Biology subjects

Briggs, T.

Publications and source records attributed to Briggs, T..

3 recordsLinked to original sources

bioRxiv2025

FuChi: A cell cycle biosensor for investigating cell-cycle kinetics during avian development.

The ability to monitor the proliferative status of live cells both in vitro and in vivo over time has revolutionised our understanding of development, growth and disease. This was first made possible by fluorescent ubiquitination-based cell cycle indicator (Fucci) technology, which distinguishes specific cell cycle phases through the reciprocal degradation of fluorescently tagged, truncated forms of human CDT1 and GMNN proteins. Fucci genetic systems have been successfully implemented in transgenic mice, zebrafish, and axolotls. To date, no viable, stably expressing Fucci line has been developed in an avian species. Although a range of continuously improving Fucci constructs have been developed in recent years, existing in vivo Fucci models remain limited because they rely on older reporter technology that fails to distinguish cells in S, G2, and M phases or to label cells in early G1. As a result, these models can be challenging to interpret and their utility for continuous cell tracking and precise analysis of cell-cycle dynamics is limited. Here, we introduce FuChi, a multicistronic Fucci-expressing chicken line incorporating a newly optimised reporter construct composed of an mCerulean-tagged Histone H1.0 linker protein fused via a self-cleaving 2A peptide to the tandem Fucci(CA) cell cycle biosensor, with additional epitope tags included for detection in fixed tissues. We show that this system accurately discriminates and permits tracking of cells in G1, S, G2, and M phases both in vitro and in vivo, enabling faithful visualisation of cell cycle status in intact tissues and organs. Using FuChi embryos, we mapped proliferation dynamics across developing tissues, analysed cell cycle states of migrating cells, and performed live imaging of early embryos. These latter experiments revealed that transition from S phase may be a key morphogenetic event during gastrulation as mesendoderm cells egress from the primitive streak to form embryonic structures including the prechordal plate. Pairing this advanced reporter with the intrinsic experimental advantages of the chicken embryo positions FuChi as a premier in vivo system for studying cell-cycle kinetics in development, delivering clear technological improvements over current Fucci models. FuChi chickens provide a powerful new resource for studying embryonic development, organ growth, tissue homeostasis, disease processes, and infection responses.

developmental biology↗

bioRxiv2024

CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress

Eukaryotic cell division is regulated by oscillating CDK activity, which must reach critical CDK threshold activity levels to progress through cell cycle stages. In low mitogen, low CDK environments cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation enhances genomic instability after release from quiescence. Here, we determined the mechanisms that promote genome instability in CIZ1 ablated cells using a combination of Fucci(CA) live cell imaging, cell-free DNA replication assays and DNA combing. Cell cycle dynamics are unaffected in CIZ1-/- (CIZ1 KO) fibroblasts; however, a specific post-quiescent phenotype is observed resulting in a reduced G1 phase and cell cycle length. The reduction in G1 length in CIZ1 KO cells is associated with increased cyclin E1/E2 and A2 expression, and enhanced phosphorylation of Rb leading to early restriction point bypass. CIZ1-/- cells are deficient in cyclin A chromatin binding and required increased cyclin-CDK activity for the initiation of DNA replication, which is associated with DNA replication stress in vitro and in vivo. Significantly, the CDK threshold for initiation of DNA replication was 2-fold higher in CIZ1 KO nuclei than parental controls. Importantly, addition of recombinant CIZ1 in vitro and in vivo promotes recruitment of cyclin A to chromatin and reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. Loss of CIZ1 is associated with dysregulated cyclin-CDK signalling, resulting in reduced G1 length, an increased CDK activity threshold required to promote initiation of DNA replication that results in DNA replication stress. These data suggest that CIZ1 facilitates recruitment of cyclin-CDK complexes to chromatin and contributes to the mechanisms that determine the threshold CDK activity required for the G1/S transition in post-quiescent cells. Taken together the data support a role for CIZ1 in the prevention of DNA replication stress and maintenance of genome stability.

cell biology↗

bioRxiv2018

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here eleven new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that six of eighteen (33%) protein-coding mutations and the two (100%) 5-UTR mutations display incomplete penetrance. Three mutations were identified in 2 independent kindreds, due to a hotspot or a founder effect. Lastly, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

genetics↗