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Results: 8

1.
Figure 2

Figure 2. Lgi4 is expressed by glia in the myenteric plexus, DRGs, and sympathetic ganglia of adult Lgi4LacZ/+ mice. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

Immunofluorescence analysis of the LacZ expression pattern in the myenteric plexus (A), DRGs (B), and sympathetic ganglia (C) of young adult Lgi4LacZ/+ mice showed overlapping staining with the glial marker BFABP but not with the neuronal markers HuC/D or NeuN. Nuclei were visualized using 4’,6-diamino-2-phenylindole dihydrocloride (DAPI) staining (blue).

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
2.
Figure 3

Figure 3. Lgi4LacZ/LacZ mice exhibit abnormal forelimb posture, peripheral nerve hypomyelination, growth retardation, and neonatal death. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) Lgi4LacZ/LacZ mice (L/L) exhibited an arthrogryposis-like forelimb posture abnormality at P0 and growth retardation relative to littermate controls (+/+) by P14.
(B) The body mass of Lgi4LacZ/LacZ mice was similar to littermate controls at E14.5 but significantly reduced at P1 and P14 (p<0.01 by Student’s T-test; n=12–18 mice/genotype at E14.5, 18–27 at P0, and 9–11 at P14)
(C) Genotyping the progeny of Lgi4LacZ/+ intercrosses revealed expected numbers of Lgi4LacZ/LacZ mice at E14.5 and E18.5 but many Lgi4LacZ/LacZ mice died immediately after birth and no Lgi4LacZ/LacZ mice survived to P21.
(D–E) Sciatic nerves from P12 Lgi4LacZ/LacZ mice were thinner (D) and hypomyelinated (E) relative to sciatic nerves from littermate controls.
(F) Immunostaining of transverse sections through P12 Lgi4LacZ/LacZ and control sciatic nerves revealed little Krox20 or Periaxin staining in the absence of Lgi4, but normal peripherin staining.

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
3.
Figure 5

Figure 5. Lgi4LacZ/LacZ mice have normal numbers of undifferentiated progenitors and neurons but fewer glia throughout the PNS. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) Transverse sections of guts from E10.5 control and Lgi4LacZ/LacZ embryos were stained for p75 (a marker of neural crest stem/progenitor cells).
(B) The number of p75+ cells that colonized the gut at E10.5 did not differ between control and Lgi4LacZ/LacZ embryos (mean±SD of number of p75+ cells per section; four sections per gut region/mouse and three mice/genotype).
(C) Transverse sections of the gut from E10.5 or E18.5 control and Lgi4LacZ/LacZ embryos were stained with antibodies against neuronal (Tuj1) and glial (BFABP) markers.
(D) The number of neurons (Tuj1+) per gut section was similar in control and Lgi4LacZ/LacZ embryos at E10.5 and E18.5. No glia (BFABP+) were observed in E10.5 gut sections, but the number of glia per section was significantly lower in Lgi4LacZ/LacZ embryos at E18.5 (*, p<0.05; 4–6 sections/gut region per mouse and 4–5 mice/genotype).
(E) Transverse sections of sympathetic ganglia from E10.5 control and Lgi4LacZ/LacZ mouse embryos were stained with antibody against Sox10 (a marker of neural crest stem/progenitor cells at this stage of development).
(F) The number of Sox10+ cells per section did not differ between E10.5 control and Lgi4LacZ/LacZ embryos in DRG or sympathetic ganglia (four sections/mouse and three mice/genotype).
(G) Transverse sections of sympathetic ganglia from E10.5 and E18.5 control and Lgi4LacZ/LacZ embryos were stained with antibodies against neuronal (Tuj1) and glial (BFABP) markers.
(H) The number of neurons (Tuj1+) per section through DRGs and sympathetic ganglia was similar in control and Lgi4LacZ/LacZ embryos at E10.5 and E18.5. No glia (BFABP+) were observed in E10.5 sections, but the number of glia per section was significantly lower in Lgi4LacZ/LacZ embryos at E18.5 (*, p<0.05; 4–6 sections/mouse and 4–6 mice/genotype).

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
4.
Figure 6

Figure 6. Lgi4 is required for glial cells to acquire a normal, mature morphology throughout the PNS in vivo. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) Whole mount X-gal staining of outer plexus/muscle layers from the P0 gut of Lgi4LacZ/LacZ mice and littermate controls. The Lgi4LacZ/LacZ gut had a sparse myenteric plexus with fewer connections among ganglia relative to the Lgi4LacZ/+ gut.
(B) Whole mount immunostaining of outer plexus/muscle layers from the P0 gut of Lgi4LacZ/LacZ mice and littermate controls with antibodies against the glial markers GFAP and BFABP, and the neuronal marker TuJ1. GFAP+ and BFABP+ staining was sparse in the Lgi4LacZ/LacZ gut. Higher magnification images of glial cells (inset, BFABP) suggested that Lgi4LacZ/LacZ glia were smaller with fewer and shorter processes
(C–D) GFAP immunostaining in DRGs (C) and sympathetic ganglia (D) in sections from P4 wild-type mice showed a honeycomb-like pattern that enwrapped neurons but in sections from P4 Lgi4LacZ/LacZ mice there were fewer glia with shorter processes that did not enwrap neurons.

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
5.
Figure 1

Figure 1. Lgi4 is expressed by neural crest stem cells and other undifferentiated neural crest cells throughout the developing PNS. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) Dorsal view of X-gal stained E9.5 Lgi4LacZ/+ and Lgi4+/+ embryos revealed Lgi4 expression (blue, arrows) by migrating trunk neural crest cells.
(B) X-gal staining of E10.5 Lgi4LacZ/+ and Lgi4+/+ embryos revealed Lgi4 expression by cranial ganglia (open arrows), DRGs (black arrowhead), sympathetic ganglia (black arrow), and migrating neural crest cells within the gut (open arrowhead). The cross section of the Lgi4LacZ/+ embryo was taken at the level of the black bar indicated in the photo at left.
(C) X-gal staining of sections from E13.5 and P0 Lgi4LacZ/+ and Lgi4+/+ mice revealed Lgi4 expression in the myenteric plexus (gut, arrow), DRGs, sympathetic ganglia (at P0, arrowhead indicates sympathetic ganglion and arrow indicates celiac ganglion), peripheral nerve, and parasympathetic ganglion. Note the bacterial LacZ activity in the lumen of the P0 gut.
(D) Virtually all neurospheres cultured from the gut, DRGs, and sympathetic ganglia of E13.5 Lgi4LacZ/+ mice stained with X-gal (mean±SD X-gal stained is indicated in each panel, n=3 independent experiments).
(E) Lgi4LacZ/+ neurospheres transferred to adherent cultures stained positively for X-gal and Nestin.

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
6.
Figure 4

Figure 4. Lgi4 is not required for neural crest stem cell formation or self-renewal but is required for normal glial differentiation in culture. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) Gut cells from E13.5 Lgi4LacZ/LacZ mice formed neurospheres with normal morphology after 10 days in non-adherent, low density (~1 cell/µl) cultures.
(B) Lgi4 deficiency did not affect the percentage of cells from E13.5 gut, DRGs, or sympathetic ganglia that formed multilineage neurospheres, neurosphere diameter, or self-renewal potential (number or percentage of cells from individual primary neurospheres that formed multipotent secondary neurospheres upon subcloning; n=5 independent experiments).
(C) Multilineage Lgi4LacZ/LacZ NCSC colonies (cultured from E13.5 gut) did not differ from control colonies in terms of neurons (peripherin+) or myofibroblasts (SMA+) but had fewer GFAP+ glia and less pronounced GFAP staining.
(D–E) Adherently cultured gut (D) and DRG (E) cells from E13.5 Lgi4LacZ/LacZ embryos formed normal numbers of total colonies, neuron (N)-containing colonies, and myofibroblast (M)-containing colonies, but significantly fewer glia (G)-containing colonies compared to littermate control cells. Cells were plated at clonal density (500 cells per 35mm dish) such that individual cells could form spatially-distinct colonies (*, p<0.05; four independent experiments.)

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
7.
Figure 8

Figure 8. ADAM22 is a physiological receptor for Lgi4 that mediates the effect of Lgi4 on PNS gliogenesis. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A–B) Lgi4 binds to ADAM11, ADAM22 and ADAM23 but not ADAM9. (A) FLAG tagged Lgi4 was co-transfected with HA-tagged ADAMs into 293T cells. Immunoprecipitation with anti-FLAG pulled down ADAM11, ADAM22, and ADAM23 but not ADAM9. Immunoprecipitation with anti-HA antibody pulled down Lgi4 when ADAM11, ADAM22, or ADAM23 were expressed but not when ADAM9 was expressed. Input: before immunoprecipitation; IP: after immunoprecipitation. (B) Immuofluorescence analysis indicated that FLAG-tagged Lgi4 over-expressed in COS7 cells was retained on the cell surface when HA-tagged-ADAM11, ADAM22, or ADAM23 were co-expressed but not when ADAM9 was expressed.
(C) In situ hybridization indicated that Adam22 is expressed in DRGs, sympathetic ganglia, and myenteric plexus of E14.5 and P0 mice. Adam11 and Adam23 were also expressed in certain locations within the PNS (Suppl. Fig. 5).
(D) At birth, Adam22−/− and Lgi4LacZ/LacZAdam22−/− mice exhibited an arthrogryposis-like forelimb posture abnormality similar to Lgi4LacZ/LacZ mice.
(E) Adam22−/− and Lgi4LacZ/LacZAdam22−/− mice exhibited postnatal growth retardation similar to Lgi4LacZ/LacZ mice. Lgi4LacZ/LacZ and Adam22−/− mutations did not have additive effects on growth.
(F) Compared to wild-type NCSC colonies, E13.5 gut NCSC colonies from Adam22−/− and Lgi4LacZ/LacZAdam22−/− mice generated smaller numbers of glial cells with less pronounced GFAP staining, similar to Lgi4LacZ/LacZ NCSCs.
(G–H) Compared to wild-type, E18.5 gut sections from Adam22−/− and Lgi4LacZ/LacZAdam22−/− mice had smaller numbers of glial cells, similar to Lgi4LacZ/LacZ NCSCs (P<0.05; 4–5 mice/genotype with 4–6 sections/mouse). Lgi4LacZ/LacZ and Adam22−/− mutations did not have additive effects on gliogenesis, suggesting that Lgi4 and ADAM22 act in the same pathway.

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.
8.
Figure 7

Figure 7. Lgi4 is required for the proliferation of glial restricted progenitors but not neuronal restricted progenitors. From: Lgi4 promotes the proliferation and differentiation of glial lineage cells throughout the developing peripheral nervous system.

(A) p75+α4+ rat sciatic nerve NCSCs were cultured at clonal density for 10 days in standard medium supplemented with control conditioned medium (293T), Lgi4 conditioned medium, or recombinant Neuregurin-1. Lgi4 did not affect colony composition, in contrast to Neuregulin-1, which instructed glial differentiation by NCSCs at the expense of multilineage differentiation.
(B–H) Dissociated muscle/plexus layer cells from P0 wild-type and Lgi4LacZ/LacZ gut were cultured at clonal density for 10 days in adherent cultures. Lgi4LacZ/LacZ cells formed smaller glia-only colonies with less GFAP staining than control cells (B) but normal neuron-only colonies (C). The number of cells per glia-only colony (D) and the frequency of glia-only colonies (E) were significantly reduced in cultures of Lgi4LacZ/LacZ cells (**, p<0.05). In contrast, the number of cells per neuron-only colony (F) and frequency of neuron-only colonies (G) was not affected by Lgi4 deficiency.
(H) The frequency of phospho-histone H3+ (pH3+) cells per glia-only colony was significantly decreased in Lgi4LacZ/LacZ colonies (**, p< 0.05; see mean±SD below images).
(I) Muscle/plexus layer cells from P0 wild-type gut were cultured for 10 days in standard medium supplemented with control conditioned medium or Lgi4 conditioned medium. Lgi4 significantly increased the number of cells per glia-only colony (**, p< 0.05; see mean±SD below images).
(J–L) E16.5 gut sections from wild-type and Lgi4LacZ/LacZ mice revealed a significantly lower frequency of pH3+BFABP+ cells (white arrows in J) in the Lgi4LacZ/LacZ gut (K; **, p<0.05) but a normal frequency of pH3+BFABP− cells (L).

Jinsuke Nishino, et al. J Neurosci. ;30(45):15228-15240.

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