A Positive Regulatory Loop between foxi3a and foxi3b Is Essential for Specification and Differentiation of Zebrafish Epidermal Ionocytes

BACKGROUND: Epidermal ionocytes play essential roles in the transepithelial transportation of ions, water, and acid-base balance in fish embryos before their branchial counterparts are fully functional. However, the mechanism controlling epidermal ionocyte specification and differentiation remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In zebrafish, we demonstrated that Delta-Notch-mediated lateral inhibition plays a vital role in singling out epidermal ionocyte progenitors from epidermal stem cells. The entire epidermal ionocyte domain of genetic mutants and morphants, which failed to transmit the DeltaC-Notch1a/Notch3 signal from sending cells (epidermal ionocytes) to receiving cells (epidermal stem cells), differentiates into epidermal ionocytes. The low Notch activity in epidermal ionocyte progenitors is permissive for activating winged helix/forkhead box transcription factors of foxi3a and foxi3b. Through gain- and loss-of-function assays, we show that the foxi3a-foxi3b regulatory loop functions as a master regulator to mediate a dual role of specifying epidermal ionocyte progenitors as well as of subsequently promoting differentiation of Na(+),K(+)-ATPase-rich cells and H(+)-ATPase-rich cells in a concentration-dependent manner. CONCLUSIONS/SIGNIFICANCE: This study provides a framework to show the molecular mechanism controlling epidermal ionocyte specification and differentiation in a low vertebrate for the first time. We propose that the positive regulatory loop between foxi3a and foxi3b not only drives early ionocyte differentiation but also prevents the complete blockage of ionocyte differentiation when the master regulator of foxi3 function is unilaterally compromised

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs–the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs

Zebrafish Egg Infection Model for Studying Candida albicans Adhesion Factors.

Disseminated candidiasis is associated with 30-40% mortality in severely immunocompromised patients. Among the causal agents, Candida albicans is the dominant one. Various animal models have been developed for investigating gene functions in C. albicans. Zebrafish injection models have increasingly been applied in elucidating C. albicans pathogenesis because of the conserved immunity, prolific fecundity of the zebrafish and the low costs of care systems. In this study, we established a simple, noninvasive zebrafish egg bath infection model, defined its optimal conditions, and evaluated the model with various C. albicans mutant strains. The deletion of SAP6 did not have significant effect on the virulence. By contrast, the deletion of BCR1, CPH1, EFG1, or TEC1 significantly reduced the virulence under current conditions. Furthermore, all embryos survived when co-incubated with bcr1/bcr1, cph1/cph1 efg1/efg1, efg1/efg1, or tec1/tec1 mutant cells. The results indicated that our novel zebrafish model is time-saving and cost effective

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs–the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs.Sin financiación3.152 JCR (2017) Q1, 2/21 Anatomy & Morphology; Q2 123/261 NeurosciencesUE

Localization of OG1 <i>Candida albicans</i> cells in zebrafish egg bath infection model.

<p>Embryos were co-incubated with 1 × 10⁶ cells/mL of OG1 <i>C</i>. <i>albicans</i>. The representative slices of confocal images (a-c) are shown. The distance between two slices was approximately 55 μm. The whole merged images are presented (d). The phase contrast photos showing <i>C</i>. <i>albicans</i> hyphae were taken by an inverted microscope (e, f). f is the enlargement of the arrow area in e. Scale bars = 200 μm.</p

Conditions for the zebrafish egg bath infection model (mean of survival rate ± standard deviation).

<p>EW: egg water; R: RPMI; S: 10% FBS; 10⁶:1 × 10⁶ cells/mL;10⁷:1 × 10⁷ cells/mL</p><p>This data are from 3 repeat experiments. Approximately 30 embryos were tested for each treatment.</p><p>Conditions for the zebrafish egg bath infection model (mean of survival rate ± standard deviation).</p

Virulence of <i>C</i>. <i>albicans</i> mutant strains in the infection model.

<p>(A) Survival rates of embryos. Embryos alone (Control) or embryos with 5 × 10⁵ cells/mL of <i>bcr1/bcr1</i>, <i>cph1/cph1 efg1/efg1</i>, <i>efg1/efg1</i>, <i>tec1/tec1</i>, <i>cph1/cph1</i>, <i>sap6/sap6</i>, or WT (SC5314) cells in RPMI/serum were incubated at 30°C for 4 h. Survival rates were determined after an additional 1 day and 2 days of incubation. (B) Representative embryos were co-incubated with (a) <i>bcr1/bcr1</i>, (b) <i>cph1/cph1 efg1/efg1</i>, (c) <i>efg1/efg1</i>, (d) <i>tec1/tec1</i>, (e) <i>cph1/cph1</i>, (f) <i>sap6/sap6</i>, or (g) WT (SC5314) cells, and photographed after an additional 2 days of incubation. Scale bar = 200 μm. The data are from 4 repeat experiments. Approximately 70 embryos were tested for each strain.</p