Cyanobacteria, also known as blue-green algae, are known to produce a number of biologically active compounds. Extracts of cultured cyanobacteria isolated from South Florida sources were screened for possible developmental toxins using the zebrafish (Danio rerio) embryo as a model of vertebrate development. A strain of cyanobacteria, Fischerella 52-1, isolated from the Florida Everglades, was found to produce metabolites that caused a consistent developmental dysfunction in embryos exposed to lipophilic extract. Initial chemical characterization of the bioactive fraction identified a series of eight apparent indole-containing compounds. The two main components were purified using the zebrafish embryo model to guide the fractionation. Chemical characterization using 1- and 2-dimensional NMR, HESIMS, HRHESIMS, and IR determined that the two main compounds were the previously identified 12-epi-Hapalindole H Isonitrile, and a novel compound 12-epi-Ambiugine B Nitrile. The major contributor of the developmental defects detected in the zebrafish embryos was 12-epi-Hapalindole H Isonitrile.

TRPM7 (Transient Receptor Potential Melastatin-like 7) is an ion channel necessary for the proper development of many cell types. Insight into the precise role of the channel in different cells has been hampered by the lethality of knocking out the gene in model organisms such as the mouse. Here I examine a zebrafish that has a loss-of-function mutation in the gene encoding Trpm7. First, I show that trpm7 is important for the function of developing dopaminergic neurons in the zebrafish. Second, I examine the interaction between trpm7 and the related gene vmat2 in order to develop a cellular mechanism of trpm7 function in presynaptic dopaminergic neurons. Finally, I investigate the necessity of the kinase and ion channel domains of trpm7 in their ability to promote pigmentation in melanophores as a model cell type. Based on the results from these experiments and observations from other researchers, I form a new hypothesis for Trpm7 function in protein sorting. These studies provide a detailed and novel analysis of the function of an ion channel that is necessary for life.

Examples of asymmetric organs are found throughout the animal kingdom. Whether it is superficial like the fiddler crab’s claw or within an organism like our visceral organs, asymmetries have repeatedly evolved in nature. However, the genetic and developmental origins for asymmetric organ development remain unclear, especially for superficially paired structures. Within zebrafish, a striking example of asymmetry occurs within the ace/fgf8 mutant. The pharyngeal cartilages of these mutants develop asymmetrically 35% of the time, with more cartilages developing on the left or right side of the head, but the origins of this asymmetry are unknown. A significant proportion of mutants also exhibit situs inversus, whereby the visceral organs develop on the opposite side of the body. Here we seek to understand the temporal window most sensitive to giving rise to this asymmetry, and to understand if there is a correlation between the developing heart field and pharyngeal cartilage with respect to the direction of the asymmetry. Wild type (WT) zebrafish were exposed to SU5402 during different periods of development, and heart position as well as cartilage development was observed within the developing larvae. The direction of asymmetry (i.e., left or right biased) was also recorded in ace/fgf8 mutant heart position and cartilage number to observe if there was a correlation between the two developing fields. SU5402 experiments revealed that the time window most sensitive to the development of cartilage asymmetries was during heart looping and pharyngeal arch segmentation. Furthermore, ace/fgf8 mutants exhibited a robust correlation between ventricle position and the side of cartilage asymmetry, with more cartilages forming on the side where the ventricle is located. Given the close proximity of the heart and pharyngeal cartilage fields we suggest that the heart field is influencing the developing cartilage, with signaling permeating from the developing heart to the pharyngeal mesoderm to provide a buffer on the side of the developing ventricle.

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

Synovial joints enable frictionless movement between the adjacent bones. Lubricating fluid enclosed within the joint reduce the pressure to allow free movement. Previously synovial joints have been associated with a terrestrial life and have mainly been studied in tetrapods, such as mammals and chicken, under the assumption that the joint did not evolve before animals adapted to a life on land. However, a recent study described the production and function of lubricin in the jaw and pectoral fin joints in ray-finned fishes, such as zebrafish, suggesting previous assumption to be questionable. The current study aim to further investigate the jaw joints in the ray-finned zebrafish, by examininge the morphology and presence of specific cell types, called synoviocytes, associated with the synovial joint. The synoviocytes are divided into two groups: macrophage-like cells and fibroblast-like cells. Tissue sections were histologically investigated with nuclear staining and immunohistochemically with antibodies to chondroitin sulphate (CS-56) and uridine diphosphoglucose dehydrogenase (UDPGD, UGDH) to investigate the presence of fibroblast-like cells. Small and circular cells were observed in the joint line and surrounding cartilage nascent to the joint. These cells could not be identified as chondrocytes or part of the perichondrium, and their UDPGD activity witness on similarities to the fibroblast-like cells. UDPGD is associated to the synthesis of hyaluronan (HA) which plays a key role in joint cavitation, hence, suggesting functional similarities in the development of zebrafish jaw joint to synovial joints. Furthermore a double transgenic zebrafish, fli1:EGFP/mpeg1:mCherry, was used to examine the presence of macrophages within the joint area. Macrophages were observed on the surface of the jaw joint in close association with blood vessels but did not show signs of incorporation within the joint. These results present further similarities of joints in ray-finned fish to the synovial joint but also the need to further examine similarities in order to understand the development and evolution of the joints. The possibility to implement the zebrafish model would facilitate future studies on the healthy synovial joint as well the diseases related to it.

No description available.

Aquaculture is currently the fastest growing animal production sector. Because the aquaculture sector is growing at rapid rates, certain materials for feed, specifically marine protein sources, are becoming increasingly expensive and unsustainable. To counteract the reliance on fishmeal (FM) and other marine protein sources in the industry plant protein (PP), specifically soybean meal (SBM), has been investigated to replace FM as a protein source. Unfortunately, SBM when given in high quantities (greater than 30%) has been shown to negatively affect fish performance including retarded growth, intestinal inflammation, reduction of spawn quality, as well as dysbiosis in the gut microbiome, most likely due to presence of antinutritional factors such as saponins and tannins in SBM. The goal of this thesis was to investigate the effect of nutritional programming (NP) with SBM-based diet on gut microbiota in broodstock and progeny fish. Three feedings trials were conducted to test the efficacy of 3 approaches towards improving the use of PP in fish.The first trial (Chapter 2), tested the effect of NP on larval zebrafish (Danio rerio). NP is the theory of introducing an early nutritional stimulus to an animal that will ‘program’ the animal to better utilize the stimuli later in its adult life. The zebrafish were programmed in their larval stages, and the trial lasted for 65 days. There was a significant effect on growth performance for the programmed group (NP-PP) in terms of weight gains, as the NP-PP group grew better compared to the non-programmed group (NP-FM) and negative control (-control). There was no significant effect on the gut microbiome in terms of alpha or beta diversity, however, there were significant changes in the relative abundance (RA) of the gut microbiome throughout time in the NP-PP and the NP-FM groups. The findings of the study support that early NP of zebrafish with SBM improves growth performance on PP diet, but the gut microbiome does not seem to be a mechanism for NP.The second feeding trial (Chapter 3) focused on NP induced in the zebrafish broodstock with dietary SBM. For two weeks, the broodstock fish were fed with either a SBM diet or a FM diet so that gametogenesis occurred with either a FM or PP diet. This phase was called the ‘broodstock programming’ stage. The broodstock were then spawned, and the larval fish were separated into four different treatments: 1) SBM broodstock progeny, fed SBM for the entire trial (PPBS-PP) 2) SBM broodstock progeny fed FM the entire trial (PPBS-FM), 3) FM broodstock progeny fed FM the entire trial (+ control, FMBS-FM), and 4) FM broodstock progeny fed SBM the entire trial (- control, FMBS-PP). The PPBS-PP group achieved similar weight gains compared to all other treatments in terms of grams, but was numerically greater than the FMBS-PP treatment. There were no differences detected in gut microbiome alpha or beta diversity in any of the groups, however, there was significant change observed of certain bacterial phyla between the ‘programmed broodstock’, larval fish, and fish at the end of the trial, 48 days post hatch. Overall, this trial suggests that parental programming does not improve PP utilization in the progeny of zebrafish. It also appears that the gut microbiome is not a mechanism of parental programming. The third feeding trial (Chapter 4), was conducted on largemouth bass (Micropterus salmoides). This chapter had a similar experimental design as the first trial (Chapter 2), and larval largemouth bass were programmed with dietary SBM. This trial had an additional group added to it, which included a dietary saponin-programmed group. The study found that the NP with SBM diet or dietary saponin did not improve PP utilization and growth performance of largemouth bass in its pre-adult age. The study also found that the NP with SBM diet or dietary saponin did not have any effect on the largemouth bass gut microbiome, and there does not seem to be any gut microbiome modification associated with the NP in this fish species. Overall, NP can be used to improve dietary PP utilization but optimal timing and PP delivery method must be well assessed to ensure successful PP exposure and adaptation in different species. Nevertheless, the gut microbiome does not seem to be affected by NP and therefore is not considered the mechanism behind NP. Finally, studies on both zebrafish and largemouth bass presented major shifts in the gut microbiome as the fish aged. In addition, the core microbiomes of both species appeared to become more pronounced as the fish become adults. There seem to be an evolutionary tie between host and its gut microbiome. More studies, however, should further investigate this and the genetic effects on gut microbiota development and its heritability.

Development and the associated cellular differentiation are some of the most fundamental processes in biology. Since the early conception of the Waddington landscape, with cells portrayed as rolling down a landscape, understanding these processes has been at the forefront of biology. Progress in tissue regeneration, organoid culture, and cellular reprogramming relies on our ability to unfold cellular decision making and its dynamics. In this thesis, we ask to what extent development follows such landscape. Secondly, we address whether cellular branching points are discrete events. Given the recent surge in single-cell genomics data, we can now address these fundamental questions. To this end, we analyzed two large-scale single-cell RNAseq time course datasets from vertebrate embryogenesis in zebrafish. From the Waddington analogy, we expect the cell-to-cell correlation to increase across development as cells specialize. Our analysis does not show a linear trend, but rather, that cell-to-cell variability is lowest during gastrulation. Interestingly, the two different datasets from two different laboratories display a qualitatively similar trend, providing internal consistency of our analysis. To uncover the branchpoint dynamics, we extended our analysis to include computations of gene-to-gene correlations. It has been shown, using PCR data, that the transition index, the ratio between cell-to-cell and gene-to-gene correlations, displays a peak during such branchpoints, suggesting discrete transitions. To this end, we tracked individual developmental trajectories, and characterized both correlations, enabling computation of the transition index. However, the cell-to-cell correlation and gene-to-gene correlation did not follow a generic inverse relationship, as previously suggested. No unique signal corresponding to the branchpoints could, thus, be detected. Therefore, our analysis does not support the view that branchpoints during vertebrate embryogenesis are discrete, well-defined transition events. In conclusion, this first large-scale single-cell based analysis of time-resolved developmental data does not support a downhill rolling ball notion where cells decide their fate at discrete transition points. The temporal organization of an undulating developmental landscape appears to be more complex than initially conceptualized by Waddington. Therefore, it is of paramount interest to extend this type of analysis to other systems and to develop techniques to compute such landscape in a data-driven manner.

<p>Iodine is considered an essential nutrient as lack can cause severe metabolic and neurological issues in adults, with the added consequence of permanent developmental damage in children and infants. However, excessive iodine intake can result in similar symptoms, with a wide variance in adverse health outcomes. The safe range of iodine intake may be relatively low, with some studies suggesting the possibility of a high frequency of subclinical cases of iodine poisoning going unnoticed or misdiagnosed. </p> <p>In this study, the zebrafish model was tested as an integrative whole animal model to demonstrate behavioral, morphological, and genetic responses to overt and subclinical iodine poisoning in developing humans. Zebrafish embryos were treated with sodium iodide (NaI) immediately after fertilization. Survivability was monitored every 24 hours until 120 hours post fertilization (hpf). Concentrations with no statistical significance on survival, plus the smallest dose of significant lethality were then examined using behavioral analysis at 120 hpf to compare both overt and subclinical outcomes. Morphology measurements of body length, head length, head width, brain length, swim bladder volume, jaw length, and ventral dissension were also recorded at 120 hpf. Gene expression of <i>slc5a5</i>, <i>tpo</i>, and <i>tshba</i> at 72 hpf was also measured using quantitative PCR (qPCR). </p> <p>A significant decrease in survival rates were observed at 24 hpf for 25, 37.5, and 50 mM NaI treatments (p<0.0001). Morphological measurements taken at 120 hpf showed a significant increase in body length, head length, head width, jaw length, and swim bladder volume in the 10 mM NaI treatment group (p<0.0001) and a significant decrease in body length, head length, jaw length, and swim bladder volume in the 25 mM treatment group (p<0.0001). A ventral distension also developed near the location of the thyroid gland exclusively in the 25 mM group. </p> <p>Behavioral analysis showed significant increases in movement for both the 10 mM and 25 mM treatment groups during dark phases (p<0.0001). The 25 mM treatment group had an increase in movement during dark phases for standard well environments (p<0.0001), but this did not hold true for larger well environments, instead trending towards a non-significant decrease (p>0.05). The 10 mM group had a significant decrease during the first light phase in standard wells (p=0.002), with a significant increase in the second light phase for large wells (p=0.005). There were no significant changes in the expression of selected genes associated with the thyroid pathway (<i>slc5a5</i>, <i>tpo</i>, or <i>tshba</i>) across all treatment groups (p>0.05). </p><p><br></p> <p>Overall, the results suggest zebrafish larvae exhibit both overt and subclinical symptoms of excess iodine intake. Future studies are needed to determine internalization, biodistribution, clearance, and further characterization of adverse outcomes along the thyroid pathway for additional exploration into subclinical thyrotoxicosis due to excess iodine intake. Researchers should express caution with time points, as the Wolff-Chaikoff effect may influence exposure windows in zebrafish. </p>

Psychostimulants are known to alter visual function acutely and on withdrawal, and can cause attention deficit following prenatal exposure. However, psychostimulants can also improve visual attention in patients with attention deficit. The mechanisms involved in these contrasting effects remain largely unknown. To determine the role of specific brain regions and the dopamine system in the impact of cocaine exposure on visual processing, we employed two-photon microscopy and a transgenic larval zebrafish expressing the calcium indicator GCaMP-HS. We documented neuronal responses to contrasting visual stimuli, red light (LF) and dark (DF) flashes. We found that in the optic tectum neuropil (TOn), both stimuli elicited similar responses, though after repeated stimulus presentation, habituation developed to dark flash only. The dorsal telencephalon (dTe) responded and habituated to LF only. Acute cocaine (0.5 μM) reduced neuronal responses to LF in both brain regions and prevented habituation of dTe neurons to LF, but did not modify responses or habituation to dark flash. Prenatal cocaine exposure (PCE) did not modify baseline responses, but it prevented the acute effects of cocaine on LF responses in both regions and habituation in dTe, with no impact on dark flash responses. PCE also significantly reduced D1 dopamine receptor expression in TOn and cerebellum, but not dTe or the eye. Fish lacking the dopamine transporter (DATKO) retained normal D1 expression throughout the brain, baseline responses to LF in both TOn and dTe, and response reduction following cocaine in TOn. However, they demonstrated abnormal swimming behavior, and neither their swimming behavior nor dTe responses to LF were modified by cocaine. We discovered that in zebrafish, a diurnal vertebrate, responses to light not only require the primary visual processing center TO (superior colliculus in mammals), but also higher level processing by dTe. Responses to light but not darkness are modified by cocaine, unless the fish lack DAT or were exposed to PCE. Together, our results demonstrate specific effects of cocaine on visual processing mediated by the dopamine system, and provide a novel animal model for further investigation of these phenomena and development of new therapeutic approaches.