PgmNr W398: Visualization and quantification of the transcriptional response to GLP-1/Notch signaling in the germline stem cell niche.

Authors:
Judith Kimble 1,2 ; ChangHwan Lee 1,2 ; Erika Sorensen 1,2


Institutes
1) Univ. Wisconsin, Madison, WI; 2) HHMI.


Keyword: Germ line stem

Abstract:

Notch signaling regulates stem cells, proliferation and differentiation throughout the animal kingdom. In the C. elegans gonad, the somatic distal tip cell (DTC) niche uses GLP-1/Notch signaling to maintain germline stem cells (GSCs), which generate, maintain and regenerate the germline tissue. We recently identified the sygl-1 gene as a direct downstream target of GLP-1/Notch transcriptional activation and key GSC regulator (1). Here we use single molecule fluorescence in situ hybridization (2) to visualize sygl-1 transcripts in germ cells of the progenitor zone, the region harboring GSCs. Using exon- and intron-specific probes, we distinguish active transcription sites (ATS) in nuclei and mature transcripts in the cytoplasm. With a custom MATLAB code, we record number, intensity and spatial coordinates of both ATS and mRNAs. This approach provides a direct and quantitative readout of the endogenous transcriptional response to GLP-1/Notch. In wild-type germlines, sygl-1 ATS occur stochastically, with little correlation between intensities in a nucleus or with cell cycle stage. Yet the response is patterned: ATS are restricted to the distal ~1/3 of the progenitor zone, and within that region, the percent nuclei with sygl-1 ATS is graded, from 65% at the distal end to <5% 30 μm or 7 germ cell diameters from the distal end. The sygl-1 transcriptional response is gone in glp-1 null mutants, expanded in glp-1 gain of function mutants, abolished within 30 minutes after shifting glp-1(ts) to restrictive temperature and less robust than wild-type in glp-1(ts) mutants at permissive temperature. Comparison of readouts in wild-type and glp-1(ts) at permissive temperature reveals how the response varies as a function of signaling strength: weaker signaling leads to fewer ATS-positive nuclei, lower ATS signal intensity and shorter ATS extent from the DTC. To learn how the graded ATS spatial pattern is generated, we used glp-1(ts) to turn off GLP-1/Notch and abolish ATS and then turn it back on to monitor how fast the ATS signals reform. Their pattern was fully regenerated within an hour, too fast for the main patterning mechanism to be signal decay as germ cells move proximally within the niche. We suggest instead that the strength of GLP-1/Notch signaling is graded in the distal progenitor zone. The major conclusions are that the transcriptional response to Notch signaling is stochastic and that signaling strength determines probability and intensity of transcriptional firing. This high resolution view of the Notch transcriptional response in stem cells will serve as a paradigm for analyzing the readout of signaling more generally.

(1) Kershner et al. (2014) PNAS 111, 3739-3744; (2) Raj and van Oudenaarden (2008) Cell 135, 216-226.



Wormbase Genetic Index
1. glp-1
2. sygl-1