The authors have declared that no competing interests exist.
Conceived and designed the experiments: EG SA. Performed the experiments: EG RLS FD. Analyzed the data: EG RL GMT MP SA. Wrote the paper: EG SA.
Current address: Department of Arboriculture and Pomology, University of Turin, Grugliasco, Italy
Congenital tufting enteropathy (CTE) is a life-threatening hereditary disease that is characterized by enteric mucosa tufting degeneration and early onset, severe diarrhea. Loss-of-function mutations of the human
Supporting information is available for this article.
EpCAM, also known as Trop-1, from the
Inactivating germ-line mutations of the human
Loss-of-function animal models have been used to tackle the
Recently, a role for the murine EpCAM/mTrop-1 protein in intercellular adhesion and cell motility and migration was shown in a mouse conditional knockout (KO) with
Hence, we used rigorous gene-replacement and gene-trapping approaches, and obtained a gene-trapped KO mouse that was devoid of a functional mTrop-1 protein. The
The exon numbering in mouse and man differs, as an additional 5′-untranslated exon has been described in the mouse (NM_008532.2), for a total of 10 exons,
The pGT1TMPFS vector was used to generate gene-trapped clones from ES cells
The feeder-independent E14Tg2A.4 ES cell line obtained from 129/Ola mice was used for gene-trapping. The RST412 and RST413
The G8.8 rat anti-mTrop-1 mAb
Procedures involving animals were conducted in compliance with institutional guidelines and with national (D.L. No. 116, G.U., Suppl. 40, Feb.18, 1992; circolare No. 8, G.U., July, 1994) and international laws and policies (UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia; EEC Council Directive 86/609, OJ L 358. 1, Dec.12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996). Experiments on animals were approved by the Interuniversity Animal Research Ethics Committee (CEISA) of Chieti–Pescara and Teramo Universities. Animals were anesthetized with ketamine/xylazine before any invasive procedures. Euthanasia was performed by CO2 inhalation followed by cervical dislocation (adult mice) or decapitation (newborn mice). All efforts were made to minimize suffering of the animals. The
Mouse genotyping was performed on genomic DNA extracted from tail biopsies or embryonic tissues (Supporting
Timed matings between fertile males and spontaneously cycling females were set up to obtain embryos at defined developmental stages. Pregnant female mice were sacrificed between E9.5 and E10.5 (vaginal plug = E0.5). The uterus was removed and quickly rinsed in cold phosphate-buffered saline (PBS). Individual embryos were isolated either within their intact decidual swelling or as dissected from the surrounding maternal tissues. The morphology of the freshly dissected embryos was analyzed under a stereo-microscope (G.M.T.). Embryos were then embedded in optimal cutting temperature (OCT) compound and snap frozen in liquid N2, for subsequent histopathology and molecular analyses. Newborn mice were sacrificed at different times after birth. Internal organs were excised, formalin-fixed, and paraffin-embedded. The gastrointestinal tract (stomach, small intestine and colon) was quickly removed as a whole, rinsed in PBS and either formalin-fixed and paraffin-embedded or frozen (snap freezing in liquid nitrogen for nucleic acid extraction, or OCT embedding for cryostatic microtome sectioning). Five-µm organ sections were stained with hematoxylin and eosin (H&E) following standard procedures, and examined (M.P. and R.L.). Mouse tail tips were processed for DNA extraction and genotyping.
Five-micrometer sections of formalin-fixed and paraffin-embedded tissues from WT and KO mice were stained using the indicated antibodies. Antigen retrieval was performed by microwave treatment at 750 W for 10 min in 10 mM sodium citrate buffer (pH 6.0). After blocking endogenous mouse immunoglobulins using the Rodent Block kit (Biocare Medical, Concord, CA), sections were incubated overnight with the anti-β-catenin (1∶30 dilution) and anti-E-cadherin (1∶200 dilution) primary antibodies. The anti-mouse and the anti-rabbit EnVision kits (Dako, Glostrup, Denmark) were used for signal amplification, as appropriate. In control sections the specific primary antibody was replaced with isotype-matched immunoglobulins (Dako).
Cell staining and flow cytometry analyses (FACScalibur, FACScan, Becton Dickinson, Sunnyvale, CA) were performed as previously described
The χ2 test was used to compare genotype ratios. Kaplan–Meier plots
We used both gene-replacement and gene-trapping approaches, and corresponding validation procedures, to obtain a KO mouse devoid of functional
Hence, we resorted to a gene-trapping approach. Two gene-trapped ES clones were identified, i.e., RST412 and RST413, where
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To investigate embryonic development defects brought about by
Confocal microscopy analysis of HET and KO embryos at E10.5. Frozen sections were stained with the G8.8 anti-mTrop-1 mAb (green); nuclei are stained with propidium iodide (PI; red) for context identification of immunofluorescent signals (merge). Matching bright field images are shown (left column) from consecutive frozen tissue sections stained with hematoxylin (A–I, E–I) or H&E (B–I, C–I, D–I). (A, B) Embryo intestine. (C, D) Embryo forelimb bud. (E) Mother uterine tissue. Embryonic tissues that express mTrop-1 at high levels are shown. HET mouse embryos show strong intestinal (A) and limb bud (C) epithelial staining localized at cell membranes, as expected. The same tissues from KO embryos (B, D) show complete absence of green signal. Uterine glands in maternal tissue express high levels of mTrop1 (E) and were used as stringent internal controls. Target tissue architecture and morphology in the HET embryos were normal. No gross differences were detected between HET and KO embryos. Scale bars: 100 µm.
These findings indicated a different pathogenetic course from that described by Nagao et al.
(A) Whole litter from a representative HET crossing. Day 0: At birth all of the 10 pups were alive; KO mice were indistinguishable from their littermates. Days 2, 3: One pup (circled in red) is appreciably smaller than the others. Bottom: genomic PCR genotyping identified the small pup as KO (star). The KO
Serial analysis of gene expression (SAGE) analyses and microarray hybridization profiles of embryonic tissues showed that
H&E staining of formalin-fixed paraffin-embedded small intestine and colon sections from WT and KO newborn mice, from day 0 to day 4. Insets: magnified areas. Villous atrophy was found throughout the small intestine of KO mice. Severity progressed from day 0 to day 4 (day of death). Red arrowheads: tufts of extruding epithelium, with surface enterocyte disorganization and focal crowding. These abnormalities were focally distributed, and increased over time, with highest tuft density at the time of death. Lymphocytes and plasma cells in the lamina propria were infrequent. KO colon crypts showed pseudo-cysts formation (black arrowheads) and abnormal regeneration with branching (block arrows). Hemorrhagic enteritis was apparent in the small intestine of KO mice from day 0 (top, right); black arrows: red blood cells in the intestinal lumen. Scale bars: 40 µm.
Small intestine from WT (left) and KO (right) newborn pups at day 0, analyzed by immunofluorescence confocal microscopy. Staining with the G8.8 anti-mTrop-1 mAb (green). Nuclei were stained with PI (red) for context identification of immunofluorescent signals (merge). Expression of the β-gal marker from the
Taken together, these findings show that
Trop-1 ablation in zebrafish embryos was shown to cause a decrease in membrane-bound E-cadherin
E-cadherin immunostaining pattern in small bowel of WT (wt) and KO mice at birth (day 0), and at day 1 and day 4 after birth. E-cadherin shows a typical membrane immunoreactivity in WT mice (A, C, E), whereas in KO mice (B, D, F) it is localized increasingly in the cytoplasm, with a prevalent cytoplasmic accumulation and membrane-disrupted pattern at day 4 after birth. (Scale bar: 20 mm).
Parallel immunohistochemistry analyses were performed for β-catenin (
β-catenin immunostaining pattern in small bowel of WT and KO mice at birth (day 0), and at day 1 and day 4 after birth. Membrane pattern is typically seen in WT mice (A, C, E). A progressive cytoplasmic immunoreactivity, with concomitant diffuse and “dot-like” perinuclear patterns, and disruption of the membrane immunoreactivity for β-catenin is seen in KO mice (B, D, F) starting from day 1 after birth.(Scale bar: 20 mm).
Our results are consistent with Trop-1 loss being a single-gene cause of CTE. The
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We thank Lorenza Ronfani and Pasquale Simeone for help during the course of this study and Paola Ascione for technical assistance.