DEV01429 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Cre-recombinase fate mapping. Wnt1-Cre transgenic mice were mated with conditional reporter mice such as loxpRosa mice (A) in which b-gal is expressed under the control of a ubiquitous promoter, but only after a floxed stop codon is removed by Cre-mediated recombination (B). Thus b-gal is not expressed in Cre^–loxpRosa⁺ mice, but is expressed in all cells derived from Cre-expressing cells in Cre⁺loxpRosa⁺ mice (C). Outside of the midbrain and the dorsal neural tube, Wnt1 is only expressed in neural crest cells (Danielian et al., 1997; Ikeya et al., 1997; Parr et al., 1993). As a result, outside of the CNS Wnt1-Cre⁺loxpRosa⁺ mice exhibit b-gal staining specifically in neural crest derived tissues (see Fig. 3).

• Supplemental Figure 2 -

  Fig. S2. Cre-recombinase fate mapping specifically marks neural crest-derived cells. Wnt1-Cre transgenic mice were mated with loxpRosa conditional b-gal reporter mice and the offspring were processed at either E13.5 (whole-mount images in A,E) or postnatal day 3 (B-D,F-J). A-D depict images from Wnt1-Cre^–loxpRosa⁺ control littermates, whereas E-J depict images from Wnt1-Cre⁺loxpRosa⁺ pups. Note the lack of X-gal staining in the littermate control (A), but the extensive X-gal staining in the Wnt1-Cre⁺loxpRosa⁺ pup (E), corresponding to expected neural crest derivatives. Note the extensive X-gal staining in peripheral nerves in the trunk and limbs. Control littermates exhibited no X-gal staining, even within transverse sections through neural tube (NT; B), dorsal root ganglia (DRG; B), oesophagus (OE; C), carotid artery (CA; C), and peripheral nerve (PN; D). By contrast, Wnt1-Cre⁺loxpRosa⁺ pups consistently exhibited strong staining in expected neural crest derivatives, including sympathetic ganglion (SG; F), peripheral nerve (PN; E- J), the smooth muscle around the subclavian artery (SA; G), the capsule around the thymus (Thy; G), the enteric ganglia around the oesophagus (OE; G), dorsal root ganglia (DRG; I), the dorsal root (DR, I) and the ventral root (VR, I). As expected, based on the expression pattern of Wnt1 (Danielian et al., 1997; Ikeya et al., 1997; Parr et al., 1993), there was also scattered X-gal staining in the dorsal neural tube (NT; I). There was no detectable staining of non-neural crest derivatives outside of the CNS, such as in the liver (red tissue, E), thymic hematopoietic cells (G), oesophageal epithelium (G), blood cells, endothelium, and smooth muscle around blood vessels in the hindlimb (V; H), skeletal muscle (SkM; J) and cartilage primordium (C; J). E13.5 Rosa26⁺ embryos showed ubiquitous b-gal expression, as expected (K), unlike the staining that was restricted to neural crest-derived tissues in Wnt1-Cre⁺loxpRosa⁺ embryos (E). E13.5 CMV-Cre⁺loxPRosa⁺ embryos (M) also ubiquitously expressed b-gal, consistent with the expression of the CMV promoter throughout the early embryo. Rosa26^– (L) littermate controls and CMV-Cre^–loxpRosa⁺ (N) littermate controls were processed in parallel and showed no X-gal staining.

• Supplemental Figure 3 -

  Fig. S3. b-gal is expressed in perineurial cells and pericytes of Rosa26⁺ sciatic nerves. Sciatic nerves were dissected from P11 Rosa26^– and Rosa26⁺ pups and processed for X-gal staining (A-C) and immunohistochemistry (D-L). In Rosa26⁺ animals, the perineurial cells and pericytes were b-gal⁺ by both methods. Sections through Rosa26⁺ X-gal-stained nerves showed that the flattened perineurial cells surrounding nerve bundles were heavily stained with X-gal (B, black arrows), as were pericytes surrounding blood vessels (C, black arrow) and other cell types. Rosa26^– nerves processed in parallel never showed X-gal staining, including in perineurial cells (arrowheads, A) and pericytes (arrow, A). Sections through paraformaldehyde-fixed nerves were stained with antibodies against smooth muscle actin (SMA, green) and b-gal (red), and counterstained with DAPI (blue). SMA⁺ pericytes were also positive for b-gal (D-F, white arrows). Panels G-I show an enlarged view of the starred blood vessel in D. The perineurium was also b-gal⁺ (E, arrowheads). Rosa26^– embryos and sections through P11 Rosa26^– nerves (A,J-L) never stained positively for b-gal. These data demonstrate that perineurial cells and pericytes can express b-gal from the unconditional Rosa26 promoter.

• Supplemental Figure 4 -

  Fig. S4. The fluorescent cell in the perineurium of the nerve in Fig. 2B was a blood cell. A DIC image of the nerve section shown in Fig. 2B (same as panel A) reveals that the red, fluorescent, circular cell in the perineurium was a blood cell, not a perineurial cell (A-C, green arrow).

• Supplemental Figure 5 -

  Fig. S5. Fate mapping of Dhh-expressing cells using Dhh-Cre. We observed no bluo-gal staining in E10.5 Dhh-Cre⁺loxpRosa⁺ (n=3) embryos (A), indicating a lack of Dhh-Cre expression in migrating neural crest cells or in cells that have started to form ganglia, such as sensory ganglia (red asterisk, B), or in the neural tube (black asterisk, C). At E12.5, we detected strong b-gal expression in developing peripheral nerves (black arrows, E,F), but not in the gut (D), neural tube (black asterisk, E,F), sensory ganglion (red asterisk, F), or non-neural tissues (D-F). Note that b-gal expression within peripheral nerves was spotty, with some segments exhibiting strong staining, and adjacent segments exhibiting weak staining (E). We never observed bluo-gal staining in Dhh-Cre^–LoxpRosa⁺ control embryos (G).

• Supplemental Figure 6 -

  Fig. S6. The combination of Bmp4, Nrg1 and Delta-Fc caused sciatic nerve NCSCs to form glia and myofibroblasts, but not neurons, whereas gut NCSCs formed neurons and myofibroblasts but not glia. E14.5 sciatic nerve NCSCs and gut NCSCs were isolated by flow-cytometry and cultured for 6 days at clonal density in standard medium supplemented with Bmp4, Nrg1 and Delta-Fc. Panels A-C show one field of view from a typical sciatic nerve NCSC colony, while panels D-F show one field of view from a typical gut NCSC colony. Note that some of the cells within the sciatic nerve NCSC colony stained positively for both GFAP and SMA (arrowheads, C).