Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

Joshua Hawley, Robert Lea, Veronica Biga, Nancy Papalopulu, Cerys Manning

Posted on: 15 November 2024

Preprint posted on 12 October 2024

Notching it up a bit: Notch signalling reception is observed in cells at a distance through cellular protrusions in the developing spinal cord.

Selected by Ankita Walvekar

Categories: cell biology, developmental biology

Background

The Notch signalling pathway is a known cell communication pathway, occurring between neighbouring cells. The current preprint addresses the question of how Notch signalling could actually affect cells that are not direct neighbours. The authors postulate a mathematical model in which there are micro clusters of cells that periodically signal to (and beyond) adjacent cells. Previous experimental work showed the existence of such micro clusters (3-5 cells) and the oscillatory expression of the Notch target HES5 in these cells (Biga et al., 2021).

Because Notch signalling requires direct membrane contact for its activation, the preprint authors looked into the importance of membrane protrusions (like cytonemes) in allowing long-range signalling. These protrusions were already shown to be important for other signalling pathways like Shh and Wnt in the neural tube (Hall et al., 2024). Hence, the authors scored for the presence of cellular protrusions when micro clusters of HES5-expressing cells were present.

Main findings

In this preprint the authors tested for and subsequently confirmed the presence of actin-based cell protrusions in radial glial cells in the mouse developing spinal cord, in fixed as well as live tissue samples.
Additionally, they confirmed the presence of the Notch ligand DLL1 in these membrane protrusions.
The authors further confirmed that the actin regulator CDC42 is involved in the formation of these protrusions.
They show that perturbing the density of these protrusions caused reduced HES5 spatial periods, but this does not affect the length of the protrusions.

Why I highlight this preprint

I chose to highlight this preprint as I closely study signalling in the neural tube and was fascinated to know that Notch signalling could also be elicited through cell protrusions (apart from Shh and Wnt signalling) in the developing neural tube.

Questions to the authors

Perturbing the formation of cellular protrusions affects DLL1, but how does it affect neural tube development? Does it cause any significant temporally-regulated developmental perturbations or is it inconsequential in the larger scheme of development?
Would you expect any differences in the reception of Notch signalling through these protrusions in dorsal versus ventral neural tube cells?
Do the cellular protrusions affect the other Notch signalling components, as HES5 is only one of the downstream targets?

References:

Biga, V., Hawley, J., Soto, X., Johns, E., Han, D., Bennett, H., Adamson, A.D., Kursawe, J., Glendinning, P., Manning, C.S. and Papalopulu, N. (2021) ‘A dynamic, spatially periodic, micro‐pattern of HES5 underlies neurogenesis in the mouse spinal cord’, Molecular Systems Biology, 17(5), pp. 1–27. Available at: https://doi.org/10.15252/msb.20209902.

Hall, E.T., Dillard, M.E., Cleverdon, E.R., Zhang, Y., Daly, C.A., Ansari, S.S., Wakefield, R., Stewart, D.P., Pruett-Miller, S.M., Lavado, A., Carisey, A.F., Johnson, A., Wang, Y.D., Selner, E., Tanes, M., Ryu, Y.S., Robinson, C.G., Steinberg, J. and Ogden, S.K. (2024) ‘Cytoneme signaling provides essential contributions to mammalian tissue patterning’, Cell, 187(2), pp. 276-293.e23. Available at: https://doi.org/10.1016/j.cell.2023.12.003.

Tags: cellular protrusions, neural tube development, notch signalling

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Author's response

Cerys Manning shared

Our previous studies described a dynamic spatial pattern of the Notch target HES5 in the spinal cord, whereby microclusters of high HES5 expressing cells were regularly spaced along the dorsoventral axis, i.e. they were spatially periodic (Biga et al. 2021). Our modelling predicted the pattern was dependent on an extended Notch signalling distance beyond neighbouring cells and controlled the spacing and rate of differentiation (Hawley et al. 2022). Here, we find dynamic protrusions emanate up to 20µm from radial glial cells of the developing spinal cord and contain the Notch ligand Dll1, supporting the idea that Notch signalling can be transduced between non-neighbouring cells. Functional perturbation of these protrusions led to a change in the HES5 pattern, although not significantly. We think this is exciting because specialised signalling protrusions, or cytonemes, are emerging as players in multiple key developmental signalling pathways and processes.

1.Perturbing the formation of cellular protrusions affects DLL1, but how does it affect neural tube development? Does it cause any significant temporally regulated developmental perturbations or is it inconsequential in the larger scheme of development?

It is challenging to assign a developmental function to protrusions as it is difficult to specifically perturb protrusions in the tissue. Currently the best evidence is from Hall et al. who used a myosinX knockout mouse to show that protrusions emanating from floorplate and roofplate cells that carried Shh and Wnt signals were required for correct dorsal-ventral patterning. In the developing chick spinal cord, Kasioulis et al. 2022 found protrusions from radial glia cells at the apical surface but could not assign a developmental function; loss of apical protrusions did not alter neural tube organisation or lead to premature differentiation. In our paper, we describe that protrusions exist all along the apical-basal axis of radial glia cells and that reducing their number led to a trend towards a shorter distance between HES5 microclusters. Therefore, although in this paper we cannot specifically define a larger function on development, the results are moving in the right direction.

2. Would you expect any differences in the reception of Notch signalling through these protrusions in dorsal versus ventral neural tube cells?

We didn’t examine this directly, but we saw protrusions from cells across the entire dorsoventral axis. Further, our mathematical model that recapitulated the observed repetitive microclusters of HES5 high cells along the D-V axis did not require any differential in Notch signalling along the D-V axis. However, the Notch ligands Dll1 and Jag1, and the Fringe proteins that modify Notch signalling, are patterned in non-overlapping stripes along the D-V axis (Marklund et al. 2010). So, although we showed the presence of Dll1 on protrusions, it would be nice to check whether Jag1 and Fringe proteins are also found on protrusions.

3. Do the cellular protrusions affect the other Notch signalling components, as HES5 is only one of the downstream targets?

We haven’t directly tested this, but we propose that Notch cleavage and NICD activation at the membrane of cells contacted by protrusions would lead to activation of multiple Notch target genes, not just HES5. We focused on HES5 because it is the main HES expressed in the ventral spinal cord at this stage.

References:

Biga V, Hawley J, Soto X, Johns E, Han D, Bennett H, Adamson AD, Kursawe J, Glendinning P, Manning CS, Papalopulu N. A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord. Mol Syst Biol. 2021 May;17(5):e9902. doi: 10.15252/msb.20209902.

Hall ET, Dillard ME, Cleverdon ER, Zhang Y, Daly CA, Ansari SS, Wakefield R, Stewart DP, Pruett-Miller SM, Lavado A, Carisey AF, Johnson A, Wang YD, Selner E, Tanes M, Ryu YS, Robinson CG, Steinberg J, Ogden SK. Cytoneme signaling provides essential contributions to mammalian tissue patterning. Cell. 2024 Jan 18;187(2):276-293.e23. doi: 10.1016/j.cell.2023.12.003.

Hawley J, Manning C, Biga V, Glendinning P, Papalopulu N. Dynamic switching of lateral inhibition spatial patterns. J R Soc Interface. 2022 Aug;19(193):20220339. doi: 10.1098/rsif.2022.0339. Epub 2022 Aug 24.

Kasioulis I, Dady A, James J, Prescott A, Halley PA, Storey KG; A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis. J Cell Sci 15 March 2022; 135 (6): jcs259897. doi: https://doi.org/10.1242/jcs.259897

Marklund U, Hansson EM, Sundström E, de Angelis MH, Przemeck GK, Lendahl U, Muhr J, Ericson J. Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression. Development. 2010 Feb;137(3):437-45. doi: 10.1242/dev.036806.

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