Notch signalling defines dorsal root ganglia neuroglial fate choice during early neural crest cell migration

Abstract

Background: The dorsal root ganglia (DRG) are a critical component of the peripheral nervous system, and function to relay somatosensory information from the body's periphery to sensory perception centres within the brain. The DRG are primarily comprised of two cell types, sensory neurons and glia, both of which are neural crest-derived. Notch signalling is known to play an essential role in defining the neuronal or glial fate of bipotent neural crest progenitors that migrate from the dorsal ridge of the neural tube to the sites of the DRG. However, the involvement of Notch ligands in this process and the timing at which neuronal versus glial fate is acquired has remained uncertain.

Results: We have used tissue specific knockout of the E3 ubiquitin ligase mindbomb1 (Mib1) to remove the function of all Notch ligands in neural crest cells. Wnt1-Cre; Mib1^fl/fl mice exhibit severe DRG defects, including a reduction in glial cells, and neuronal cell death later in development. By comparing formation of sensory neurons and glia with the expression and activation of Notch signalling in these mice, we define a critical period during embryonic development in which early migrating neural crest cells become biased toward neuronal and glial phenotypes.

Conclusions: We demonstrate active Notch signalling between neural crest progenitors as soon as trunk neural crest cells delaminate from the neural tube and during their early migration toward the site of the DRG. This data brings into question the timing of neuroglial fate specification in the DRG and suggest that it may occur much earlier than originally considered.

Keywords: Dorsal root ganglia; Fate restriction; Mib1; Neural crest; Notch signalling.

Fig. 1

Loss of Mib1 in neural crest cells causes severe DRG hypoplasia. Longitudinal sections over the forelimb region of E12.5 wildtype, Wnt1-Cre; Mib1^fl/+; Z/EG, and Wnt1-Cre; Mib1^fl/fl; Z/EG embryos were co-immunostained for Tuj1 (a) and EGFP (b) n = 3/genotype. Blue, DAPI. Colour channels are separated for clarity. nt, neural tube; spn, spinal nerve. Scale bar = 100 μm

Fig. 2

Loss of Mib1 causes a reduction in glial progenitors and DRG cell death. a Transverse sections over the forelimb region of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E10.5 co-immunostained for the glial marker Sox10 and the neuronal marker Isl1. b Serial section of A co-immunostained for the axonal marker Tuj1 and cell death marker TUNEL. Scale bar = 50 μm. c Quantitation of Isl1-positive and Sox10-positive cells per DRG at E10.5. Number represents mean of n = 3 embryos, with over 20 individual sections counted per embryo, ns not significant. ****p < 0.0001. d Transverse sections over the forelimb region of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E11.5 co-immunostained for Sox10, Isl1 and the cell death marker cleaved-Caspase 3 (Casp3). e Serial section of D co-immunostained for Tuj1 and TUNEL. Scale bar = 50 μm. f Quantitation of Isl1-positive, Sox10-positive and Casp3-positive cells per DRG at E11.5. Number represents mean of n = 3 embryos, with over 20 individual sections counted per embryo, nt neural tube, spn spinal nerve. *p < 0.05, ***p < 0.001

Fig. 3

Premature differentiation and aberrant cell death of Trk-positive neuronal subtypes in the DRG of Wnt1-Cre; Mib1^fl/fl embryos (a–f). Transverse serial sections over the forelimb of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E10.5 co-immunostained for the neuronal marker Isl1, cell death marker cleaved-Caspase 3 (Casp3), and the neurotrophin receptors TrkA (a, b), TrkB (c, d) and TrkC (e, f). Scale bar = 20 μm. g Quantitation of Trk and Isl1-double-positive cell bodies per DRG at E10.5. Number represents mean of n = 3 embryos, with 8 individual sections counted per embryo. *p < 0.05, **p < 0.01, ****p < 0.0001. h–m Transverse serial sections over the forelimb of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E11.5 co-immunostained for Isl1, Casp3, and TrkA (h, i), TrkB (j, k) and TrkC (l, m). Scale bar = 20 μm. n Quantitation of Trk and Isl1-double-positive cell bodies per DRG at E11.5. Number represents mean of n = 3 embryos, with 8 individual sections counted per embryo, ns not significant, *p < 0.05, ****p < 0.0001

Fig. 4

Sox10 is rapidly downregulated in Wnt1-Cre; Mib1^fl/fl embryos during early neural crest cell migration. a Transverse sections over the forelimb region of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E9.25 (20 somite pairs) co-immunostained for the migrating neural crest cell markers AP2α and Sox10. Inset images in lower panels show higher magnification of the boxed region in upper panels. Scale bar = 50 μm. b Transverse sections of embryos at E9.5 (23 somite pairs) co-immunostained for AP2α and Sox10. Scale bar = 50 μm. c Transverse sections at E9.75 (26 somite pairs) co-immunostained for Isl1 and Sox10. Scale bar = 50 μm. d Quantitation of AP2α-positive and Sox10-positive cells per migrating neural crest stream at E9.25. Number represents mean of n = 3 embryos, with over 20 individual sections counted per embryo. ns = not significant. e Quantitation of E9.5 embryos, n = 4 embryos. *p < 0.05. f Quantitation of E9.75 embryos, n = 3 embryos, nt neural tube, **p < 0.01

Fig. 5

DLL1 accumulation in Wnt1-Cre; Mib1^fl/fl embryos reveals timing of DLL1-Notch signalling during early neural crest cell migration. a Transverse sections through the forelimb region of wildtype embryos at E9.25 (20 somite pairs) co-immunostained for DLL1 and the neural crest cell marker p75. Lower panel shows DLL1 staining alone for clarity. Inset image is a higher magnification of the boxed region. b Sections of Wnt1-Cre; Mib1^fl/fl embryos at E9.25 co-immunostained for DLL1 and p75. Arrowheads indicate regions with increased DLL1 accumulation compared to wildtype. c, d Wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E9.5 (23 somites) co-immunostained for DLL1 and p75. e, f Wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E9.75 (26 somites) co-immunostained for DLL1 and the axonal/neuronal marker Tuj. g, h Wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E10.5 co-immunostained for DLL1 and Tuj, n = 3 embryos/genotype. nt neural tube; spn spinal nerve. All scale bars = 50 μm

Fig. 6

Notch signalling is active in neural crest cells at the earliest stages of migration. a Transverse sections through the forelimb region of wildtype embryos at E9.25 (20 somite pairs) co-immunostained for N1ICD and the neural crest cell marker AP2α. Inset images in lower panels show higher magnification of the boxed region in upper panel. Solid arrowheads highlight neural crest cells undergoing active Notch signalling (AP2α and N1ICD positive). Open arrowheads highlight non-neural crest cell types within the migrating neural crest stream that are undergoing active Notch signalling (AP2α-negative, N1ICD-positive). nt, neural tube; da, dorsal aorta. b Sections of Wnt1-Cre; Mib1^fl/fl embryos at E9.25 co-immunostained and labelled for N1ICD and AP2α. Scale bar = 50 μm. c, d Sections of wildtype and Wnt1-Cre; Mib1^fl/fl embryos at E9.5 (23 somite pairs) co-immunostained for N1ICD and AP2α. n = 3 embryos/genotype. Scale bar = 50 μm