Biology subjects

Rainger, J.

Publications and source records attributed to Rainger, J..

11 recordsLinked to original sources

bioRxiv2026

The regulatory landscape of optic fissure closure in the vertebrate eye

PurposeOptic fissure closure (OFC) is a critical tissue fusion event in normal eye development and its failure results in ocular coloboma (OC), an inferonasal ocular tissue defect that persists throughout life. Most OC cases lack a genetic diagnosis, reflecting limited understanding of the genes and pathways that regulate OFC. We aimed to determine if OFC gene expression is regulated by the non-coding genome and identify novel candidate loci for OFC/OC. MethodsUsing the embryonic chicken eye, we performed integrated profiling of accessible chromatin and gene expression patterns during OFC. Matched RNA-seq and ATAC-seq from distinct spatial and temporal eye tissues were utilised with bioinformatic tools to identify regulatory genomic elements and infer gene regulatory networks, and to map synonymous loci in the human genome. ResultsWe revealed domains of accessible chromatin during active fusion and the broader ventral retina that were distinct from those in the dorsal retina, implicating these loci for gene regulation during development and fusion of the optic fissure. In silico analysis using de novo motif enrichment and transcription factor (TF) foot-printing revealed TEAD, ZIC, and SOX TF activity during OFC, and retinoic acid signalling related TF activity in the dorsal eye. A subset of chicken OFC-specific peaks mapped to human cis-regulatory elements near known coloboma genes and OFC candidate genes identified in this study. ConclusionsThis provides the first evidence for dynamic cis-regulatory activity during OFC, identifies candidate loci for future mutational analysis, and offers new genetic leads for OC cases without a genetic diagnosis.

developmental biology↗

bioRxiv2025

FuChi: A cell cycle biosensor for investigating cell-cycle kinetics during avian development.

The ability to monitor the proliferative status of live cells both in vitro and in vivo over time has revolutionised our understanding of development, growth and disease. This was first made possible by fluorescent ubiquitination-based cell cycle indicator (Fucci) technology, which distinguishes specific cell cycle phases through the reciprocal degradation of fluorescently tagged, truncated forms of human CDT1 and GMNN proteins. Fucci genetic systems have been successfully implemented in transgenic mice, zebrafish, and axolotls. To date, no viable, stably expressing Fucci line has been developed in an avian species. Although a range of continuously improving Fucci constructs have been developed in recent years, existing in vivo Fucci models remain limited because they rely on older reporter technology that fails to distinguish cells in S, G2, and M phases or to label cells in early G1. As a result, these models can be challenging to interpret and their utility for continuous cell tracking and precise analysis of cell-cycle dynamics is limited. Here, we introduce FuChi, a multicistronic Fucci-expressing chicken line incorporating a newly optimised reporter construct composed of an mCerulean-tagged Histone H1.0 linker protein fused via a self-cleaving 2A peptide to the tandem Fucci(CA) cell cycle biosensor, with additional epitope tags included for detection in fixed tissues. We show that this system accurately discriminates and permits tracking of cells in G1, S, G2, and M phases both in vitro and in vivo, enabling faithful visualisation of cell cycle status in intact tissues and organs. Using FuChi embryos, we mapped proliferation dynamics across developing tissues, analysed cell cycle states of migrating cells, and performed live imaging of early embryos. These latter experiments revealed that transition from S phase may be a key morphogenetic event during gastrulation as mesendoderm cells egress from the primitive streak to form embryonic structures including the prechordal plate. Pairing this advanced reporter with the intrinsic experimental advantages of the chicken embryo positions FuChi as a premier in vivo system for studying cell-cycle kinetics in development, delivering clear technological improvements over current Fucci models. FuChi chickens provide a powerful new resource for studying embryonic development, organ growth, tissue homeostasis, disease processes, and infection responses.

developmental biology↗

bioRxiv2024

A stable Netrin-1 fluorescent reporter chicken reveals cell-specific molecular signaturesduring optic fissure closure.

NTN1 is expressed in a wide range of developmental tissues and is essential for normal development. Here we describe the generation of a Netrin-1 reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the NTN1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1T2A-eGFP/+) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression in the optic fissure and neural tube floorplate. Furthermore, all NTN1T2A-eGFP/+ embryos and hatched birds appeared phenotypically normal. We applied this resource to a pertinent developmental context - coloboma is a structural eye malformation characterised by failure of epithelial fusion during optic fissure closure (OFC) and NTN1 is specifically expressed in fusion pioneer cells at the edges of the optic fissure. We therefore optimised the isolation of GFP expressing cells from embryonic NTN1T2A-eGFP/+ eyes using spectral fluorescence cell-sorting and applied transcriptomic profiling of pioneer cells, which revealed multiple new OFC markers and novel pathways for developmental tissue fusion and coloboma. This work provides a novel fluorescent NTN1 chicken reporter line with broad experimental utility and is the first to directly molecularly characterise pioneer cells during OFC.

developmental biology↗

bioRxiv2023

Cell adhesion marker expression dynamics during fusion of the optic fissure

Tissue fusion is a critical process that is repeated in multiple contexts during embryonic development and shares common attributes to processes such as wound healing and metastases. Ocular coloboma is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause of childhood blindness. Coloboma results from fusion failure between opposing ventral retinal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, ZO-1, and the EMT transcriptional activator and cadherin regulator SNAI2. We find that fusion pioneer cells at the edges of the fusing optic fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression of SNAI2. This highlights the unique properties of these cells and indicates that regulation of cell adhesion factors is a critical process in optic fissure closure.

developmental biology↗

bioRxiv2022

Identification of novel coloboma candidate genes through conserved gene expression analyses across four vertebrate species

Ocular coloboma (OC) is a failure of complete optic fissure closure during embryonic development and presents as a tissue defect along the proximal distal axis of the ventral eye. It is classed as part of the clinical spectrum of structural eye malformations with microphthalmia and anophthalmia, collectively abbreviated to MAC. Despite deliberate attempts to identify causative variants in MAC, many patients remain without a genetic diagnosis. To reveal potential candidate genes, we utilised transcriptomes experimentally generated from embryonic eye tissues derived from human, mouse, zebrafish, and chicken at stages coincident with optic fissure closure. Our in-silico analyses found 10 genes with optic fissure specific enriched expression: ALDH1A3, BMPR1B, EMX2, EPHB3, NID1, NTN1, PAX2, SMOC1, TENM3, and VAX1. In situ hybridization revealed that all 10 genes were broadly expressed ventrally in the developing eye, but that only PAX2 and NTN1 were expressed in cells at the edges of the optic fissure margin. Of these conserved optic fissure genes, EMX2, NID1, and EPHB3 have not previously been associated with human MAC cases. Targeted genetic manipulation in zebrafish embryos using CRISPR/Cas9 caused the developmental MAC phenotype for emx2 and ephb3. We scrutined available whole genome sequencing datasets from MAC patients and identified a range of variants with plausible causality. In combination our data suggest that expression of genes involved in ventral eye development are conserved across a range of vertebrate species, and that EMX2, NID1, and EPHB3 are candidate loci that should be adopted into clinical diagnostic screens for patients with structural eye malformations.

genetics↗

bioRxiv2022

Characterization of an Eye Field-like State during Optic Vesicle Organoid Development

Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors (TFs). This critical event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate. Using optic vesicle organoids to model the onset of the EF, we generate timecourse transcriptomic data allowing us to identify dynamic gene-expression programs that characterise this cellular-state transition. Integrating this with chromatin accessibility data suggests a direct role of canonical EFTFs in regulating these gene-expression changes, and high-lights candidate cis-regulatory elements through which these TFs act. Finally, we begin to test a subset of these candidate enhancer elements, within the organoid system, by perturbing the underlying DNA sequence and measuring transcriptomic changes during EF activation.

developmental biology↗

bioRxiv2022

Robust genetic analysis of the X-linked anophthalmic (Ie) mouse

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a [~] 6Mb region on the X chromosome. Short and long read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.

genetics↗

bioRxiv2020

Functional Predictors of Causative Cis-Regulatory Mutations in Mendelian Disease

Undiagnosed neurodevelopmental disease is significantly associated with rare variants in cis-regulatory elements (CRE) but demonstrating causality is challenging as target gene consequences may differ from a causative variant affecting the coding region. Here, we address this challenge by applying a procedure to discriminate likely diagnostic regulatory variants from those of neutral/low-penetrant effect. We identified six rare CRE variants using targeted and whole genome sequencing in 48 unrelated males with apparent X-linked intellectual disability (XLID) but without detectable coding region variants. These variants segregated appropriately in families and altered conserved bases in predicted CRE targeting known XLID genes. Three were unique and three were rare but too common to be plausibly causative for XLID. We compared the cis-regulatory activity of wild-type and mutant alleles in zebrafish embryos using dual-color fluorescent reporters. Two variants showed striking changes: one plausibly causative (FMR1CRE) and the other likely neutral/low-penetrant (TENM1CRE). These variants were "knocked-in" to mice and both altered embryonic neural expression of their target gene. Only Fmr1CRE mice showed disease-relevant behavioral defects. FMR1CRE is plausibly disease-associated resulting in complex misregulation of Fmr1/FMRP rather than loss-of-function. This is consistent both with absence of Fragile X syndrome in the probands and the observed electrophysiological anomalies in the FMR1CRE mouse brain. Although disruption of in vivo patterns of endogenous gene expression in disease-relevant tissues by CRE variants cannot be used as strong evidence for Mendelian disease association, in conjunction with extreme rarity in human populations and with relevant knock-in mouse phenotypes, such variants can become likely pathogenic.

genetics↗

bioRxiv2019

The transcriptional signature associated with human motile cilia

Cilia are complex microtubule-based organelles implicated in the aetiology of numerous diseases. Accordingly, many cilia-associated proteins have been described, while those distinguishing cilia subtypes are poorly defined. Here, we characterise the gene signature associated with human motile cilia that captures both known and unknown components of this class of cilia. To define the signature, we performed network deconvolution of transcriptomics data derived from tissues possessing motile ciliated cell populations. For each tissue, genes coexpressed with the motile cilia-associated transcriptional factor, FOXJ1, were identified. The consensus across tissues provided a transcriptional signature of 248 genes. For validation, we examined the literature, databases, single cell RNA-Seq data, and the localisation of mRNA and proteins in motile ciliated cells. To validate some of the many poorly characterised genes, we performed new localisation experiments on ARMC3, EFCAB6, FAM183A, MYCBPAP, RIBC2 and VWA3A. In summary, we report a highly validated set of motile cilia-associated genes that helps shape our understanding of these complex cellular organelles.\n\nSummaryThis work defines a conserved transcriptional signature associated with human motile cilia, including many genes with little or no previous association with these structures. These genes were compared with existing resources and a number of poorly characterised genes validated.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC=\"FIGDIR/small/817072v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (74K):\norg.highwire.dtl.DTLVardef@152f5d6org.highwire.dtl.DTLVardef@189eba6org.highwire.dtl.DTLVardef@1cde94forg.highwire.dtl.DTLVardef@1906450_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential and conserved mediator of epithelial fusion during vertebrate embryogenesis.

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including strong evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin at the fusion plate but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and fish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have a major role in palate fusion. Our data reveal that NTN1 is a new locus for human coloboma and other multi-system developmental fusion defects, and that chick OFC is a powerful model for epithelial fusion research.

developmental biology↗

bioRxiv2018

Arginine to glutamine mutation in olfactomedin-like 3 (OLFML3) is a candidate for severe goniodysgenesis and glaucoma in the Border Collie dog breed.

Goniodysgenesis is a developmental abnormality of the anterior chamber of the eye. It is generally considered to be congenital in dogs (Canis lupus familiaris), and has been associated with glaucoma and blindness. Goniodysgenesis and early-onset glaucoma initially emerged in Border Collies in Australia in the late 1990s and has subsequently been found in Europe and the USA. The objective of the present study was to determine the genetic basis of goniodysgenesis in Border Collies. Clinical diagnosis was based on results of examinations by veterinary ophthalmologists of affected and unaffected dogs from eleven different countries. Genotyping using the Illumina high density canine SNP chip and whole genome sequencing were used to identify candidate genetic regions. Expression profiles and evolutionary conservation of candidate genes were assessed using public databases. Analysis of pedigree information was consistent with an autosomal recessive mode of inheritance for severe goniodysgenesis (potentially leading to glaucoma) in this breed. There was a highly significant peak of association over chromosome 17, with a p-value of 2 x 10-13. Whole genome sequences of three dogs with glaucoma, three severely affected and three unaffected dogs identified a missense variant in the olfactomedin like 3 (OLFML3) gene in all six affected animals. This was homozygous in all nine cases with glaucoma and 12 of 14 other severely affected animals. Of 67 reportedly unaffected animals, only one (offspring of two homozygous affected parents) was homozygous for this variant. The identification of a candidate genetic region and putative causative mutation will aid breeders to reduce the frequency of goniodysgenesis and the risk of glaucoma in the Border Collie population.

genetics↗