Biology subjects

Heffner, C.

Publications and source records attributed to Heffner, C..

4 recordsLinked to original sources

bioRxiv2026

Splicing of ultraconserved poison exons controls mitotic fidelity and stem cell viability

SR proteins are essential splicing regulators whose expression is controlled in part through poison exons (PEs) -- ultraconserved non-coding exons that trigger nonsense-mediated decay -- yet the biological functions of these elements remain undefined. Here, we show that homozygous deletion of SRSF3-PE or TRA2{beta}-PE is selected against in mouse embryos and human induced pluripotent stem cells (iPSCs), and that conditional PE deletion causes apoptotic death in iPSCs but is tolerated in post-mitotic neurons, revealing a proliferative-state-specific requirement. Mechanistically, PE deletion elevates SR protein levels, triggers widespread splicing dysregulation, and disrupts the correct splicing of a mitotic gene network associated with spindle defects and mitotic errors. These findings establish ultraconserved poison exons as essential regulators of mitotic splicing fidelity and stem cell viability.

molecular biology↗

bioRxiv2026

Novel mouse reporter models for the detection of genome editing events in vivo

With the expansion of therapeutic gene editing technology, small animal models provide essential platforms to evaluate the function of these new approaches in vivo. As part of the Somatic Cell Genome Editing (SCGE) Consortium, we developed next-generation murine reporters that overcome current model limitations and broaden detectable in vivo editing outcomes. These include two mouse models built on the "traffic light" reporter concept. This system enables fluorescent detection of both gene repair (green) and CRISPR-generated indels (red) events following editing by a single guide and either dsDNA or single-stranded oligonucleotide donor. We also generated a third reporter model that efficiently detects A-base editor activity. Reporters were validated in cultured embryos, via germline editing, and through activation in vivo by AAV transduction or direct ribonucleoprotein delivery. Together, these new models provide a valuable resource for improved detection of genome editing events in vivo.

genomics↗

bioRxiv2026

Modeling patient variants of Cnot1 and Cdc42bpb results in distinct forms of congenital diaphragmatic hernia in mice

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly characterized by impairment of both diaphragm and lung development in utero. CDH presents as a spectrum of forms and severities, with diaphragm defects arising in the dorsal/posterior region typically correlating with more severe pulmonary disease and higher risk of mortality than those appearing in ventral/anterior regions. The genetic etiology underlying CDH is complex, with many genes implicated showing variable expressivity and incomplete penetrance in both human patients and mouse models. Here we present in vivo validation of two genes previously unassociated with CDH: the CDC42-interacting kinase CDC42BPB; and CNOT1, a scaffolding protein of the CCR4-NOT protein complex, critical for mRNA regulation through modifications such as deadenylation. Each gene was found to have a damaging, de novo missense variant in a recent large-scale CDH patient sequencing screen. Loss of Cdc42bpb leads to ventral diaphragmatic hernias, heart septal defects and minor lung epithelial differentiation defects in mouse embryos. Installation of the orthologous patient-specific missense variant through CRISPR/Cas9 editing leads to less severe ventral diaphragm defects. Mouse embryos with either one or two copies of the orthologous Cnot1 variant, c.1867C>T (p.R623W), develop dorsal diaphragmatic hernias with low (<50%) penetrance, and mutants showed alterations in mRNA isoform expression consistent with the molecular role of Cnot1 in RNA splicing. These results underscore the power of in vivo functional modeling to validate genes and patient-specific variants uncovered by patient sequencing, reveal two previously unrecognized genetic causes of CDH, and highlight the heterogeneity of different patient anatomic presentations.

genetics↗

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↗