Biology subjects

Moosajee, M.

Publications and source records attributed to Moosajee, M..

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↗

bioRxiv2024

Dominant RDH12-retinitis pigmentosa impairs photoreceptor development and cone function in retinal organoids

Retinal dehydrogenase 12 (RDH12) is a photoreceptor NADPH-dependent retinal reductase enzyme, converting all-trans-retinal to all-trans-retinol. Heterozygous variants in RDH12 cause a rare autosomal dominant (AD) retinitis pigmentosa. As no disease models exist, we generated human induced pluripotent stem cell derived retinal organoids (RO) from a RDH12-AD patient (with pathogenic c.759del p.(Phe254Leufs*24) variant), alongside a healthy control (WT). RDH12-AD RO exhibited correct localisation of RDH12 to the photoreceptor inner segments up to week 44; transmission electron microscopy at week 37 showed photoreceptors were less abundant and shorter in length compared to WT. Visual cone function, retinol biosynthesis and the vitamin A pathway were also highly disrupted at week 44. Our study is the first to describe a RDH12-AD disease model with pathology at later stages of photoreceptor differentiation, in keeping with the milder disease course seen in humans. It provides insights into the aetiology and possible targets for future therapeutic development.

pathology↗

bioRxiv2024

Metabolic and neuroactivity imbalances in plasma from aniridia patients with PAX6 haploinsufficiency

PAX6 is a transcription factor crucial for the development of the eye, pancreas, and brain. Heterozygous variants resulting in PAX6 haploinsufficiency are the main genetic cause of congenital aniridia, characterized by both anterior and posterior ocular defects and sight loss. The extra-ocular features of PAX6 haploinsufficiency are becoming more widely recognised, with systemic manifestations like obesity, diabetes, and neurological/behavioural disorders being reported. In this study, we uncovered the metabolomic profile of the blood plasma from 25 PAX6-related aniridia patients compared to gender and age-matched controls. We found significant disruptions in lipid and energy metabolism, increased oxidative stress and neurotransmitters imbalances, as well as alterations linked to the gut microbiome. This study identified novel metabolic changes associated with PAX6 haploinsufficiency, providing evidence for the systemic aetiology of congenital aniridia and emphasizing the need for multidisciplinary management and further exploration into ocular and systemic therapeutic approaches.

genetics↗

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

Loss of REP1 impacts choroidal melanogenesis in choroideremia

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, however, the involvement of the choroid in disease progression is not fully understood. CHM is caused by mutations in the CHM gene, encoding the ubiquitously expressed Rab escort protein 1 (REP1). REP1 plays an important role in intracellular trafficking of vesicles, including melanosomes. In this study, we examined ultrastructure of the choroid in chmru848 fish and Chmnull/WT mouse models using transmission electron microscopy. Significant pigmentary disruptions were observed, with a lack of melanosomes in the choroid of chmru848fish from 4 days post fertilisation (4dpf). Total melanin and expression of melanogenesis genes tyr, tryp1a, mitf, dct and pmel were also reduced from 4dpf. In Chmnull/WT mice, choroidal melanosomes were significantly smaller at 1 month and at 1 year, eumelanin was reduced, and the choroid was thicker. The choroid in CHM patients was also examined using optical coherence tomography (OCT) and OCT- angiography (OCT-A) and the area of preserved choriocapillaris (CC) was found to be smaller than that of overlying photoreceptors, indicating that the choroid is degenerating at a faster rate. Histopathology of an enucleated eye from a 74-year-old CHM male patient revealed isolated areas of RPE but no associated underlying CC. Significant degenerative changes in the choroid of CHM patients and animal models are seen, highlighting the importance of administrative routes involving the choroid, such as suprachoroidal delivery. Pigmentary disruptions in CHM animal models reveal an important role for REP1 in melanogenesis, and drugs that improve melanin production represent a potential novel therapeutic avenue.

molecular 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

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

Aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough inducing compounds (TRIDs) have the ability to weaken the recognition of in-frame premature stop codons (PTCs), permitting full-length protein to be translated. We have established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from an aniridia patient with a prevalent PAX6 nonsense mutation. Both in vitro models show reduced PAX6 protein levels, mimicking the disease. Repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP), and positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models, and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESC. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

genetics↗

bioRxiv2022

Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis, and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.

developmental biology↗

bioRxiv2022

Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein

Point mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs, BE4max-WTCas9, and evoCDA-SpCas9-NG) as a proof-of-concept therapeutic option. We observed the KCNJ13-related retinopathy phenotype in patients harboring L144P mutation. Our in-silico prediction and in vitro validation demonstrated that L144P mutation affects the channel function. We observed high on-target efficiency in the CBEs treated L144P mutant gene expressing HEK-293 cells. Strikingly, our evaluation of base editing efficacy using electrophysiology showed negligible channel function. We found that the editing bystander Cs in the protospacer region led to a missense change (L143F) in evoCDA edited cells and only silent changes in BE4max edited cells. Upon investigation of the effect of the synonymous codon, our extended analysis revealed distortion of mRNA structure, altered half-life, and/or low abundance of the cognate tRNA. We propose that KCNJ13-L144P mutation or other genes that share similar genetic complexity may be challenging to correct with the current generation of CRISPR base editors, and a combinational therapy using CRISPR base editors with a tighter editing window and requisite cognate-tRNA supplementation could be an alternative therapeutic approach to restore Kir7.1 channel function in LCA16 patients. Other options for hard-to-rescue alleles could employ homology-directed repair using CRISPR/Cas9 nucleases, Prime editing, and AAV-mediated gene augmentation.

genetics↗

bioRxiv2021

Fission of megamitochondria into multiple smaller well-defined mitochondria in the ageing zebrafish retina

Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. When investigating temporal gene expression of several mitochondrial-related markers, they indicated fission as the dominant mechanism contributing to these changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction.

cell biology↗

bioRxiv2020

Age-related degeneration leads to gliosis but not regeneration in the zebrafish retina

Ageing is a significant risk factor for degeneration of the retina. Harnessing the regenerative potential of Muller glia cells (MG) in the retina offers great promise for the treatment of blinding conditions. Yet, the impact of ageing on MG regenerative capacity has not yet been considered. Here we show that the zebrafish retina undergoes telomerase-independent age-related neurodegeneration. Yet, this progressive neuronal loss in the ageing retina is insufficient to stimulate the MG regenerative response. Instead, age-related neurodegeneration leads to MG gliosis and loss of vision, similarly to humans. Nevertheless, gliotic MG cells retain Yap expression and the ability to regenerate neurons after acute light damage. Therefore, we identify key differences in the MG response to acute versus chronic damage in the zebrafish retina and show that aged gliotic MG can be stimulated to repair damaged neurons in old age. SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/174821v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1a184daorg.highwire.dtl.DTLVardef@1193750org.highwire.dtl.DTLVardef@7e65c0org.highwire.dtl.DTLVardef@4a2fe3_HPS_FORMAT_FIGEXP M_FIG C_FIG Our data suggest there are key differences between mechanisms driving regeneration in response to acute damage versus age-related chronic damage. It may be that either the number of cells dying in natural ageing is not enough to stimulate MG to proliferate, or the low number of microglia and respective signals released are not sufficient to trigger MG proliferation. Importantly, we show that gliotic MG cells can be stimulated to repair damaged neurons in old zebrafish retina.

neuroscience↗