Gaia Gestri

University College London | UCL · Division of Biosciences

Appropriate patterning of the retina during embryonic development is assumed to underlie the establishment of spatially localised specialisations that mediate the perception of specific visual features. For example, in zebrafish, an area involved in high acuity vision (HAA) is thought to be present in the ventro-temporal retina. Here, we show that...

Neurons on left and right sides of the nervous system frequently show asymmetric properties but how these differences arise is poorly understood. Through a forward genetic screen in zebrafish, we find that loss of function of the transmembrane protein Cachd1 results in right-sided habenular neurons adopting left-sided character. Cachd1 is expressed...

Appropriate patterning of the retina during embryonic development is assumed to underlie the establishment of spatially localised specialisations that mediate the perception of specific visual features. For instance, in zebrafish, an area involved in high acuity vision (HAA) is thought to be present in the ventro-temporal retina. Here we show that...

Appropriate patterning of the retina during embryonic development is assumed to underlie the establishment of spatially localised specialisations that mediate the perception of specific visual features. For example, in zebrafish, an area involved in high acuity vision (HAA) is thought to be present in the ventro-temporal retina. Here, we show that...

Zebrafish provide a unique opportunity for drug screening in living animals, with the fast-developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-...

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotyp...

Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed a easy-t...

Background: Shaping the vertebrate eye requires evagination of the optic vesicles. These vesicles subsequently fold into optic cups prior to undergoing neurogenesis and allocating a population of late progenitors at the margin of the eye. mab21l2 encodes a protein of unknown biological function expressed in the developing optic vesicles, and loss...

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes reveal...

ELife digest Left and right eyes develop independently, yet they consistently grow to roughly the same size in humans and other creatures. How they do this remains a mystery, though scientists have learned that both eyes originate from a single group of cells in the developing nervous system called the eye field. As development progresses, the eye...

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes reveal...

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mappi...

Misregulation of expression of genes potentially implicated in midline axon guidance in slbpty77e mutants. Views of heads/brains of wildtype (A-H) and slbpty77e (A’-H’) embryos showing expression of genes indicated to the left of each row. Genotype is indicated at top of each column. Lateral views (A,A’,C,C’,E,E’); dorsal views (B,B’,D,D’,F,F’,G,G’...

List of transcripts with differential expression between wildtype and slbpty77e mutants. Unprocessed transcript list derived from the differential expression analysis performed on the BAM files from all three biological replicates and the merged transcript dataset using Cuffdiff. (XLSX)

Gene list used for GO term enrichment analysis for “Biological Process” on all of the upregulated genes showing a significant change in expression (q value ≤ 0.01) in our RNAseq data. (Sheet 1) Upregulated genes sorted by q value. (Sheet 2). Upregulated genes sorted by log2(fold change). (Sheet 3) List of GO terms related to “Biological Process” ge...

Manually curated list of genes showing significant changes in expression level related to nervous system development, cell cycle and histones. (Sheet 1) Downregulated genes with a log2(fold change >-2) related to neural Development, axon pathfinding and synaptogenesis. (Sheet 2) Upregulated genes related to cell cycle. (Sheet 3) Histone related gen...

Morpholino, Rescue and supplementary phenotype data. (A-A’) Analysis of live 50hpf Control (A) and Morpholino injected embryos (A’) show that the morphological dent caudal to the MHB (white arrow), indentations in the retina (black arrow) and coloboma are successfully phenocopied. (B) Electrophoresis gel of RT-PCR analysis confirmed that several mi...

Upregulation of cell cycle markers in slbpty77e mutants. Images of wildtype (wt, A-F) and slbpty77e (A’-F’) heads (A-C’; E,E’) and eyes (D-F’) at 60hpf showing expression of genes indicated to the left of each row. Genotypes indicated at top of each column. Lateral (A,A’; C-F’) and dorsal view (B,B’). Scale bars: 100μm. (TIF)

Gene list used for GO term enrichment analysis for “Biological Process” on all of the downregulated genes showing a significant change in expression (q value ≤ 0.01) in our RNAseq data. (Sheet 1) Downregulated genes sorted by q value. (Sheet 2). Downregulated genes sorted by log2(fold change). (Sheet 3) List of GO terms related to “Biological Proce...

Table S1. PTMscan phosphoSerine Dataset of HDMECs si-NRP1 or si-Control, Related to Figures 3 and S2

The transmembrane protein neuropilin-1 (NRP1) promotes vascular endothelial growth factor (VEGF) and extracellular matrix signaling in endothelial cells (ECs). Although it is established that NRP1 is essential for angiogenesis, little is known about its role in EC homeostasis. Here, we report that NRP1 promotes mitochondrial function in ECs by prev...

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte ( ele ) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic map...

The vertebrate eye originates from the eyefield, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7la is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eyefield in tcf7l1a mutants, these fish develop normal eyes revealing...

Coloboma is a defect in the morphogenesis of the eye that is a consequence of failure of choroid fissure fusion. It is among the most common congenital defects in humans and can significantly impact vision. However, very little is known about the cellular mechanisms that regulate choroid fissure closure. Using high-resolution confocal imaging of th...

Loss of α-catenin-labeled adherens junctions from the fusing choroid fissure. Time lapse movie of the choroid fissure from a Tg(ctnna:citrine)ct3aGT embryo from 38 to 48 hpf. Maximum projection of 10 Z-sections per frame with an interval between frames of 10 min.

Cells lining the choroid fissure can show protrusive activity. Time-lapse movie of the choroid fissure in which all nuclei are labeled with nuclear-localized GFP (green) and transplanted cells are labeled with membrane targeted RFP (magenta). Z projections of sections covering 6 μm with an interval between frames of 10 min.

Cells lining the choroid fissure cells are proliferative. Time-lapse movie of the ventral eye in which all cells express membrane targeted RFP and one cell in the fissure margin expressing cytoplasmic GFP undergoes cell division. Z projections of sections covering 6 μm with an interval between frames of 10 min.

Transplanted optic vesicles are depleted of neural crest. Time-lapse movie of Tg(sox10:GFP)zf77 neural crest cells (green) around the transplanted optic vesicle labeled in red (mRFP-positive), showing the presence of few neural crest cells. The olfactory placode (to the left in the movie) has also been transplanted. Z projections of sections coveri...

Recruitment of host neural crest cells to a transplanted optic vesicle. Time-lapse movie of Tg(sox10:GFP)zf77 cells (from the host) approaching from the right and interacting with a transplanted optic vesicle (the transplanted optic vesicle is visible in red; mRFP-positive) on the yolk. Z projections of sections covering 20 μm with an interval betw...

Optic vesicle transplantation and POM migration. (A) Schematic showing the transplant procedure. Optic vesicles (ov) from Tg(sox10:GFP)zf77 embryos injected at the 1-cell stage with RNA encoding mRFP were transplanted at between 7 and 18 somites (ss, about 12–18 hpf) under the skin above the yolk of age-matched Tg(sox10:GFP)zf77 host embryos. The l...

Failure of choroid fissure fusion in eyes derived from optic vesicles with reduced POM. (A–F) Methylene blue stained sagittal sections through ectopic eyes at 5 days post-fertilization. (A) Image of a section through a wild-type eye at 72 hpf. (B,C) Low resolution images of sections through an embryo with an ectopic eye on the yolk at the level of...

Some cells lining the choroid fissure can move toward the inner surface of the retina. Time lapse movie from 38 to 50 hpf of the choroid fissure in an embryo injected with RNA encoding H2B:RFP and Pard3:GFP. Individual nuclei are manually tracked showing that as Pard3 labeling retracts toward the outer retina, some nuclei of cells initially lining...

Periocular mesenchyme cells are actively motile in the choroid fissure. Time-lapse movie of an eye with all cell membranes labeled by the Tg(β-actin:HRAS-EGFP)vu119 transgene. Z projections of sections covering 18 μm with an interval between frames of 10 min.

The hyaloid artery is displaced superficially during choroid fissure fusion. Time-lapse movie of an eye with cell membranes labeled by mGFP injection. Z projections of sections covering 30 μm with an interval between frames of 10 min. Lateral view (rotated to look into the fissure) of the hyaloid vasculature.

Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the cili...

Sprouting blood vessels are led by filopodia-studded endothelial tip cells that respond to angiogenic signals. Mosaic lineage tracing previously revealed that NRP1 is essential for tip cell function, although its mechanistic role in tip cells remains poorly defined. Here, we show that NRP1 is dispensable for genetic tip cell identity. Instead, we f...

The earliest known determinants of retinal nasotemporal identity are the transcriptional regulators Foxg1, which is expressed in the prospective nasal optic vesicle, and Foxd1, which is expressed in the prospective temporal optic vesicle. Previous work has shown that, in zebrafish, Fgf signals from the dorsal forebrain and olfactory primordia are r...

The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate spec...

During ocular morphogenesis the retinal pigment epithelium (RPE) and neural retina segregate fates from a bi-potential progenitor pool of cells comprising the optic vesicle. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specifica...

Vertebrate eye formation is a multistep process requiring coordinated inductive interactions between neural and non-neural ectoderm and underlying mesendoderm. The induction and shaping of the eyes involves an elaborate cellular choreography characterized by precise changes in cell shape coupled with complex cellular and epithelial movements. Conse...

Sprouting blood vessels are led by filopodia-studded endothelial tip cells that respond to angiogenic signals. Mosaic lineage tracing previously revealed that NRP1 is essential for tip cell function, although its mechanistic role in tip cells remains poorly defined. Here, we show that NRP1 is dispensable for genetic tip cell identity. Instead, we f...

Background Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elabora...

Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex w...

tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt-Oram syndrome (HOS), a highly...

tbx5 and miR-218 are co-expressed in mouse tissues. A-D, relative expression of tbx5, slit2, slit3 and miR-218 as evaluated by q-RT-PCR in different newborn mouse tissues. Results are standardized against GAPDH for genes, and against U6 for miRNAs. Values represent the averages and standard deviations of at least two independent experiments. (TIF)

Expression of pre-miR-218-1 parallels that of mature miR-218 during mouse differentiation and tbx5 modulation. Q-RT-PCR detection of pre-miR-218-1 and pre-miR-218-2 relative expression in P19CL6 cells during differentiation (A), or 48 h after plasmids or siRNA transfection (B). In B fold changes of CMV-Tbx5 and siRNA-Tbx5 are relative to CMV-empty...

Rescue of cardiac defects induced by miR-218a over-expression was accomplished by co-injecting MO-218a. A, qRT-PCR analysis of miR-218a relative expression in 24 hpf embryos microinjected with 12 ng of control morpholino (MO-Ct) or MOD-218a and with 260 pg of miR-Ct or miR-218a mimic. miR-218a relative expression was calculated as the ratio between...

miR-218 dysregulation does not affect vascular integrity. Confocal images of representative 72 hpf Tg(flk1:eGFP) embryos injected with 260 pg of miR-Ct (A), 260 pg of miR-218 mimic (B) or 8 ng MOD-218 (C). Black and white scale bars: 100 µm. (TIF)

Injection of miR-218a in Tbx5a morphants increases the severity of heartstring phenotype. A, phenotypic analysis of Tbx5a morphants co-injected with 1 ng of MO-Tbx5a and either 130 pg of miR-218a mimic or 130 pg of miR-Ct. B, representative images of 48 hpf embryos showing the edema expansion caused by the co-injection of miR-218a mimic. C, phenoty...

Schematic representation of the bioinformatic tool developed to identify in silico Tbx5-controlled miRNAs. Ensembl was used to obtain information about chromosome location, position and segment of the selected genes. Ensembl is a joint scientific project between the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/clustalw/) and the Wel...

tbx5 and miR-218a misexpression does not alter amhc and vmhc cardiac marker expression in zebrafish embryos. Ventral views of 48 hpf embryos injected with the indicated miRNA mimics (260 pg) or MOs (12 ng MO-Ct and MOD-218a, 3 ng MO-Tbx5a) after mRNA in situ hybridization. Scale bar 100 µm. (TIF)

miR-218 targets the 3′ UTR of robo1 in zebrafish embryos. Top: schematic representation of sensors and miRNAs used for in vivo sensor assay. Bottom: examples of 24 hpf embryos microinjected with 40 pg of RFP mRNA, 400 pg of 3′UTR robo 1 sensor and 160 pg of miR-Ct (a,c,e) or miR-218a (b,d,f). In figures C and D the percentage of the relative phenot...

MO-Tbx5a and MO-Tbx5b effectively knockdown the two zebrafish tbx5 isoforms. A-D, 35 pg of pCS2 plasmid expressing GFP fused with MO-Tbx5a or MO-Tbx5b target sequences were injected in one-cell stage embryos in the absence (A and C) or in the presence (B and D) of 1,5 ng of the relative morpholino. Representative fluorescent images of 24 hpf embryo...

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger a...

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence, there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually driven behaviors in the newly hatched larvae.The ability to exploit the genetic and embryon...

Retinoic acid receptor (RAR) signaling is required for morphogenesis of the ventral optic cup and closure of the choroid fissure, but the mechanisms by which this pathway regulates ventral eye development remain controversial and poorly understood. Although previous studies have implicated neural crest-derived periocular mesenchyme (POM) as the cri...

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish devel...

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screen...

Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add ne...

Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37...

To gain insight into the mechanisms of Lmx1b function during ocular morphogenesis, we have studied the roles of lmx1b.1 and lmx1b.2 during zebrafish eye development. In situ hybridization and characterization of transgenic lines in which GFP is expressed under lmx1b.1 regulatory sequence show that these genes are expressed in periocular tissues and...

Although it is well established that Six3 is a crucial regulator of vertebrate eye and forebrain development, it is unknown whether this homeodomain protein has a role in the initial specification of the anterior neural plate. In this study, we show that exogenous Six3 can expand the anterior neural plate in both Xenopus and zebrafish, and that thi...

In Xenopus neuroectoderm, posterior cells start differentiating at the end of gastrulation, while anterior cells display an extended proliferative period and undergo neurogenesis only at tailbud stage. Recent studies have identified several important components of the molecular pathways controlling posterior neurogenesis, but little is known about...

Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to in...

We investigated the function of Xrx1 during Xenopus retinogenesis. Xrx1 overexpression lengthens mitotic activity and ectopically activates the expression of markers of undifferentiated progenitors in the developing retina. We assayed Xrx1 ability to support proliferation with a cell-autonomous mechanism by in vivo lipofection of single retinal pro...

vax2 is a recently isolated homeobox gene, that plays an important role in controlling the dorso-ventral patterning of the retina. In this paper we present a thorough description of the Xvax2 expression pattern all along Xenopus embryogenesis, and compare this pattern in detail to that shown by Xvax1b and Xpax2, two genes also involved in ventral e...

Vertebrate eye formation is a complex process which involves early specification of the prospective eye territory, induction events, patterning along the polarity axes and regional specification, to bring about the proper morphogenetic movements, cell proliferation, cell differentiation and neural connections allowing visual function. The molecular...