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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001388

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Conditional ablation of GFR1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype

¹ Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
² Departments of Medicine, Renal Division, Washington University School of Medicine, St Louis, MO 63110, USA.
³ Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
⁴ Department of Cell Biology and Neurosciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
⁵ Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. GDNF expression is restricted to the distal gut after colonization of the entire gut by ENS progenitors. (A) GDNF expression in gut at E15.5. The gut isolated from GDNFlacZ/+ mouse embryos was stained with X-gal histochemistry. Intense X-gal staining was observed in the cecum and colon. (B) Wholemount preparations of the small intestine (upper panels) and colon (lower panels) of E15.5 GFR1GFP/+ embryos (obtained by crossing GFR1flox/+ mice to ß-actin Cre mice: see Materials and methods). GFR1-expression (as revealed by GFP) was confirmed in almost all PGP9.5+ neurons in the colon, whereas only a small neuronal population expressed GFR1 in the small intestine. (C) Quantitative analysis of GFR1-expressing neuronal population. Percentage of GFP+-PGP9.5+ neurons / total PGP9.5+ neurons is presented. Scale bar: 10 µm in B. Co, colon; SI, small intestine.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Conditional inactivation of the GFR1 gene in mouse. (A) Schematic drawing of the GFR1 locus, the floxed GFR1 and GFP knock-in (null) alleles. Floxed GFR1 allele expresses GFR1 cDNA, thereby serving as a functional allele. Activation of Cre recombinase results in a removal of floxed GFR1, simultaneously generating GFP knock-in (GFR1-null) allele. (B) Schematic diagram of breeding strategy. GFR1+/-:CAGGCre-ERTM mice were mated to GFR1flox/flox mice to obtain GFR1flox/+:CAGGCre-ERTM (control) and GFR1flox/-:CAGGCre-ERTM (knockout, cKO) embryos in the same litter. Pregnant mice were given an intraperitoneal injection of 4-OHT at the desired time point to induce Cre activity. Following recombination, GFP reporter gene was expressed in the control and cKO embryos. (C) 4-OHT-induced Cre recombination in enteric neurons. Minimal `leaky' recombination in the colon of E15.5 GFR1flox/+ embryo was observed in the absence of 4-OHT treatment (left panel). Administration of 4-OHT induced GFP expression in a large number of neurons in the colon, indicating that Cre recombination occurred in the majority of enteric neurons in the colon (right panel). (D) Histological analysis of cKO enteric plexus showing no GFR1-immunoreactivity (red) in GFP+ cells (green). Note the expression of GFR1 (red) in neighboring un-recombined cells. (E) Absence of pERK in GFR1-deficient enteric neurons. Activation of ERK (pERK) was detected in GFP-negative-TuJ1-positive cells (arrowhead). By contrast, no pERK signal was observed in GFP-positive-TuJ1-positive cells (asterisk). Scale bars: 20 µm in C; 5 µm in D,E.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Inactivation of GFR1 depletes enteric neurons in the colon. (A) PGP9.5 immunostaining of the distal colon in E15.5 control and cKO mouse embryos before injection of 4-OHT. Comparable ENS staining was observed in control and cKO colon, indicating normal development of the ENS before induction of GFR1 inactivation. (B) Wholemount AChE histochemical analysis of E18.5 gut 3 days after inactivation of GFR1. The highly organized meshwork-like pattern of ENS network observed in control (top) was dramatically disrupted in cKO (bottom). ENS structure in the small intestine was well maintained in both embryos (insets). (C) Toluidine Blue staining revealed the absence of enteric ganglia (Ga) in the myenteric layer (between Lm and Cm) of the colon in cKO embryos. Cm, circular muscle; Co, colon; Ga, enteric ganglia; Lm, longitudinal muscle; SI, small intestine. Scale bars: 20 µm in A; 200 µm in B; 10 µm in C.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Spatiotemporally specific effects of GFR1 inactivation on ENS development in mouse. (A) Confocal microscopic images of wholemount preparations of midgut from E13.5 control and cKO embryos in which GFR1 inactivation was induced at E11.5. GFP+ cells were less dense in the cKO than control midgut. (B) Wholemount PGP9.5 staining of small intestine from E18.5 control and cKO embryos subjected to 4-OHT treatment at E13.5. Although total colon aganglionosis was observed in cKO embryos (bottom right), the ENS structure in the small intestine was well maintained (top right). (C) Wholemount preparation of myenteric plexus of the colon from P14 mice subjected to GFR1 inactivation at P5. No obvious abnormalities were found in enteric neurons (PGP9.5+) or GFP+ cells of cKO myenteric plexus (upper panels). Even multiple 4-OHT injection did not affect ENS structure (lower panels). Recombination efficiency by single 4-OHT injection was estimated as 50% for A and B, and 40% for C. S, sacrificed. Scale bars: 10 µm in A; 50 µm in B; 100 µm in C.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Absence of caspase activation during ENS degeneration induced by inactivation of GFR1. (A) Timecourse analysis of ENS degeneration after administration of 4-OHT (at 0 h). The gut samples collected at different time points were doubly stained by GFP immunohistochemistry and TUNEL. Note the progressive loss of GFP-positive cells in cKO colon 24-36 hours after administration of 4-OHT. TUNEL staining was observed in the colon 24-27 hours after 4-OHT treatment. GFP staining persisted in control colon (bottom). (B) Immunostaining of GFP (green) and Phox2b (red). Nearly complete overlap between GFP and Phox2b signals indicates that GFP predominantly labels enteric neurons and glia, but not smooth muscle cells, in the E16 distal colon. (C) Histochemical visualization of activated caspase-3-(green) and TUNEL-positive (red) cells. In embryonic DRGs, activated-caspase-3-positive cells outnumbered TUNEL-positive cells, and there was a significant overlap between those two cell populations. Caspase activity was not detected in any of the TUNEL-positive cells in cKO colon at any point during the timecourse of loss of GFP+ cells. Scale bars: 20 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Caspases are not required for the death of GDNF-deprived enteric neurons. (A) Enteric neurons from distinct gut regions respond differentially to GDNF deprivation. Enteric neurons isolated from mouse E15.5 colon and small intestine were cultured for 2 days and then switched to GDNF-deprived conditions. The ratio of surviving neuron numbers (48 hours after GDNF deprivation) to initially plated cell numbers is shown. (B) Timecourse analysis of colonic neuron survival. TuJ1-positive neurons were counted at 0, 12, 24 and 48 hours after GDNF deprivation (-) or in the presence of GDNF (+). (C) Nuclear staining by Hoechst 33342 (upper panels) and TUNEL (lower panels) of enteric neurons 12 hours after GDNF deprivation (left panels) or of SCG neurons 24 hours after NGF deprivation (right panels). Condensed chromatin and nuclear fragmentation associated with strong TUNEL-reactivity, salient features in dying SCG neurons (arrowheads in right panels), were never observed in GDNF-deprived enteric neurons (arrowheads in left panels). (D) Treatment with the broad-range caspase inhibitor zVAD-fmk (100 µM) rescues NGF-deprived SCG neurons (right) but not GDNF-deprived enteric neurons (left). Survival of enteric neurons and sympathetic neurons was examined 2 days after GDNF deprivation or 5 days after NGF deprivation, respectively. (E) Responses of Bax-deficient enteric neurons to GDNF withdrawal. No significant differences were observed in the survival of colonic neurons between Bax-deficient and wild-type embryos (E15.5). Values represent the percentages of living cells normalized to the GDNF-maintained neurons. (F) Bcl-XL overexpression rescues the survival of GDNF-deprived enteric neurons. Enteric neurons were infected with lentivirus expressing Bcl-XL or vector alone several hours after plating. Surviving neurons (TuJ1+) were counted 48 hours after GDNF deprivation. Scale bar: 10 µm in C. Co, colon; SI, small intestine; WT, wild type.

View larger version (77K): [in this window] [in a new window]   Fig. 7. Morphological characteristics of dying enteric ganglion cells. (A) Longitudinal section of enteric ganglia in a segment of the colon from E16.5 control and cKO mouse embryos 1 day after inactivation of GFR1. Many nuclei exhibited constricted and irregularly shaped morphologies in cKO embryos (top right). (Bottom) Schematic figures depicting the location of the myenteric ganglia (circled by dotted lines) and the positions of the nuclei in myenteric ganglion cells (indicated by N). Multiple nuclear profiles observed in a single cell plane are marked by pink and green. (B) A parallel section series of a single cell in cKO colon, revealing abnormal lobulation of the nucleus, but not fragmentation. (C) High-magnification view of perinuclear regions of control and cKO cells. Note that electron-dense structures in the nucleus (N) are more prominent in cKO than in control cells (right, arrows). No significant changes were observed in mitochondrial morphology (arrowheads). (D) Clearance of dying cells by large vacuoles (arrowheads) in cKO enteric ganglia. Conditional KO colon 27 hours after inactivation of GFR1 shown as an example (upper panel). A phagocytotic vacuole containing cell debris of high electron density, possibly representing a condensed nucleus of the engulfed cell (lower panel). (E) Left: a typical degenerating cell with severely depleted cytoplasm in the cKO distal colon 27 hours after inactivation of GFR1. Middle: representative image of a single-membraned vacuole containing cellular components (arrowhead) in a degenerating ganglion cell. Right: a degenerating cell with high electron-dense cytoplasm throughout the entire cell body. Note that the cell also contains multivesicles (arrowhead). Broken lines depict cell margins. Scale bar: 5 µm in A; 1 µm in B,D,E; 0.5 µm in C. Cm, circular muscle; Ga, enteric ganglia; Lm, longitudinal muscle; N, nucleus.