The Dlx homeobox genes encode homeodomain transcription factors that are involved in multiple developmental aspects. In the brain, these genes take part in neuronal migration and differentiation, more precisely in the migration and differentiation of GABAergic neurons. Dysfunctions in the GABAergic system can lead to various pathological conditions, where impaired inhibitory function is one of the main causes of several neuropathies characterized by neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly conserved cis-regulatory elements have been previously characterized to be fundamental for the regulation of Dlx expression in developing embryos of different vertebrates. The activity of these regulatory elements and the Dlx genes has been well studied in developmental stages of mice and zebrafish, but little is known about their activity in the adult brain. The extensive neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the visualization of mechanisms involving dlx genes during adulthood and their possible involvement in adult neurogenesis. Here we show novel information concerning the expression of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity of cells that express dlx. We also demonstrate the involvement of dlx genes in brain regeneration and through lineage tracing, their fate determination in the adult zebrafish brain. Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the forebrain and midbrain throughout adulthood and expression is found in almost all areas presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal identity in the adult forebrain where, in some areas they were identified as the Calbindin subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing cell co-localized with a marker for neural stem cells. However, cells expressing dlx iii genes did not co-localize with markers for proliferating cells or for glia. Investigations during brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more specifically, dlx5a, is up regulated during the peak of regeneration response proposing a possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas of the adult brain where they seem to become mature neurons. Our observations provide a better understanding about the role of dlx genes during adulthood, further contributing to the general knowledge of the molecular pathways involved in adult neurogenesis and regeneration in the zebrafish adult brain.

Wnt signaling is an evolutionarily conserved pathway that is essential for the development of the metazoan embryo. Wnt signaling controls essential developmental processes including cell fate, cell polarity, dorsal-ventral patterning and tissue movement. Misregulated Wnt signaling can have disastrous effects on the developing human embryo, leading to potentially fatal congenital malformations including anencephaly and spina bifida. In addition to embryonic development, misregulated Wnt signaling has been implicated in human pathologies including colon and breast cancers and skeletal malformations. Wnt signaling is divided into two main pathway branches, canonical or beta-catenin dependent, and non-canonical, sometimes referred to as the planar cell polarity (PCP) pathway. The former branch activates the transcription of the downstream target genes leading to the patterning the dorsal-ventral axis of the developing embryo, whilst the latter has no downstream transcriptional targets but rather acts upon the cytoskeleton to control cell and tissue polarity and movement. Wnt signaling bifurcates into these two branches at the level of the protein Dishevelled (Dvl). The Dishevelled-associated activator of morphogenesis 1 (Daam1) protein was identified via a yeast-two hybrid screen using Dvl as bait. Daam1 interacts directly with Dvl and mediates activation of the small GTPase Rho, a key player in non-canonical Wnt signaling necessary for proper gastrulation in the Xenopus (frog) embryo. In addition to Daam1, vertebrates possess a second Daam, Daam2, originally identified via an in silico screen in humans. Similar to Daam1, frog Daam2 participates in non-canonical Wnt signaling, contributing to proper formation of the embryonic neural tube. However, conflicting opinions on the function of Daam2 have led to discrepancies regarding its position in Wnt signaling and function in development. Daam1/2 have not been extensively investigated at the genetic level, therefore, I employed the genetic model zebrafish (Danio rerio) to further clarify their role in Wnt signaling. Using techniques such as the latest gene-editing system CRISPR/Cas9 and other well-established molecular methods including in situ hybridization, RT-PCR and knockdown using morpholino oligonucleotides, I sought to further establish the role of the Daam protein family in vertebrate embryonic development. Together, my results indicate that the zebrafish Daam1a/b and Daam2 behave similarly to Daam1 and Daam2 in frog, respectively, by participating in the non- canonical Wnt signaling pathway and mediating morphology in the developing zebrafish embryo. / Biology

Rag1-/- mutant zebrafish lack lymphocytes and were used to study the basis of acquired protective immunity in the absence of lymphocytes to the intracellular bacterium Edwardsiella ictaluri. This study morphologically identified and quantified lymphocyte like cells (LLCs) present in the liver, kidney and spleen of these fish. LLCs included Natural Killer (NK) cells and non-specific cytotoxic cells (NCCs) and were discriminated by size, and by the presence of cytoplasmic granules. The antibodies anti-NITR9, anti-NCCRP-1 (5C6) and anti-MPEG-1 were used to evaluate these cell populations by flow cytometry. Gene expression profiles in these tissues were evaluated after the Rag1-/- mutants were intra coelomically injected with the toll like receptor (TLR)-2 ligand, β glucan, TLR3 ligand, Poly I:C, or TLR 7/8 ligand, R848. The genes interferon y (infγ), expressed by activated NK cells and macrophages, tumor necrosis factor α (tnfα), expressed by activated macrophages, myxovirus resistance (mx) expressed by cells induced by IFNα, T-cell transcription factor (t-bet) expressed by NK cells and novel immune type-receptor 9 (nitr-9) expressed by NK cells were evaluated. The TLR ligands induced different patterns of expression and stimulated both macrophages and NK cells. Then fish were vaccinated with an attenuated mutant of E. ictaluri (RE33®) with or without the TLR ligands then challenged with WT E. ictaluri to evaluate protection. RE33® alone and each TLR ligand alone provided protection. Coministration of β glucan and RE33® or R848 and RE33® resulted in survival higher than RE33® alone showing an adjuvant effect. Tissue specific gene expression of ifnγ, t-bet, nitr9, NK cell lysin a (nkla), nklb, nklc and nkld were correlated to protection. The final component of this study was the development of tools to discriminate NK cell populations and evaluate the contribution of macrophages. Rag1-/- zebrafish were modified to express cherry red in lymphocyte like cells using the Lymphocyte specific tyrosine kinase (lck) promotor. Also, rag1-/- zebrafish were modified so that the gene encoding the proto-oncogene serine/threonine-protein kinase that is involved in macrophage training (raf1) is disrupted. This study indicated that the acquired protection in the absence of lymphocytes likely involves NK cells with possible contribution by macrophages.

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Butylparaben (Butyl p-hydroxybenzoic acid) is a widely used cosmetic and pharmaceutical preservative that has been recently shown to induce oxidative stress and have endocrine disrupting effects in rodents, and promote adipocyte conversion of human adipose cells. Embryonic development is extremely sensitive to oxidative stress due to changes in cell growth, development and differentiation that occur during this life stage. Fluctuations in redox potentials play critical roles in normal embryonic development by guiding these cell signaling, cell-fate decisions and apoptosis. The most prevalent endogenous antioxidant that defends against oxidative stress is glutathione (GSH), which scavenges reactive oxygen species. The low antioxidant capacity of pancreatic beta cells suggests that they are sensitive target tissues of oxidative stress; this has yet to be investigated during embryonic development. Here, we aim to 1) determine whether embryonic exposure to butylparaben prompts structural and functional changes in the developing endocrine pancreas and 2) determine whether oxidative stress may be involved. Transgenic insulin-GFP zebrafish embryos were treated daily with 250, 500, 1,000 and 3,000 nM butylparaben starting at 3 hours post fertilization (hpf). Pancreatic islet and whole embryo morphological development were examined daily until 7 days post fertilization (dpf). Redox potentials were measured at 24 and 28 hpf using HPLC. Area of the pancreatic islet increased over time with increasing butylparaben exposure in a dose-dependent manner by as much as a 55% increase in islet area at 3 dpf when compared to controls. Butylparaben concentrations of 500 and 1,000 nM increased GSH by 10 and 40%, respectively, and decreased oxidized glutathione disulfide by 37 and 59%. GSH redox potentials were only significant in embryos collected at 28 hpf and became more reduced with 500 and 1,000 nM butylparaben exposure, decreasing redox potentials by 7 and 18 mV, respectively. Cysteine redox potentials also became more reduced, decreasing by 17 and 28 mV. Our data show that butylparaben-induced redox potential disruptions that may be responsible for the effects on pancreatic islet structure and function, but further studies are needed to determine how and if that directly affects pancreas development.