Epithelial organization and cyst lumen expansion require efficient Sec13-Sec31-driven secretion

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPII-dependent secretion, notably assembly of Sec13–Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture

Phenotypic analysis of images of zebrafish treated with Alzheimer's γ-secretase inhibitors

<p>Abstract</p> <p>Background</p> <p>Several γ-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer's disease (AD). This enzyme mediates the proteolytic cleavage of amyloid precursor protein (APP) to generate amyloid β protein, Aβ, the pathogenic protein in AD. The γ-secretase also cleaves Notch to generate Notch Intracellular domain (NICD), the signaling molecule that is implicated in tumorigenesis.</p> <p>Results</p> <p>We have developed a method to examine live zebrafish that were each treated with γ-secretase inhibitors (GSI), DAPT {N- [N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine <it>t</it>-Butyl Ester}, Gleevec, or fragments of Gleevec. These compounds were first tested in a cell-based assay and the effective concentrations of these compounds that blocked Aβ generation were quantitated. The mortality of zebrafish, as a result of exposure to different doses of compound, was assessed, and any apoptotic processes were examined by TUNEL staining. We then used conventional and automatic microscopes to acquire images of zebrafish and applied algorithms to automate image composition and processing. Zebrafish were treated in 96- or 384-well plates, and the phenotypes were analyzed at 2, 3 and 5 days post fertilization (dpf). We identified that AD95, a fragment of Gleevec, effectively blocks Aβ production and causes specific phenotypes that were different from those treated with DAPT. Finally, we validated the specificity of two Notch phenotypes (pigmentation and the curvature of tail/trunk) induced by DAPT in a dose-dependent manner. These phenotypes were examined in embryos treated with GSIs or AD95 at increasing concentrations. The expression levels of Notch target gene <it>her6 </it>were also measured by <it>in situ </it>hybridization and the co-relationship between the levels of Notch inhibition by DAPT and AD95 and the severity of phenotypes were determined.</p> <p>Conclusion</p> <p>The results reported here of the effects on zebrafish suggest that this newly developed method may be used to screen novel GSIs and other leads for a variety of therapeutic indications.</p

Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of origin recognition complex factors

Meier–Gorlin syndrome (MGS) is a rare, congenital primordial microcephalic dwarfism disorder. MGS is caused by genetic variants of components of the origin recognition complex (ORC) consisting of ORC1–6 and the pre-replication complex, which together enable origin firing and hence genome replication. In addition, ORC1 has previously been shown to play a role in ciliogenesis. Here, we extend this work and investigate the function of ORC1 and two other members of the complex on cilia at an organismal level. Knockdown experiments in zebrafish confirmed the impact of ORC1 on cilia. ORC1-deficiency confers defects anticipated to arise from impaired cilia function such as formation of oedema, kidney cysts, curved bodies and left–right asymmetry defects. We found ORC1 furthermore required for cilium formation in zebrafish and demonstrate that ciliopathy phenotypes in ORC1-depleted zebrafish could not be rescued by reconstitution with ORC1 bearing a genetic variant previously identified in MGS patients. Loss-of-function of Orc4 and Orc6, respectively, conferred similar ciliopathy phenotypes and cilium shortening in zebrafish, suggesting that several, if not all, components of the ORC regulate ciliogenesis downstream to or in addition to their canonical function in replication initiation. This study presents the first in vivo evidence of an influence of the MGS genes of the ORC family on cilia, and consolidates the possibility that cilia dysfunction could contribute to the clinical manifestation of ORC-deficient MGS

Regulation of Early Zebrafish Embryogenesis by Calcium Signaling and Dachsous1b Cadherin

Early animal embryogenesis entails a dynamic combination of embryonic cleavages, axial patterning, and gastrulation movements to shape a basic body plan. The underlying molecular signaling responsible for regulating this process remains poorly understood. In this thesis work, I first review recent progress in understanding of gastrulation movements in various model organisms brought by advances in imaging techniques. The externally developing and optically translucent zebrafish embryo is an ideal model organism to study vertebrate embryonic development by in vivo imaging. The objective of my thesis research is to leverage experimental advantages in the zebrafish model to uncover novel regulators and elucidate the molecular mechanisms involved in early vertebrate embryogenesis. Calcium signaling has been implicated in the control of many aspects of embryonic development. However, the spatiotemporal dynamics of calcium signaling during embryogenesis are not well characterized. By generating stable transgenic zebrafish lines ubiquitously expressing GCaMP6s, a genetically encoded calcium indicator, I demonstrated higher activities of calcium signaling during cleavage and blastula stages compared to previous reports. In addition, I showed that superficial dorsal-biased calcium signaling during blastula and gastrula stages was strongly correlated with and dependent on the dorsal organizer establishment. In the developing gastrulae, I directly visualized calcium activity in the dorsal forerunner cells and showed it was modulated by Nodal signaling in a cell non-autonomous manner. The GCaMP6s transgenic lines revealed with unprecedented spatiotemporal resolution the dynamic calcium signaling during early zebrafish embryogenesis and provide a superior tool for future studies. In zebrafish, mutations in atypical cadherin dachsous1b/dchs1b cause pleiotropic embryonic defects, including abnormal cleavages. Using the GCaMP6s transgenic reporter to examine the furrow-associated calcium activity in zebrafish dchs1b mutants, I showed that abnormal cleavages in dchs1b mutants were due to furrow progression defects during cytokinesis. These defects were likely caused by misregulated microtubules, as in vivo imaging of fluorescently marked microtubules during cleavage stages revealed reduced microtubule dynamics and impaired midzone microtubule assembly in dchs1b mutants. I further identified Ttc28 cytoplasmic protein as a molecular link between Dchs1b and microtubule dynamics. My biochemical experiments revealed that Dchs1b physically interacts via its intracellular domain with the tetratricopeptide repeat domain of Ttc28, and controls its subcellular distribution. Moreover, genetic inactivation of ttc28 resulted in increased microtubule dynamics and suppressed the microtubule defects in dchs1b mutants, suggesting a mechanism through which Dchs1b controls embryonic cleavages. In the last part of my thesis, I aimed to determine whether the chemokine ligand Ccl19.a1, a potential upstream regulator of calcium signaling, is required for axial patterning in zebrafish. I demonstrated that TALEN-generated ccl19a.1 mutations produce mildly dorsalized phenotypes and partially suppress the ventralized ichabod/ctnnb2 mutant phenotypes to influence axis formation, providing a genetic evidence for Ccl19.1 acting as a negative regulator of β-catenin and axis formation. Together, my work make several advances in understanding early vertebrate embryogenesis: it characterizes dynamic calcium signaling during zebrafish embryogenesis with a superior spatiotemporal resolution, reveals that Dchs1b regulates microtubule dynamics and embryonic cleavages by interacting with Ttc28 and regulating its subcellular distribution, and provides genetic evidence that Ccl19a.1 is necessary to limit β-catenin activity and consequently axis formation in zebrafish

What makes cilia beat and how do different laterality mutations impact on heart morphology and physiology?

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, 2023, Universidade de Lisboa, Faculdade de CiênciasDespite their external bilateral symmetry, vertebrates are not as symmetrical as we think. Under their skin, several organs and corresponding vasculature display an asymmetric arrangement in the body cavity. This archetypal visceral patterning is the outcome of the left-right (LR) axis formation during embryonic development and is critical to ensure optimal organ functioning. For example, patients with laterality disorders tend to develop congenital cardiovascular malformations. In several species, in a transient organ known as LR organizer (LRO), directional fluid flow yielded by motile cilia beating has been reported to trigger asymmetric Nodal signaling expansion, which is critical for differentiating the left and right sides of the embryo. Zebrafish LRO comprises motile and immotile cilia and their balance is critical to develop an efficient fluid flow that promotes the correct positioning of visceral organs and, subsequently, healthy vertebrate development. Intriguingly, both types of cilia express cilia motilityrelated genes and are equipped with similar motility apparatus, and yet some beat and others do not. A previous study determined that the transcription repressor Her12 was a potential candidate to mediate this motility fate decision, and this hypothesis underpinned the beginning of this master thesis. We first aimed to ascertain the correlation between Her12 and cilia behavior by assessing her12 expression in the cilia population and identifying her12 targets. However, many pitfalls were encountered, and no conclusions could be drawn. Experiments must be repeated to solve this outstanding question. We also investigated how error-prone execution of asymmetric LR patterning in motile cilia and non-cilia mutants, ccdc40-/- and dand5-/- mutants, respectively, affected heart situs and morphogenesis. We characterized, for the first time, similar cardiovascular defects in both mutants. However, how the disrupted LR spatial-temporal signals impaired coordination between the heart situs and development remains to be enlightened and further studies should be conducted

Regulation of cardiogenesis by putative WNT signalling pathways

PhD ThesisThe Wnt/ -catenin and the Wnt/planar cell polarity (Wnt/PCP) signalling pathways have been shown to play important roles in cardiogenesis and their disruption has been shown to cause severe disturbances in heart development. Spatially and temporally complex interplays between the two pathways have been described. One component of the PCP pathway is Jnk, a member of the highly conserved mitogenactivated protein kinase (MAPK) family. This stress responsive mitogen is known to control a variety of cellular behaviours such as proliferation, apoptosis and cell migratory behaviour and as such, is likely to be of pivotal importance in cardiac development. The aim of this study was to investigate the role played by Jnk in vertebrate heart formation and the relationships between Jnk signalling and canonical Wnt signalling, using in silico and in vivo approaches in zebrafish and an in vitro approach on a mouse embryonic stem (ES) cell model of cardiogenesis. Firstly, using a range of bioinformatic methods, an analysis of jnk genes, splice variants and proteins, and an investigation of their phylogenetic relation with other species was undertaken. This suggested conservation of Jnk family members, but suggested that there were additional orthologues of jnk1 present in the zebrafish transcriptome. The spatial and temporal expression profiles of these genes were then examined by semi-quantitative PCR and in situ hybridisation. The functional role of Jnk proteins during zebrafish development was subsequently investigated using a specific chemical inhibitor, SP600125. Inhibition of Jnk signalling during gastrulation and somitogenesis caused a convergence extension-like phenotype and severe cardiac defects, including looping anomalies and alterations in atrial versus ventricular cell numbers. ES cells have the capacity to differentiate in vitro and give rise to cells of many different lineages, including cardiomyocytes. Canonical Wnt and Jnk components were manipulated during specific windows of differentiation as ES cells formed beating embryoid bodies. Examination of the spontaneous contractile behaviour of differentiating ES cells as they entered the cardiogenic lineage, and analysis of their developmental gene expression profiles, showed the beating behaviour of ES cellderived cardiac cells was enhanced in a temporally specific manner after inhibition of the non-canonical Wnt/Jnk pathway, while there was marked alteration of canonical Wnt signalling. To investigate whether there were reciprocal interactions between the two pathways, analysis of the system after activation of the canonical pathway was also undertaken. These studies indicated that the beating behaviour of ES cell-derived cardiac cells was enhanced in a temporally specific manner after inhibition of Jnk, while after activation of canonical Wnt/ -catenin signalling, the cardiogenic potential of differentiating ES cells was severely suppressed. The findings of this study extend our understanding of the role played by canonical and non-canonical Wnt signalling pathways in heart morphogenesis and highlight the interacting effects of related signalling pathways activity in cardiogenesis

Zebrafish as a Model for Determining the Mechanisms Causing Deafness in MYH9-Related Disease

Approximately 1 in 500 infants are diagnosed with hearing loss, and about half of these cases can be traced to genetic defects. Several hundred genes have been implicated in deafness, including MYH9, which codes for the conventional motor protein non-muscle myosin IIA (NMIIA). Mutations in MYH9 lead to syndromic MYH9-related diseases, which include deafness as a variable symptom, as well as non-syndromic autosomal deafness DFNA17. Despite its identification as a deafness gene, the functions of MYH9 in ear development and hearing remain unknown. To study this role, we will use zebrafish as a model. Zebrafish offer significant advantages including established genetic tools, large clutch sizes, and transparent embryos that develop externally. In addition, non-muscle myosin genes, including MYH9, are highly conserved from zebrafish to human, and the development and function of the vertebrate ear is also highly conserved. Zebrafish share many inner ear structures with humans including the sensory hair cells and cilia that make vestibular and auditory function possible. We hypothesize that myh9 plays a significant role in the developing zebrafish ear as suggested by myh9 knockdown experiments, which resulted in the phenotype of an abnormal number of otoliths. Because otoliths nucleate from precursor particles distributed in the fluid of the otic vesicle by motile cilia, we further hypothesize that the otolith phenotype is caused by a cilia defect. Our work has demonstrated that NMIIA colocalizes with ciliary basal bodies in the otic vesicle, suggesting a possible role in establishing basal body orientation

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

Regulated dicing of pre-mir-144 via reshaping of its terminal loop.

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs