We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.

Understanding what factors influence plastic and genetic variation is valuable for predicting how organisms respond to changes in the selective environment. Here, using gene expression and DNA methylation as molecular phenotypes, we study environmentally induced variation among Arabidopsis lyrata plants grown at lowland and alpine field sites. Our results show that gene expression is highly plastic, as many more genes are differentially expressed between the field sites than between populations. These environmentally responsive genes evolve under strong selective constraint – the strength of purifying selection on the coding sequence is high, while the rate of adaptive evolution is low. We find, however, that positive selection on cis-regulatory variants has likely contributed to the maintenance of genetically variable environmental responses, but such variants segregate only between distantly related populations. In contrast to gene expression, DNA methylation at genic regions is largely insensitive to the environment, and plastic methylation changes are not associated with differential gene expression. Besides genes, we detect environmental effects at transposable elements (TEs): TEs at the high-altitude field site have higher expression and methylation levels, suggestive of a broad-scale TE activation. Compared to the lowland population, plants native to the alpine environment harbor an excess of recent TE insertions, and we observe that specific TE families are enriched within environmentally responsive genes. Our findings provide insight into selective forces shaping plastic and genetic variation. We also highlight how plastic responses at TEs can rapidly create novel heritable variation in stressful conditions.

Interspecific F1 hybrids were obtained from a cross between a male sterile Brassica napus (2n = 4x = 38, AA (20) and CC (18) genomes) and an inbreeding line B. rapa (Purple Cai-Tai inbred line 9418, 2n = 2x = 20, AA (20) genome) to introgress male sterility from a tetraploid into a diploid through backcrossing. The morphological characteristics of F1 plants were more like the female parent B. napus and segregated considerably in BC1 when backcrossed to the recurrent parent Purple Cai-Tai. The progeny became stable and more similar to Purple Cai-Tai by BC4. Most C genome chromosomes were found to be eliminated, based on cytogenetic analysis. The majority of chromosomes were eliminated at very early backcross stages, with only 20–26 chromosomes in BC1 plants, and some chromosomes were eliminated gradually with increased backcross generations. The BC4 plants were generally stable with exactly 20 chromosomes. Analysis by AFLP indicated that 49.5–68.7% of the total bands eliminated from F1 to BC4 were female parent specific, and ~12% of B. napus bands were retained with increased backcrossing. The genetic materials controlling sterility from the female parent B. napus were introgressed successfully into the BC4 plants even though most B. napus chromosomes/genetic materials were eliminated during the backcross process.

Summary: Neurodevelopmental including autistic regression in children is often associated with inconclusive diagnostic investigations, leaving the underlying pathophysiology largely unexplored and poorly understood. To address this, we conducted peripheral blood multi-omics profiling in 15 children with neurodevelopmental regression (median age 9 (4-14) years, 60% males), and 15 healthy controls (median age 12 (7-16) years, 60% males). Our comprehensive analysis included bulk RNA sequencing (8 regression vs. 8 controls), single-cell RNA sequencing (two cohorts of 4 regression vs. 4 controls), bulk proteomics (4 regression vs. 4 controls), and phosphoproteomics (4 regression vs. 4 controls). Despite clinical heterogeneity, we identified convergent pathophysiological processes, including dysregulated pathways in epigenetic pathways (chromatin remodelling, histone modifications, transcription factors) and immune responses, as well as downregulated ribosomal and translational pathways. These disruptions in epigenetic, immune and translational processes may arise from complex genetic and environmental interactions, offering opportunities for therapeutic interventions that specifically target these pathways.

Realisation of the immense therapeutic potential of epigenetic editing requires the development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With the rapid development of epigenetic editing technology reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Towards this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers in which clusters of primary human hepatocytes were either OTC-positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and FACS analysis on intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice that correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient AAV gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

Retinoic acid (RA) is a key morphogen in human retinal development, activating transcriptional programs that drive retinal progenitor differentiation and photoreceptor development, ensuring proper spatial organisation within the neural retina, essential for vision. Despite its well-established role in retinal patterning, the concentration-dependent effects of RA on human retinal cell fate specification and the regional definition of the primate macula and peripheral retina remain poorly understood. Here, we show that temporal and dosage-dependant modulation of RA during human retinal organoid differentiation induces distinct changes in retinal cell abundance, maturation, and organisation. Single-cell transcriptomics and protein analysis reveal that RA dosage influences the relative abundance and maturation of photoreceptors and retinal interneurons. Spatial transcriptomics analyses demonstrates that low RA levels biases retinal organoids toward a macular-like regional identity, whereas high RA levels promotes peripheral-like development. Collectively, our findings emphasise the critical role of RA signalling in retinal maturation and regional specification. This study elucidates mechanisms involved in human retinal development and we anticipate that controlled RA modulation in retinal organoids provides a strategy to refine disease modelling of inherited retinal disorders and enhance the specific generation of photoreceptors suitable for transplantation and regenerative therapies.