Constitutively active Notch1 converts cranial neural crest-derived frontonasal mesenchyme to perivascular cellsin vivo

Abstract

Perivascular/mural cells originate from either the mesoderm or the cranial neural crest. Regardless of their origin, Notch signalling is necessary for their formation. Furthermore, in both chicken and mouse, constitutive Notch1 activation (via expression of the Notch1 intracellular domain) is sufficient in vivo to convert trunk mesoderm-derived somite cells to perivascular cells, at the expense of skeletal muscle. In experiments originally designed to investigate the effect of premature Notch1 activation on the development of neural crest-derived olfactory ensheathing glial cells (OECs), we used in ovo electroporation to insert a tetracycline-inducible NotchΔE construct (encoding a constitutively active mutant of mouse Notch1) into the genome of chicken cranial neural crest cell precursors, and activated NotchΔE expression by doxycycline injection at embryonic day 4. NotchΔE-targeted cells formed perivascular cells within the frontonasal mesenchyme, and expressed a perivascular marker on the olfactory nerve. Hence, constitutively activating Notch1 is sufficient in vivo to drive not only somite cells, but also neural crest-derived frontonasal mesenchyme and perhaps developing OECs, to a perivascular cell fate. These results also highlight the plasticity of neural crest-derived mesenchyme and glia.

Olfactory ensheathing cells abutting the embryonic olfactory bulb express Frzb, whose deletion disrupts olfactory axon targeting

“…Embryos were immersion‐fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) overnight at 4°C, then embedded in OCT (Thermo Fisher Scientific) for cryosectioning at 10 μm. In situ hybridization was performed on sections as previously described (Miller, Perera, & Baker, ). The mouse Frzb clone was a gift of Christine Hartmann (Westfälische Wilhelms‐Universität Münster, Münster, Germany).…”

Neural crest Notch/Rbpj signaling regulates olfactory gliogenesis and neuronal migration

“…Two days after doxycycline injection, at E5.5 (n 5 2), most control EGFP-targeted neural crest-derived cells on the olfactory nerve expressed the OEC marker Sox10, as expected (Barraud et al, 2010) ( Figure 2a (Joseph et al, 2004) and/or perivascular cells, which are neural crest-derived in the region of the forebrain (Etchevers, Vincent, Le Douarin, & Couly, 2001); indeed, the perivascular cell marker Pdgfrb is detectable in some cells on the olfactory nerve at E6.5 (Miller, Perera, & Baker, 2017).] Immunostaining for the neuronal RNAbinding proteins Elavl3/Elavl4 (HuC/D, hereafter Elavl3/4; Okano & Darnell, 1997;Pascale, Amadio, & Quattrone, 2008) Overall, these data show that blocking Notch/Rbpj signaling in cranial neural crest-derived cells for two days from E3.5 (when Mpzpositive cells in the OEC lineage can first be identified on the olfactory nerve; Drapkin & Silverman, 1999) resulted in a majority of the targeted cells on the olfactory nerve adopting a neuronal fate (distinct from migrating GnRH neurons, which are olfactory placode-derived; Barraud et al, 2010;Sabado, Barraud, Baker, & Streit, 2012).…”

“…Two days after doxycycline injection, at E5.5 ( n = 2), most control EGFP ‐targeted neural crest‐derived cells on the olfactory nerve expressed the OEC marker Sox10 , as expected (Barraud et al, ) (Figure a–b 3 ). [The presence of Sox10 ‐negative EGFP ‐targeted cells could reflect the formation of non‐glial neural crest derivatives, such as endoneurial fibroblasts (Joseph et al, ) and/or perivascular cells, which are neural crest‐derived in the region of the forebrain (Etchevers, Vincent, Le Douarin, & Couly, ); indeed, the perivascular cell marker Pdgfrb is detectable in some cells on the olfactory nerve at E6.5 (Miller, Perera, & Baker, ).] Immunostaining for the neuronal RNA‐binding proteins Elavl3/Elavl4 (HuC/D, hereafter Elavl3/4; Okano & Darnell, ; Pascale, Amadio, & Quattrone, ) revealed many Elavl3/4‐positive neuron cell bodies on the olfactory nerve, but, as expected (Barraud et al, ), EGFP ‐targeted neural crest‐derived cells were not neurons (Figure c–c 3 ).…”

“…Previously, we used the Tol2 transposase/‘Tet‐on’ in ovo electroporation system (Sato et al, ; Watanabe et al, ) to drive NotchΔE , encoding a constitutively active form of mouse Notch1 (Kopan, Schroeter, Weintraub, & Nye, ; Sato et al, ), in cranial neural crest‐derived cells from E4 (Miller et al, ). This proved to be sufficient to convert both frontonasal mesenchyme cells, and perhaps also developing OECs, to a different cranial neural crest cell fate, namely Pdgfrb ‐positive perivascular cells (Miller et al, ). Here, we found that most targeted cranial neural crest‐derived cells on the olfactory nerve formed neurons when Notch/Rbpj signaling was blocked from E3.5.…”

“…Mouse Gnrh1 was previously cloned as described (Barraud et al, ). Digoxigenin‐labeled antisense riboprobes were generated as described (Henrique et al, ) and in situ hybridization on sections, followed by immunohistochemistry, performed as described (Miller et al, ). Primary antibodies used were: anti‐EGFP (rabbit, Life Technologies, 1:500; or mouse IgG1, Roche, 1:500); anti‐Elavl3/Elavl4 (anti‐HuC/D; mouse IgG2b, Invitrogen, 1:400); anti‐Tubb3 (neuronal class III beta‐tubulin; clone TUJ1, mouse IgG2a, Covance, 1:500).…”