Biology subjects

Papanastasiou, A. S.

Publications and source records attributed to Papanastasiou, A. S..

3 recordsLinked to original sources

bioRxiv2022

Unique functions of two overlapping PAX6 retinal enhancers

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene expression. Sequence variants in enhancers have been implicated in disease, however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development, and has a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive, redundant, or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA sequencing in dual enhancer-reporter zebrafish embryos, we find significant differences in the spatiotemporal activity of these enhancers, and show that HS5 and NRE are active in distinct cell types of the developing retina. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely non-redundant functions. This work demonstrates that unique cell type-specific functions can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.

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↗