating over feeding behavior resulting in strongly reduced learning performance. Under short photoperiod, where medaka do not mate, male performance improved to a level similar to that of females. Thus, photoperiod has sex-specific effects on the learning performance of a seasonal vertebrate.

Development of Open-Field Behaviour in the Medaka, Oryzias latipes.

The use of juvenile and larval fish models has been growing in importance for several fields. Accordingly, the evaluation of behavioural tests that can be applied to larvae and juveniles is becoming increasingly important. We tested medaka at four different ages (1, 10, 30, and 120 dph) in the open field test, one of the most commonly used behavioural assays, to investigate its suitability for larvae and juveniles of this species. We also explored ontogenetic variation in behaviour during this test. On average, adult 120-day-old medaka showed higher locomotor activity in terms of distance moved compared with younger fish. Our analysis suggests that this effect was derived from both quantitative changes in locomotion related to the ontogenetic increase in fish size as well as qualitative changes in two aspects of locomotor behaviour. Specifically, time spent moving was similar between 1- and 10-day-old medaka, but progressively increased with development. In addition, we revealed that adult medaka showed constant levels of activity, whereas younger medaka progressively reduced their activity over the course of the entire experiment. The thigmotaxis behaviour typically used to assess anxiety in the open field test emerged at 120 days post-hatching, even though a difference in the temporal pattern of spatial preference emerged earlier, between 10 and 30 days post-hatching. In conclusion, some measures of the open field test such as total distance moved allow behavioural phenotyping in the medaka of all ages, although with some degree of quantitative and qualitative developmental variation. In contrast, immature medaka appear not to exhibit thigmotactic behaviour.

Automated high-throughput heartbeat quantification in medaka and zebrafish embryos under physiological conditions.

Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology. However, dependence on anaesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the use of high-throughput heartbeat analysis. To overcome these limitations, we established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software with graphical user interface for automated quantification of heart rate and rhythm. Sensitivity of the assay was demonstrated by profiling heart rates during entire embryonic development. Our analysis revealed rapid adaption of heart rates to temperature changes, which has implications for standardization of experimental layout. The assay allows scoring of multiple embryos per well enabling a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, we captured concentration-dependent effects of nifedipine and terfenadine over time. Our novel assay permits large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development.

DIY Automated Feeding and Motion Recording System for the Analysis of Fish Behavior.

Fish species such as medaka or zebrafish are widely used as animal models to study physiology, disease development, and treatment efficacy. They are also used to study the rapidly growing field of behavior research, such as social interactions, anxiety, and the influence of environmental factors. Here we describe an automated experimental setup allowing the recording of general locomotor activity in combination with a food-on-demand system. It can simply be built with some basic electronic knowledge. Our setup enables the recording of locomotor and feeding activity of several fish for long-term studies, excluding disturbing external influences. A description of the automated recording system is given, as well as examples of recordings to illustrate its applicability for the study of fish behavior. The construction manual and operation instructions can be downloaded for free.

Quantitative morphometric analysis of adult teleost fish by X-ray computed tomography.

Vertebrate models provide indispensable paradigms to study development and disease. Their analysis requires a quantitative morphometric study of the body, organs and tissues. This is often impeded by pigmentation and sample size. X-ray micro-computed tomography (micro-CT) allows high-resolution volumetric tissue analysis, largely independent of sample size and transparency to visual light. Importantly, micro-CT data are inherently quantitative. We report a complete pipeline of high-throughput 3D data acquisition and image analysis, including tissue preparation and contrast enhancement for micro-CT imaging down to cellular resolution, automated data processing and organ or tissue segmentation that is applicable to comparative 3D morphometrics of small vertebrates. Applied to medaka fish, we first create an annotated anatomical atlas of the entire body, including inner organs as a quantitative morphological description of an adult individual. This atlas serves as a reference model for comparative studies. Using isogenic medaka strains we show that comparative 3D morphometrics of individuals permits identification of quantitative strain-specific traits. Thus, our pipeline enables high resolution morphological analysis as a basis for genotype-phenotype association studies of complex genetic traits in vertebrates.

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions.

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2'-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.