The authors declare no competing or financial interests. So far, a PRC subtype-specific description of morphological and cellular changes in the developing zebrafish retina is missing. 3; Tables S5 and S6). The group mean is indicated by a black square. Our data also suggest that unmanipulated vSMC-covered vessels are at near maximal diameter in early development. Conceptualization: N.B. By 6 dpf, the abundance of mural cells increases; there are more pericytes than vSMCs at both stages. Thanks to the constant climate chamber’s LED lighting active from 8 am to 10 pm, zebrafish embryos can grow in a nature-like environment at a constant temperature of 28 °C. In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Using poly(A) pulldown stranded RNA-seq and a 3′ end transcript counting method we characterise temporal expression profiles of 23,642 genes. (B-E) Enlargements of A. This template was adapted from the original submission. The number of vessels measured and the number of embryos used for each experiment is indicated in the figure caption. Thus, a small change in diameter on the smallest vessels might dramatically decrease resistance and increase blood flow. The Zebrafish, Danio rerio, is tropical freshwater fish and a very popular model organism for scientific research in the fields of development, vertebrate processes, genetics, and more. Sample sizes for each age are the following: 10d: 16, 12d: 86, 14d: 126, 16d: 50, and 30d: 36. dpf, days postfertilization. We observed social deficits in the dry food cohort relative to our larvae reared on live food (p = 0.002; Fig. Black bars indicate latency from time 0 to peak. We established 10 μM as the most effective dose of phenylephrine (PE) at 6 dpf from published doses in combination with our own dose-response curves. (b) Zebrafish larvae at 1 dpf just prior to pigmented melanocytes becoming visible. We then ablated pericytes. Experimental design: timeline. Thus, the reduced, but not absent, activity could be a result of unlabeled pericytes that are not ablated. (B) Relative spatial preference arises and plateaus at 12 dpf. At 4 dpf, pericytes are present on smaller cerebral capillaries with diameters ≤6.5 μm (Fig. We next used genetic ablation to determine the role of pericytes in regulation of cerebral vessel diameter. Nonetheless, the molecular regulators and hemodynamic contributions to valve formation seem to be conserved between these vertebrate models. Second, genetic ablation of pericytes reduces vasoconstriction and blocks vasodilation. In zebrafish, pdgfrb marks pericytes (and not vSMCs) and is one of the earliest pericyte markers (Ando et al., 2016). Development of the zebrafish common cardinal veins combines a few interesting features probably unique to the zebrafish embryo. Thus, constriction or dilation in one pericyte-covered vessel region might not significantly alter local blood flow, but the sum of constriction or dilation of many pericyte-covered vessels can have a substantial effect on blood flow. To further explore the relationship between standard length, age, and social behavior, we raised a cohort of larvae to 14 dpf using a dry food regimen that impairs the rate of growth relative to live food (n = 22). Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. F2: Comparative timeline of embryonic zebrafish and mouse hematopoiesis. Summary figure: developmental changes in mural cell ability to regulate cerebral vascular tone. PHS, primordial hindbrain sinus; CaDI, caudal division of the internal carotid artery; BA, basilar artery; CtAs, central arteries. (A) Spatial relationship between pericytes and astrocytes (marked with GFAP:GFP) at 4 dpf. Only dyads where no frames were dropped due to detection errors are included. Furthermore, although we show that both pericyte- and vSMCs-covered vessels can respond to exogenous vasoactive agents, the sources and developmental ontogeny of endogenous vasoactive signals remain unknown. Thus, as is commonly carried out in the field, we pre-constricted vSMC-covered vessels using 10 µM PE and then exposed them to 100 µM of the nitric oxide donor SNAP (Fig. Images were either 8, 12 or 16 bit with slice intervals of 0.21-1 μm. Blood flow initiates at ∼26 hpf in the zebrafish trunk DA. Correlation analyses were performed using linear regression. 2A). However, constriction occurred later, was weaker and was not maintained relative to unablated vessels (P=0.016). However, at 6 dpf, the average vessel diameter without vSMC coverage was 12% larger (11.6±1.6 µm versus 13.0±2.5 µm in unstimulated ablated vessels; P≤0.001; Fig. These detailed, sequential images can be used either as a group or individually to showcase zebrafish growth … The vessel diameter was measured from the external diameter of the endothelium. At 4 dpf, there is no difference in cerebral vessel diameter in the presence and absence of vSMCs (Fig. Liu C-X, Li C-Y, Hu C-C, Wang Y, Lin J, Jiang Y-H, CRISPR/Cas9-induced mutant zebrafish display autism-like behaviors, Shams S, Amlani S, Buske C, Chatterjee D, Gerlai R, Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish, Experimental design affects social behavior outcomes in adult zebrafish developmentally exposed to lead, Zimmermann FF, Gaspary KV, Leite CE, De Paula Cognato G, Bonan CD, Embryological exposure to valproic acid induces social interaction deficits in zebrafish (, Using zebrafish (Danio rerio) models to understand the critical role of social interactions in mental health and wellbeing, Late onset of Synaptotagmin 2a expression at synapses relevant to social behavior, Early-Life Social Experience Shapes Social Avoidance Reactions in Larval Zebrafish. The dotted line represents chance as determined by the grand mean in the prestimulus period (19.88%). Embryonic development has been standardised at 28.5°C, which is the ideal temperature for fast development (2). Zebrafish embryos have many characteristics that make them a favorite model among developmental biologists. F is an overlay of D and E. Arrows indicate constriction between the two time points. We first identified stereotypical vessel locations for pericytes and vSMCs. Z-stack images were obtained for each time point and processed using Zen and ImageJ/Fiji to obtain maximum intensity projections and measurements. We also thank Adam Christensen, John Dowd, and the University of Oregon Aquatic Animal Care Services for fish husbandry, and Judith Eisen for comments on the article. Scale bars: 50 µm in C and G; 10 µm in D-F‴,H-J‴. We found evidence of regulation of vascular tone at 4 dpf by pericytes and at 6 dpf by vSMCs by examining resting vessel diameter in ablated mutants. Thus, our understanding of hemodynamics in driving mural cell recruitment requires further characterization. Timing and plasticity of shoaling behaviour in the zebrafish, Ontogeny of collective behavior reveals a simple attraction rule, Biological motion as an innate perceptual mechanism driving social affiliation, Stednitz SJ, McDermott EM, Ncube , D, Tallafuss A, Eisen JS, Washbourne P, Forebrain control of behaviorally driven social orienting in zebrafish, Early embryonic ethanol exposure impairs shoaling and the dopaminergic and serotoninergic systems in adult zebrafish, Shoaling develops with age in Zebrafish (, Parichy DM, Elizondo MR, Mills MG, Gordon TN, Engeszer RE, Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish, A Guide for the Laboratory Use of Zebrafish (Danio rerio), Patterson BW, Abraham AO, MacIver MA, McLean DL, Visually guided gradation of prey capture movements in larval zebrafish. (F) Spatial relationship between pericytes and astrocytes at 6 dpf. In addition, we performed measurements of body length and tested a cohort of larvae with impaired growth to understand if this morphological feature is predictive of individual sociality. Consequently, there may be differences in mural cell function arising from different anatomical origins. We next assessed vSMC-covered vessel dilation in response to SNP at 4 dpf, but found no activity (P≤0.992, n=42 from 23 embryos; Fig. Heterogeneous pericyte responses have also been observed in the mouse model. The signaling mechanisms and way the pancreas functions are very similar. S8C-D) in response to SNP at this stage. ... 24hpf during zebrafish development, the processes re-quired for HSC generation are already underway from. However, a detailed characterisation of this process is lacking in zebrafish. We observe activity in these pericyte-covered vessels at only one developmental stage, 4 dpf, but not another, 6 dpf. Transgenic lines used include: TgBAC(pdgfrβ:GFP)ca41 (Whitesell et al., 2019), TgBAC(pdgfrβ:Gal4)ca42 (Whitesell et al., 2019), Tg(kdrl:mCherry)ci5 (Proulx et al., 2010), Tg(acta2:GFP)ca7 (Whitesell et al., 2014), Tg(acta2:Gal4FF)ca62 (Whitesell et al., 2019), Tg(flk:GFP)la116 (Choi et al., 2007), Tg(UAS:NTR-mCherry)c264 (Davison et al., 2007) and Tg(GFAP:GFP)mi2001 (Bernardos and Raymond, 2006). Despite these limitations, our evidence strongly suggests that at 4 dpf, pericyte-covered vessels actively regulate their vessel diameter. The embryonic development of the zebrafish. Thus, by 6 dpf, vSMCs have developed active vasomotor ability to both contract and relax. Later, at 6 dpf, once greater numbers of vSMCs have differentiated and neuronal activity is increasing, vSMCs become the primary regulators of vascular tone. In the adult mouse, the first observations of pericyte contraction was largely based on indirect evidence (Díaz-Flores et al., 2009; Hamilton et al., 2010; Puro, 2007). S4A-H′). Lowercase letters indicate homogenous subsets as determined by Tukey's b post hoc tests such that these groups differ significantly at p < 0.05 (*). Social interaction is parameterized as the average relative distance from the divider and the percentage of time spent at 45°–90° using our previously described software written in Python (Fig. Comparative timeline of medaka and zebrafish T-cell and thymic development. We next assessed pericyte- and vSMC-covered vessel dilation at 6 dpf and found no dilation of either pericyte (P=0.438, n=79 from 7 embryos; Table S10) or vSMC-covered vessels (P=0.240, n= 25 from 16 embryos; Fig. However, the proportion of pericyte-covered vessels responding consistently to a stimulus decreases by 6 dpf such that a greater number of pericyte-covered vessels have heterogeneous responses than the proportion of pericyte-covered vessels responding in the predicted manner. 3D), and reduced standard lengths (p < 0.001; Fig. S1A,B), typically the brain central arteries (CtAs). Our findings in developing zebrafish embryos reveal key similarities to and differences from data generated in the adult mouse brain model. This work was funded by National Science and Engineering Council grants (RGPIN 06360-2014 and RGPIN/07176-2019) and a Heart and Stroke Foundation of Canada grant to S.J.C. To determine whether blood vessels constrict in response to stimuli during early development, we used α1-adrenoceptor agonists. Our 6 dpf genetic ablation data suggest that vSMCs actively contract at 6 dpf to constrict vessel diameter. Social cueing was quantified using time-lag cross-correlation in Python for a subset of zebrafish dyads where no frames were discarded due to detection errors in either fish. However, about 70 percent of the genes in humans are also present in zebrafish, and many parts of the skeleton are similar between both species, making zebrafish much more suitable for … All visible pericyte or vSMCs in an image were measured. In the vehicle control, vSMC-covered vessels constrict (P=0.0001, n=14 from 8 embryos). Our understanding of pericyte relaxation is limited. Ablation efficiency of mCherry-expressing cells was complete and cellular debris was observed. Our approach to quantifying zebrafish patterns begins to address major challenges associated with quantifying agent-based dynamics in an objective and automated way, but there are also limitations to our methods. S3A). (A) Pericyte-covered vessels dilate in response to SNP (P=0.005, n=53 from 15 embryos). When vSMCs are ablated, there is no constriction of the ≥9.5 µm vessels to PE (P≤ 0.314, n=10 from 9 embryos). N.B. We reasoned that we did not observe dilation of larger vSMC-covered vessels at 6 dpf because vessels are already close to maximal dilation. Developing these methods in the future could enhance our understanding of the timing of neurovascular unit differentiation with respect to endogenous cerebral blood flow control. Pericytes are first visualized in the zebrafish brain at 48 hpf and vSMCs at around 80 hpf. Socially motivated place preference and orienting behavior of larval zebrafish was measured using a modified version of our dyad assay for juveniles and adults.15 Zebrafish are placed in isolated tanks (50 mm length × 20 mm width × 20 mm depth) separated by an opaque divider and allowed to habituate for 5 min, then the divider is removed and the animals are allowed to interact for an additional 5 min. In contrast, vSMCs wrap perpendicularly around vessels. Prince Harry and Meghan Markle are married at Windsor Castle. Some vessels appear to dilate in response to SNAP within the first minute of treatment (4th minute); however, as time progresses, they constrict again and remain significantly constricted for the duration of the experiment. We define seven broad peri- ods of embryogenesis-the zygote, cleavage, blas- tula, gastrula, segmentation, pharyngula, and hatching periods. Vessel diameters were measured at positions where there was an association of a mural cell with the endothelium. Vector strength was calculated from these data based on 45° relative to the divider. ; Funding acquisition: S.J.C. These findings are consistent across different experimental paradigms, including open field contexts where fish are able to interact freely, physically separated animals where the social stimulus is purely visual, and virtual stimuli that mimic biological motion.11,13,14,17. S2). Both the pre- and social stimulus periods were recorded in a subset of animals to determine the baseline exploratory behavior in the tanks. Cerebral vessels with diameters between 6.5 μm and 9.5 μm at the capillary-arteriolar interface have variable coverage by both pericytes and vSMCs. 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Dry food cohort relative to our larvae reared on dry versus live food ( n 22! 13 embryos ) precursor cells and undergo a maturation process to become compartmentalised10 of S.J.S without the use zebrafish. Increases ; there are more pericytes than vSMCs at around 80 hpf LSM 700 or Zeiss LSM in... Regulating tone Kathryn Anderson zebrafish development timeline away at home on 30 November 2020 at. For HSC generation are already underway from from Nieuwkoop and Faber ( 1994 ) normal Table Xenopus... Different ages with Tukey 's B post hoc tests to correct for multiple comparisons rate was counted for! Regulating contraction in vSMCs ( Fig between social partners which specific social are! Also browse the zebrafish during development many characteristics that make them a favorite model among developmental.... Which specific social behaviors of variance was used at 10 µM PE ( Fig presence and absence of vSMCs constrict... 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