National Avian Research Facility
National Avian Research Facility

Notch-reporter

Transient fluorescent reporter of Hes5-mediated Notch gene activity

Summary & Utility

The Notch-reporter line can be utilised to study Notch signalling during development in the chick embryo. Notch signalling is a well-known principal regulator of neural stem cells and neural development. Notch activity and its relationship to the behaviour of individual cells can be investigated. To accurately report on endogenous Notch activity in cells the reporter has been designed to be transient. The Notch-reporter is based on the chick Hes5 promoter coupled to a short-lived fluorescent protein1. Previous, GFP based Notch reporters did not accurately report on Notch activity because they continued to produce a fluorescent signal long after Notch signalling was extinguished. The utility of these embryos has been shown by Dr Samuel Tozer, Dr Xavier Morin and colleagues (Institute of Biology of the École Normale Supérieure)2.

In mammals, Notch expression persists into adulthood and is present in the adult brain and in mature differentiated cells in the central nervous system3. Notch signalling also regulates osteogenic differentiation and skeletal development4. However, Notch signalling in these contexts has not been explored in birds.

 

Line origin

The generation of the Notch-reporter line was funded by the Wellcome Trust. The Notch-reporter line carries a lentivirus/transgene designed to report on Hes5-mediated Notch gene activity. The transgene was developed by Dr Lucy Freem and Professor Helen Sang after discussion and advice from Professor Kate Storey (University of Dundee), Dr Filipe Vilas-Boas and Dr Domingos Henrique (Universidade de Lisboa), using their previously published transgene1. The transgene was inserted in reverse orientation in an HIV vector and a line established carrying a single copy of the transgene. The transgene contains the promoter of the Hairy and Enhancer of Split 5 (Hes5) gene, a target of the Notch pathway, upstream of a destabilized nuclear Venus coding sequence (Venus-NLS-PEST (VNP)). Note that the expression of the fluorescent protein Venus is transient and only expressed when notch activity results in transcription from the Hes5-1 promoter. Due to this, the fluorescent signal is consequently faint, and immunostaining has been used to investigate the intensity of the VNP signal. Direct visualization methods are still to be developed.

Notch Reporter
(A) Schematic representation of the Hes5-VNP sequence that was inserted in the Notch reporter transgenic chick line. (B) Left: Transverse sections of the neural tube of the Hes5-VNP transgenic line at E3 and E4 immunostained for Venus (green) and HuCD (red) to label neurons. Middle: Colour coded map of Hes5-VNP intensity. The red line separates HuCD− from HuCD+ cells. The black dotted lines delineate the ventral limit of the roof plate and dorsal limit of the motor neuron domain. Right: Distribution of the Hes5-VNP signal intensity in HuCD− and HuCD+ cells. Note that cells within the limits of the black dotted lines of the colour code panel were labeled in black in the HuCD− population. Figure taken from Beak et al. 2018.

For publications please reference; Baek, C. et al. "Mib1 prevents Notch Cis-inhibition to defer differentiation and preserve neuroepithelial integrity during neural delamination." PLoS biology 16(4), e2004162 (2018).

 

Publications

  1. Vilas-Boas, F., Fior, R., Swedlow, J. R., Storey, K. G. & Henrique, D. A novel reporter of notch signalling indicates regulated and random notch activation during vertebrate neurogenesis. BMC Biol. 9, (2011).
  2. Baek, C. et al. Mib1 prevents Notch Cis-inhibition to defer differentiation and preserve neuroepithelial integrity during neural delamination. PLoS Biol. 16, (2018).
  3. Ables, J. L., Breunig, J. J., Eisch, A. J. & Rakic, P. Not(ch) just development: Notch signalling in the adult brain. Nat. Rev. Neurosci. 12, 269–283 (2011).
  4. Pakvasa, M. et al. Notch signaling: Its essential roles in bone and craniofacial development. Genes Dis. (2020).