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First published online 6 October 2004
doi: 10.1242/dev.01421

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Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis

¹ Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
² Department of Urologic Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
³ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
⁴ Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA

View larger version (44K): [in a new window]   Fig. 1. RetDN/+ mice fail to thrive. (A) Mutant or wild-type human RET9 cDNA was introduced into exon 1 (black box) of the mouse Ret locus (black triangles, loxP sites). The restriction sites (K, KpnI; N, NcoI) and length of the expected fragments in Southern blots are indicated. (B) Schematic of Ret knocked-in alleles. Open box, RET9 cDNA; black line, SV40 3'UTR; ECD, extracellular domain; TK, tyrosine kinase domain; triangle, loxP sites; L985P and Y1062F, RET mutations in the DN allele. (C) Southern blot analysis confirmed successful generation of Ret mutant mouse lines. (D) Growth curves of RetDN/+ and wild-type (wt) littermates at indicated postnatal time points (n=3 for each time point; *=P<0.01; error bars, mean±s.d.). (E) Photograph showing the typical size difference between 3-week-old RetDN/+ and wild-type littermates. (F) Expression of Ret mutant alleles in mouse brain RNA (P0) by RT-PCR. Total, endogenous mouse and knocked-in human RET; knockin, mice harboring the indicated human RET cDNA (Ret9 or RetDN) in the Ret locus generate a knockin-specific product; knockout, RetTGM mice where the ret locus is inactivated with the TGM allele. Note that retention of the neomycin resistance gene (RetDNneo/+) results in a marked reduction in expression of the knocked-in RetDN allele. wt, wild type. cDNAs knocked-in in the ret locus: RetDN, mutant human RET9 (L985P, Y1062F); Ret9, human wild-type RET9; RetTGM, tauEGFP-myc; RetDNneo, retained neomycin resistance gene in RetDN allele. `+' denotes one copy of the endogenous mouse allele.

View larger version (67K): [in a new window]   Fig. 2. Defects in enteric nervous system development in RetDN/+mice. (A) The intestines from a P21 RetDN/+ mouse with a contracted distal colon (double arrow) and a dilated distal small bowel (triple arrowheads). The intestines of wild-type (wt), Ret9/+ and RetTGM/+ mice are normal (arrow, ileocecal junction; Es, esophagus; Re, rectum). (B) Intestinal aganglionosis and hypoganglionosis in P21 RetDN/+ mice. Hematoxylin and Eosin (HE) staining revealed the presence of neurons (arrowheads) in the muscularis externa of the colon in wild-type (wt) but not RetDN/+ mice. Acetylcholinesterase (AChE) staining demonstrated neuronal (black arrows) and nerve fiber (black arrowheads) loss in RetDN/+ colon (the brown fibers in the RetDN/+ colon are extrinsic innervations characteristic of HSCR). Both AChE- and nicotinamide adenine dinucleotide phosphate (NADPH)-stained RetDN/+ proximal small intestines display a reduced number of neurons (arrows) and nerve fibers (arrowheads), when compared with an identical region from wild-type mice. Scale bars: 100 µm (HE); 400 µm (AChE); 200 µm (NADPH). (C) The aganglionosis is fully penetrant but variable in RetDN/+ mice. The schematic shows the extent of aganglionosis determined by AChE staining in various RetDN/+ mice (each red mark represents one RetDN/+ mouse, n=16). The numbers correspond to the percentage of the respective intestinal segment (small or large intestine) successfully colonized by neurons. Ret9/+ (n=7), RetDNneo/+ (n=3) and RetTGM/+ (n=3) intestines were normal, and RetTGM/TGM (Ret null) had total intestinal aganglionosis. (D) Quantitative analysis of neuronal number and fiber density in the ENS of P21 wild-type and RetDN/+ mice demonstrated a dramatic reduction in the number of neurons and neuronal fiber density in the proximal small bowel (n=3, *=P<0.01, mean±s.e.m.).

View larger version (99K): [in a new window]   Fig. 3. Abnormal development of parasympathetic, but not sympathetic, ganglia in RetDN/+ mice. (A) The number, size and target organ innervation of sphenopalatine ganglia (SPG) parasympathetic neurons was decreased in RetDN/+ mice, when compared with wild-type (wt), Ret9/+ and RetTGM/+ mutant animals. (Top left) Thionin-stained SPG neuron number per animal was decreased in RetDN/+ mice (P0). (Bottom left) The size of SPG neurons in RetDN/+ mice was 40-50% smaller than wild-type SPG neurons (n=3, *=P<0.01, mean± s.e.m.). (Top right) Thionin-stained RetDN/+ SPG neurons appear smaller than wild type (wt) (P21; scale bar: 50 µm). (Bottom right) The SPG target organ, the intraorbital harderian gland, showed a decrease of PGP9.5-stained nerve fibers in RetDN/+ mice compared with wild-type littermates where nerve fibers surround almost every acinus (arrowheads) Scale bar: 70 µm. (B) The sympathetic nervous system developed normally in RetDN/+ mice, as revealed by whole-mount tyrosine hydroxylase (TH) immunostaining (P0). (Top) The superior cervical ganglion (SCG) was normally located, and had normal projections to the eye (vertical arrowheads) and submandibular gland (sm, horizontal arrowheads) in RetDN/+ and wild-type (wt) mice (scale bar: 400 µm). (Bottom) The sympathetic chain ganglia and their neurite outgrowths (arrowheads) also appeared normal in RetDN/+ mice (scale bar: 300 µm). Abnormal development of the SCG (located more caudally near the stellate ganglion, stg; black circle indicates the expected SCG location), SCG projections (top right; scale bar: 800 µm), and sympathetic chain (bottom right; scale bar: 600 µm) in Ret-null mice (RetTGM/TGM) is shown for reference.

View larger version (63K): [in a new window]   Fig. 4. Urogenital abnormalities in RetDN/+ mice. (A, top row). Compared with wild-type (wt) mice, RetDN/+ mice showed a spectrum of renal defects, including bilateral small kidneys and unilateral renal agenesis (k, kidney; a, adrenal; ub, urinary bladder, t, testis; o, ovary; adrenal gland, arrowhead). (A, bottom row) Bar graph shows the decreased total number of glomeruli in RetDN/+ kidney at birth (n=3 animals, 5 kidneys), when compared with wild-type litter mates (n=3 animals, 6 kidneys) (*=P<0.001, mean±s.e.m.). HE-stained sections show tubulocystic degeneration (arrows) in approximately 50% of 3- to 4-week-old RetDN/+ mice (6 out of 12) (co, cortex; me, medulla; pe, pelvis). Scale bar: 5 mm in top row; 600 µm in bottom row. (B) HE-stained sections show atrophic, degenerating seminiferous tubules with multinucleated giant cells (arrows) in RetDN/+ mice (P28). Wild-type (wt), Ret9/+, RetTGM/+ and RetDNneo/+ mice show age-appropriate germ cell maturation. Scale bar: 100 µm for wild-type and RetDN/+ mice; 130 µm for Ret9/+, RetTGM/+ and RetDNneo/+ mice. (C) At P28, a significant decrease in the percentage of RetDN/+ seminiferous tubules containing round spermatids is noted, and no tubules contain elongated spermatids (n=3 for each genotype, *=P<0.01, mean±s.e.m.).

View larger version (45K): [in a new window]   Fig. 5. Defective spermatogenesis in RetDN/+ mice is due to maturation delay and reduced germ cell number. (A) Representative picture depicting increased apoptosis by TUNEL staining (brown nuclei) in RetDN/+ seminiferous tubules at 3 to 4 weeks of age. (B) Increased apoptosis (TUNEL positive) in RetDN/+ seminiferous tubules was noted as early as P17 (n=3 for each time point, *=P<0.01, mean±s.e.m.). (C) RetDN/+ mice had a reduced number of germ cells by GCNA-immunostaining (left, n=3, *=P<0.01, mean±s.e.m.), and reduced cell proliferation (measured by BrdU incorporation, right, n=2, mean±s.d.), relative to wild-type (wt) mice at P10. Note the similar germ cell number between RetDN/+ and wild-type testes at birth. (D) Representative cell ploidy analysis of postnatal (P10) testes in RetDN/+ mice shows a higher 2n:4n ratio than in wild-type mice, indicating a delay in sperm maturation in RetDN/+mice. In the left panel, the 2n and 4n peaks are indicated, the numbers on the peaks represent the percentage of cells in each peak. The bar graph on right summarizes the ploidy results, which indicate a spermatogenesis defect at P10.

View larger version (31K): [in a new window]   Fig. 6. The RetDN mutant has decreased intrinsic kinase activity and inhibits wild-type Ret activity. (A) Western blots of extracts from CHP126 cells infected with the indicated Ret lentiviruses in the presence or absence of GDNF (25 ng/ml) were probed with the indicated antibodies. Wild-type Ret9 showed ligand-dependent autophosphorylation (pY20 and pY1062), and phosphorylation of downstream AKT and MAPK (pAKT and pMAPK, respectively), but RetDN and a kinase-inactive mutant with a K758M mutation (RetKD) lack these activities. (B) Immunoprecipitation studies demonstrated an interaction between RetDN and Ret9. Wild-type Ret9 (FLAG tagged) and RetDN (HA tagged) were co-expressed in 293T cells (which lack endogenous Ret). Lysates were immunoprecipitated with either FLAG- or HA-epitope specific antibodies, followed by western blotting (WB) using a pan-Ret antibody or a FLAG antibody to detect Ret9 and RetDN complexes. (C) RetDN inhibited ligand-dependent AKT phosphorylation. The human RET9 or RetDN were expressed in GDNF-responsive Neuro2A1 cells using lentivirus infection. The cells were grown in the presence or absence of GDNF, and western blots containing these cell lysates were probed with the indicated antibodies (pMAPK, phosphor-MAPK; pAKT, phosphor-AKT). Cells infected with RetDN, but not with wild-type Ret9, had markedly decreased levels of GDNF-dependent AKT phosphorylation, whereas MAPK phosphorylation was minimally affected. An antibody to AKT indicated equivalent total AKT levels in each sample. Control lanes represent lysates from cells infected with virus expressing only the Venus reporter. (D) Quantification of decreased AKT phosphorylation. The RetDN inhibition of AKT phosphorylation was quantified by normalizing the samples according to the total AKT levels and then comparing the GDNF-stimulated phospho-AKT levels. RetDN inhibited GDNF-mediated AKT phosphorylation by approximately 70% (n=3, *=P<0.01, mean±s.e.m.). (E) RetDN inhibited endogenous mouse wild-type Ret activity in SCG neurons. Immunoblots of extracts from primary SCG neurons (cultured for 8 days) show decreased GDNF-dependent phosphorylation of Ret Y1062, AKT and MAPK in RetDN/+ mice, when compared with that of RetDNneo/+ and wild-type mice (WT). The blot was re-probed with tubulin antibody to ensure equal loading.