Rook, V. | Search bioRxiv

bioRxiv2021

Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.

genetics↗

bioRxiv2020

BMP signalling facilitates transit amplification in the developing cerebellum.

The external granule layer (EGL) is a transient proliferative layer that gives rise to cerebellar granule cell neurons. Extensive EGL proliferation characterises the foliated structure of amniote cerebella, but the factors that regulate EGL formation, amplification within it, and differentiation from it, are incompletely understood. Here, we characterise bone morphogenic protein (BMP) signalling during cerebellar development in chick and human and show that while in chick BMP signalling correlates with external granule layer formation, in humans BMP signalling is maintained throughout the external granule layer after the onset of foliation. We also show via Immunohistochemical labelling of phosphorylated Smad1/5/9 the comparative spatiotemporal activity of BMP signalling in chick and human. Using in-ovo electroporation in chick, we demonstrate that BMP signalling is necessary for subpial migration of granule cell precursors and hence the formation of the external granule layer (EGL) prior to transit amplification. However, altering BMP signalling does not block the formation of mature granule neurons but significantly disrupts that pattern of morphological transitions that accompany transit amplification. Our results elucidate two key, temporally distinct roles for BMP signalling in vivo in organising first the assembly of the EGL from the rhombic lip and subsequently the tempo of granule neuron production within the EGL. Significance statementImproper development of cerebellar granule neurons can manifest in a plethora of neurodevelopmental disorders, including but not limited to medulloblastoma and autism. Many studies have sought to understand the role of developmental signalling pathways in granule cell neurogenesis, using genetic manipulation in transgenic mice. To complement these insights, we have used comparative assessment of BMP signalling during development in chick and human embryos and in vivo manipulation of the chick to understand and segregate the spatiotemporal roles of BMP signalling, yielding important insights on evolution and in consideration of future therapeutic avenues that target BMP signalling.

developmental biology↗