Biology subjects

Busch, T.

Publications and source records attributed to Busch, T..

5 recordsLinked to original sources

bioRxiv2026

Zfp750 prevents oral adhesions and promotes temporary epithelial fusions

The differentiation cascade that converts basal keratinocytes into suprabasal layers, including periderm, depends on the activity of transcription factors. Mutations in the genes encoding many of these transcription factors, including TP63, IRF6 and GRHL3, disrupt periderm development. Such mutations can also interfere with embryonic fusion and septation events that depend on periderm development, including palatogenesis, digit separation and the formation of temporary epithelial fusions between digits, between eyelids, and between pinnae and the scalp. ZNF750 (Zfp750 in the mouse) is a transcription factor required for keratinocyte differentiation, but whether mutations in ZNF750 contribute risk for orofacial cleft, and the role of Zfp750 in periderm development, are unknown. To address these questions we sequenced ZNF750 in 5,659 individuals including 2,125 with nonsyndromic OFC. We identify 33 rare missense variants with frequencies less than 0.1% in gnomAD. Of these, about half are predicted to be damaging with in silico tools. Collectively, these missense variants are not overtransmitted from parents to children with OFCs. Two of the variants have lower activity than the reference variant in a zebrafish embryo-based assay but no phenotype in the corresponding murine model. However, in murine embryos homozygous for a frame-shift mutation in Zfp750 (Zfp750fs) that we generated, palatal shelves are fused but intra-oral adhesions are present, a phenotype seen in murine mutants of several bonafide OFC genes. In addition, temporary epithelial fusions are absent in Zfp750fs neonates. RNA sequencing of forelimbs from Zfp750fs embryos reveals decreased expression of epidermal terminal differentiation genes, and both increased and decreased expression of distinct periderm genes. Immunofluorescence shows the consistent presence of periderm proteins within the oral adhesions in Zfp750fs/fs embryos. Together these studies suggest that while mutations in ZNF750 are not a major contributor to OFC risk, Zfp750 does contribute to periderm-dependent morphogenic events.

genetics↗

bioRxiv2025

Deep learning-based image quantification of epithelial cell shapes and its application to polycystic kidney disease

Cell shape is a fundamental determinant of tissue architecture and organ function. In epithelial tissues, cytoskeletal organization and tight junctions regulate cell geometry, shaping functional tissue units. Disruption of these mechanisms may cause diseases such as autosomal dominant polycystic kidney disease (ADPKD), in which cyst formation is characterized by abnormal regulation of epithelial cell shape. The mechanisms of cystogenesis remain incompletely understood, highlighting the need for robust, high-throughput methods to quantify the morphology of epithelial cells. Here, we present a fully automated, deep learning-based image analysis pipeline to quantify epithelial cell shape and tight junction morphology from immunofluorescence images. Our approach employs a U-Net convolutional neural network for accurate segmentation of fluorescence labeled tight junctions. We introduce novel algorithms to quantify overall cell shape and tight junction morphology, as well as to estimate cytoskeletal traction at shared cell borders. Our analysis pipeline objectively identifies subtle morphogenetic changes associated with disease-related mutations, applied to a genetically modified Madin-Darby Canine Kidney cell model of ADPKD. The method enables high-throughput, standardized analysis, reduces observer bias, and facilitates comparison across experiments. We further demonstrate the pipelines generalizability by applying it to Drosophila egg chamber epithelia. Our results establish a robust and scalable framework for analyzing cell shape and mechanical interactions in epithelial tissues, with broad applications in phenotypic screening, disease modeling, and morphogenesis research. Author SummaryThe shape of epithelial cells is critical for organ function. In the kidney, properly shaped epithelial cells assemble to tubules ensuring efficient waste excretion as well as body electrolyte and water balance. Disruption of cell shape regulation can lead to diseases such as autosomal dominant polycystic kidney disease (ADPKD), characterized by cyst formation and displacement of normal kidney tissue. Traditionally, analysis of epithelial cell morphology has relied on manual, low-throughput methods, which are time-consuming and prone to error. To overcome these limitations, we developed a fully automated, artificial intelligence-based pipeline that rapidly and reliably quantifies cell shape and junctional organization from microscopic images. We validated our approach using a cellular model of ADPKD, demonstrating clear differences in cell shape and junctional structure between normal and mutant cells harboring mutations in PKD-related genes. Our method enables efficient, objective analysis of large datasets and provides a powerful tool for understanding the mechanisms underlying cell shape regulation in health and disease.

bioinformatics↗

bioRxiv2024

Cilia to basement membrane signalling is a biomechanical driver of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD), which affects around 4 million patients worldwide, is characterized by the formation of multiple tubule derived cysts, which grossly enlarge both kidneys and progressively compromise renal function. ADPKD mainly results from mutations in PKD1, leading to the loss of polycystin-1 protein, which localizes to primary cilia. Primary cilia are required for cyst formation but the biomechanical changes underlying cystogenesis upon loss of polycytin-1 are unknown. We find that cilia and polycystin-1 shape the tubular basement membrane (TBM). Combining orthologous mouse models with a tubule-on-chip approach allowing manipulations of TBM stiffness, we find that cilia regulate the composition and biomechanical properties of the TBM. In the setting of polycytin-1 loss, reduced TBM stiffness and increased luminal pressure act as biomechanical drivers of cyst formation. These findings suggest a novel biomechanical model for ADPKD and unveil that cilia to TBM signalling controls kidney shape.

physiology↗

bioRxiv2024

The Role of the Co-Chaperone DNAJB11 in Polycystic Kidney Disease: Molecular Mechanisms and Cellular Origin of Cyst Formation

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), which are required for the regulation of the renal tubular diameter. Loss of polycystin function results in cyst formation. Atypical forms of ADPKD are caused by mutations in genes encoding endoplasmic reticulum (ER)-resident proteins through mechanisms that are not well understood. Here, we investigate the function of DNAJB11, an ER co-chaperone associated with atypical ADPKD. We generated mouse models with constitutive and conditional Dnajb11 inactivation and Dnajb11-deficient renal epithelial cells to investigate the mechanism underlying autosomal dominant inheritance, the specific cell types driving cyst formation, and molecular mechanisms underlying DNAJB11-dependent polycystic kidney disease. We show that biallelic loss of Dnajb11 causes cystic kidney disease and fibrosis, mirroring human disease characteristics. In contrast to classical ADPKD, cysts predominantly originate from proximal tubules. Cyst formation begins in utero and the timing of Dnajb11 inactivation strongly influences disease severity. Furthermore, we identify impaired PC1 cleavage as a potential mechanism underlying DNAJB11-dependent cyst formation. Proteomic analysis of Dnajb11- and Pkd1-deficient cells reveals common and distinct pathways and dysregulated proteins, providing a foundation to better understand phenotypic differences between different forms of ADPKD.

molecular biology↗

bioRxiv2021

RELATIONSHIP BETWEEN PARENTAL AGE AND SEVERITY OF OROFACIAL CLEFTS

OBJECTIVESThis study aims to investigate the relationship between paternal age, maternal age, and both on the severity of orofacial clefts. DESIGNThis was a retrospective study of cases which were subjects clinically diagnosed with non-syndromic cleft lip and/or palate (CL/P). Data was obtained from the AFRICRAN project database on Nigerian non-syndromic orofacial cleft cases. SETTINGThe samples for cases in this study were obtained at the Cleft clinic of Oral and Maxillofacial surgery at the Lagos University Teaching Hospital, Lagos. OUTCOMEPrimary outcome measure is severity of orofacial clefts and secondary outcome measure is to evaluate the effect of parental age in determining the incidence of left or right sided orofacial clefts. RESULTSThere is no statistical significant association between type of CL {+/-} P and parental age in young fathers (p=0.93). When old fathers are considered, percentage of complete (more severe) CL {+/-} P cases increases especially in old mothers and this was statistically significant (p=0.036). In old fathers, the risk of CL {+/-} P is increased (OR: 2.66, CI: 1.04-6.80) and also there is increased risk of developing right sided CL {+/-} P (OR: 1.61, CI: 1.0-2.59). There is reduced risk of isolated cleft palate in young fathers (OR: 0.36, CI: 0.07-1.71) but the risk increases when considering complete types (more severe) of isolated cleft palates (OR: 1.63, CI: 0.71-3.7) CONCLUSIONThe study shows a higher risk of CL {+/-} P is associated with increase fathers age.

genetics↗