Biology subjects

Stephen, L. A.

Publications and source records attributed to Stephen, L. A..

6 recordsLinked to original sources

bioRxiv2026

Prpf8N1531S homozygous mutant mouse embryos have multiple defects in cardiac development and show aberrant splicing of the cardiac transcription factor Tead1

Mutations in the spliceosomal gene PRPF8 are associated with a range of human diseases. Studies in mouse and zebrafish suggest that Prpf8 also has a developmental function. Here, using a Prpf8 mutant mouse line isolated from a chemical induced mutagenesis screen, we uncover a previously unrecognised and essential role for Prpf8 in heart development, consistent with the embryonic lethality observed in Prpf8N1531S homozygous mutants. Prpf8N1531S mutant embryos display severe defects in ventricular trabeculation and compact zone formation, accompanied by increased cardiomyocyte proliferation specifically in the compact zone. Mutant embryonic hearts also exhibit disrupted cellular organisation, altered cytoskeletal architecture and changes in extracellular matrix protein expression. Notably, these cardiac abnormalities were exacerbated in embryos exhibiting cardiac looping defects. Transcriptomic analysis identified multiple aberrantly spliced transcripts in Prpf8N1531S mutant embryos, among which the cardiac transcription factor Tead1 was selected as a key functional candidate due to it known role in cardiac ventricle wall developemnt. Tead1 mis-splicing generated an in-frame, lower molecular weight protein isoform, associated with reduced overall TEAD1 expression. The Tead1 mis-spliced isoform showed altered nuclear localisation and dysregulation of TEAD1-dependent gene network important for heart development, including known cardiac sarcomeric genes. In addition, we observed reduced levels of the intracellular domain of the NOTCH1 receptor (NICD1), indicating impaired Notch signalling.. These findings suggest that impaired TEAD1-dependent transcription and Notch signalling contribute to abnormal cardiac trabeculation and compact zone development, highlighting a critical role for Prpf8 in maintaining proper heart development through the regulation of cardiac transcription factor expression and associated signalling networks. This study offers new mechanistic insights into congenital heart diseases linked to spliceosomal gene mutations.

developmental biology↗

bioRxiv2025

Proteomic characterisation of matrix vesicles from primary osteoblasts indicates a mixed population with both exosomal and ectosomal routes of biogenesis.

Matrix Vesicles are a crucial step in producing a mineralised, healthy skeleton. Released from chondrocytes and osteoblasts, they concentrate calcium and phosphate to establish the deposition of hydroxyapatite around and within the collagen fibrils of the extracellular matrix, becoming embedded in the matrix in the process. In the 55 years since H. Clarke Anderson first described them, blebbing from the surface of chondrocytes, a consensus on their role, contents and their biogenesis, has yet to be reached. This is in part due to the range of cell types from which they are released and the multitude techniques that can be employed to isolate them. In this study, we, for the first time, characterise the proteome of matrix vesicles isolated from primary osteoblasts. By focussing on those vesicles that have become embedded within the matrix, we are able to avoid bias for specific modes of biogenesis and focus only on osteoblast-released vesicles that are associated with the matrix. Moreover, by studying these vesicles over a time course of mineralising activity, we are able to identify changes in the properties of these vesicles, and develop a more accurate picture of which proteins are involved specifically in mineralisation. In particular we identify the presence of markers associated with the ectosomal and exosomal release of matrix vesicles, as well as identifying proteins required for the maintenance and mineralisation of the extracellular matrix. These data portray a heterogeneous population of matrix vesicles, with different roles to play in bone development.

cell biology↗

bioRxiv2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia function and determination of cardiac left-right asymmetry via regulation of Arl13b splicing

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephaloph[o]nus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

developmental biology↗

bioRxiv2023

Mitochondrial dysfunction and mitophagy blockade contribute to renal osteodystrophy in chronic kidney disease-mineral bone disorder

Chronic kidney disease-mineral and bone disorder (CKD-MBD) presents with extra-skeletal calcification and renal osteodystrophy (ROD). The origins of ROD likely lie with elevated uremic toxins and/or an altered hormonal profile but the cellular events responsible remain unclear. Here, we report that stalled mitophagy contributes to mitochondrial dysfunction in bones of a CKD-MBD mouse model, and also human CKD-MBD patients. RNA-seq analysis exposed an altered expression of genes associated with mitophagy and mitochondrial function in tibia of CKD-MBD mice. The accumulation of damaged osteocyte mitochondria and the expression of mitophagy regulators, p62/SQSTM1, ATG7 and LC3 was inconsistent with functional mitophagy, and in mito-QC reporter mice with CKD-MBD, there was a 2.3-fold increase in osteocyte mitolysosomes. Altered expression of mitophagy regulators in human CKD-MBD bones was also observed. To determine if uremic toxins were possibly responsible for these observations, indoxyl sulfate treatment of osteoblasts revealed mitochondria with distorted morphology and whose membrane potential and oxidative phosphorylation were decreased, and oxygen-free radical production increased. The altered p62/SQSTM1 and LC3-II expression was consistent with impaired mitophagy machinery and the effects of indoxyl sulfate were reversible by rapamycin. In conclusion, mitolysosome accumulation from impaired clearance of damaged mitochondria may contribute to the skeletal complications, characteristic of ROD. Targeting mitochondria and the mitophagy process may therefore offer novel routes for intervention to preserve bone health in patients with ROD. Such approaches would be timely as our current armamentarium of anti-fracture medications has not been developed for, or adequately studied in, patients with severe CKD-MBD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/573355v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1f23caforg.highwire.dtl.DTLVardef@6e3e94org.highwire.dtl.DTLVardef@1ce6adeorg.highwire.dtl.DTLVardef@ca5558_HPS_FORMAT_FIGEXP M_FIG C_FIG TRANSLATIONAL STATEMENTRenal osteodystrophy (ROD) remains the major skeletal complication of chronic kidney disease-mineral and bone disorder (CKD-MBD). As a disease characterised by biochemical and hormone abnormalities, ROD is exacerbated by osteocyte mitochondrial dysfunction. Advances in our understanding of the mitophagy pathway are vital to improving the clinical management of ROD. The dysregulation of mitophagy in murine and human CKD-MBD bone provided evidence of delayed clearance of damaged mitochondria, which was also observed in uremic toxin-treated-osteoblasts but reversible upon rapamycin treatment. This study reveals the therapeutic potential of managing ROD by restoring defective mitophagy in osteocytes.

genomics↗

bioRxiv2021

Differential Expression of Alkaline Phosphatase and PHOSPHO1 in Bone from a Murine Model of Chronic Kidney Disease

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities; a condition known as renal osteodystrophy (ROD). While Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are recognized to be critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both wild-type and Phospho1 knockout (P1KO) mice using dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (p < 0.05) but decreased in trabecular bone (p < 0.05). These changes were accompanied by decreased TNAP (p < 0.01) and increased PHOSPHO1 (p < 0.001) expression in wild-type CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expression to be down-regulated in response to phosphate and PTH. While matrix mineralization was increased with phosphate (Pi), it decreased with PTH and FGF23 had no effect. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism in an attempt to protect the bone from hypomineralization. We propose that increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

cell biology↗

bioRxiv2018

Single cell transcriptomic analysis of the adult mouse pituitary reveals a novel multi-hormone cell cluster and physiologic demand-induced lineage plasticity

The anterior pituitary gland drives a set of highly conserved physiologic processes in mammalian species. These hormonally-controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current models, largely built upon candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex models of hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with a set of orthogonal mRNA and protein imaging studies, to systematically map the cellular composition of adult male and female mouse pituitaries at single-cell resolution and in the setting of major physiologic demands. These analyses reveal sex-specific cellular diversity associated with normal pituitary homeostasis, and identify an array of cells with complex complements of hormone-enrichment as well as a series of non-hormone producing interstitial and supporting cell lineages. These scRNA-seq studies identify a major cell population that is characterized by a unique multi-hormone gene expression profile. The detection of dynamic shifts in cellular representations and transcriptome profiles in response to two well-defined physiologic stresses suggests corresponding roles of a number of these clusters in cellular plasticity within the adult pituitary. These studies point to an unanticipated complexity and plasticity in pituitary cellular composition that expands upon current models and concepts of pituitary gene expression and hormone production.

genomics↗