Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters - PubMed (original) (raw)

. 2020 Jun;158(8):2236-2249.e9.

doi: 10.1053/j.gastro.2020.02.034. Epub 2020 Feb 26.

Alexander W Coutts 2, Izumi Kaji 3, Paula Rodriguez 2, Felipe Ongaratto 2, Milena Saqui-Salces 4, Ramya Lekha Medida 4, Anne R Meyer 3, Elena Kolobova 3, Melinda A Engevik 5, Janice A Williams 3, Mitchell D Shub 6, Daniel F Carlson 2, Tamene Melkamu 2, James R Goldenring 7

Affiliations

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters

Amy C Engevik et al. Gastroenterology. 2020 Jun.

Abstract

Background & aims: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology.

Methods: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs).

Results: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver.

Conclusions: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

Keywords: Malabsorption; Missense Mutation; Motor Protein; Plasma Membrane.

Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1:

Figure 1:. Changes in small intestinal cell morphology resulting from MYO5B(P663L) mutation.

(A) H&E of WT and MYO5B(P663L) pig duodenum. Scale bars= 50 μm. (B) SEM of pig small intestine demonstrating uniform villi in WT pigs. The cells along the villi of WT pig duodenum appeared well aligned with uniform microvilli packing. MYO5B(P663L) pigs had broad, stunted villi with prominent cell rounding evident at the tips with disorganized microvilli packing. (C) TEM of the duodenum of neonatal WT and MYO5B(P663L) pigs demonstrated well-formed brush border in WT pig enterocytes. MYO5B(P663L) enterocytes had shortened microvilli, densely packed subapical vesicles, lateral microvilli in gaps between cells (indicated by yellow arrows) and the presence of intracellular microvillus lined inclusions. (D) Prominent F-actin positive inclusions were observed in the small intestine of MYO5B(P663L) pigs. No inclusions were observed in WT pigs. (E) DPPIV immunostaining of WT pig duodenum showed apical localization, in contrast MYO5B(P663L) pig duodenum had diffuse subapical DPPIV Scale bars= 2 μm.

Figure 2:

Figure 2:. Localization of apical transporters in the duodenum of neonatal pigs.

(A) Confocal imaging demonstrated the presence of diffuse SGLT1 below the apical brush border in MYO5B(P663L) enterocytes compared to WT. (B) NHE3 was observed below the apical membrane in MYO5B(P663L) enterocytes with reduced apical localization compared to WT. (C) CFTR was observed on the apical membrane of MYO5B(P663L) enterocytes as in WT. (D) Alkaline phosphatase immunostaining showed intracellular localization of alkaline phosphatase in MYO5B(P663L) pigs compared to WT. Scale bars=50 and 2 μm in low and high magnification respectively.

Figure 3:

Figure 3:. Alterations in brush border markers persist along the length of the small intestine in MYO5B(P663L) pigs.

(A) Phosphorylated ezrin-radixin-moesin (P-ERM) immunostaining showed normal apical localization in WT enterocytes. In contrast, MYO5B(P663L) enterocytes had prominent inclusions that were P-ERM positive. P-ERM immunostaining also demonstrated decreased microvilli height in MYO5B(P663L) enterocytes compared to WT enterocytes. (B) CD10 has been used to diagnose MVID in patients. CD10 immunostaining had apical localization in WT enterocytes. In MYO5B(P663L) enterocytes, CD10 was observed diffusely below the apical membrane. (C & E) Immunofluorescence staining showed P-ERM positive inclusions throughout the jejunum and ileum in MYO5B(P663L) pigs. (D & F) CD10 localized subapically in MYO5B(P663L) enterocytes in the jejunum and ileum. Scale bars=50 and 2 μm in low and high magnification respectively.

Figure 4:

Figure 4:. Pig MYO5B(P663L) enteroids mimic in vivo findings.

(A) Brightfield image of WT and MYO5B(P663L) 3D enteroids appeared similar. Immunofluorescence staining for P-ERM and p120 showed disorganized cell structure in MYO5B(P663L) enteroids and aberrant expression of P-ERM. (B) F-actin staining showed the presence of subapical inclusions in MYO5B(P663L) derived enteroids. (C) Schematic of the development of 2D enteroid monolayer cultured using air liquid interface. (D) Immunostaining of enteroid monolayers demonstrated well developed brush border in WT enterocytes that expressed NHE3 on the apical membrane. Cultured MYO5B(P663L) enterocytes appeared disorganized with subapical accumulation of NHE3. (E) SGLT1 immunostaining showed apical expression in many WT enterocytes. In MYO5B(P663L) enterocytes SGLT1 was observed diffusely below the apical membrane. Scale bars=50 μm in (A), 2 μm in (B), 10 μm in (C & D).

Figure 5:

Figure 5:. Functional CFTR in MYO5B(P663L) enteroids.

(A) In enteroid monolayers cultured using an air liquid interface CFTR was observed on the apical membrane of MYO5B(P663L) enterocytes. (B) Brightfield micrograph of differentiated WT and MYO5B(P663L) pig derived 3D enteroids before and 1 hour after administration of forskolin. (C) Forskolin swelling, measured as percent change in diameter, after 1 hour forskolin administration. MYO5B(P663L) swelled to a greater degree compared with WT enteroids. *P<0.05, n=155 WT, n=156 MYO5B(P663L) enteroids, performed in three separate experiments. (D) Fecal chloride measures from WT and MYO5B(P663L) piglets as determined by a chloride probe performed in triplicate.

Figure 6:

Figure 6:. Expression of MYO5B and MDR1 in WT and MYO5B(P663L) hepatocytes.

(A) H&E of WT and MYO5B(P663L) pig liver showed the presence of lipid droplets in WT hepatocytes but no other difference was observed. Scale bars=50 μm. (B & C) In WT pigs, MYO5B appeared closely associated with the canalicular membrane, delineated by MRP2 immunostaining. MYO5B also appeared throughout the cytoplasm of hepatocytes. In MYO5B(P663L) hepatocytes MYO5B appeared in dense clusters more distant from the canalicular membrane compared to WT hepatocytes, distance of MYO5B from MRP2 is indicated by arrows. Less cytoplasmic MYO5B was observed in MYO5B(P663L) pigs compared to WT pigs. Scale bars=50 and 5 μm, respectively. (D) MDR1 in WT and MYO5B(P663L) hepatocytes demonstrated decreased expression of MDR1 in MYO5B(P663L) hepatocytes compared with WT. MDR1 appeared diffusely below the canalicular membrane in MYO5B(P663L) hepatocytes.

Figure 7:

Figure 7:. MYO5B(P663L) pigs exhibit alterations in BSEP localization in hepatocytes.

(A) Z-stack projections of WT liver showed well developed canaliculi with apical BSEP localization. MYO5B(P663L) presented canaliculi that appeared stunted and thickened with BSEP present below the canalicular membrane in hepatocytes. (B) MRP2 was observed on the canalicular membrane of WT and MYO5B(P663L) hepatocytes, however MYO5B(P663L) hepatocyte canaliculi appeared less developed. Scale bars=10 μm.

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