Pickett MA , Dush MK , Nascone-Yoder NM .

R01 DK085300 NIDDK NIH HHS , T32 GM008776 NIGMS NIH HHS

             cell adhesion
             digestive tract morphogenesis
             determination of intestine left/right asymmetry

	Fig. 1. AChE plays a non-neuronal, non-esterase role in intestine organogenesis. Normal intestinal elongation and rotation are observed in DMSO-exposed control tadpoles (A,A′). Exposure to malathion (MTH; B,B′), chlorpyrifos-methyl (CPF; C,C′) or Huperzine A (HupA; D,D′) increases the percentage (E) of tadpoles with short/malrotated intestines. AChE activity assays (F) confirm that the applied compounds inhibit AChE in vivo. RT-PCR (G) indicates that isolated intestinal endoderm (indicated by the expression of ifabp, but not foxf1) expresses ache. −RT, control lacking reverse transcriptase. At NF 41, AChE (red) colocalizes with E-cadherin (green) at endoderm cell membranes (H-H′, arrows). By NF 46, AChE is apically enriched (I-I′, arrowheads), with reduced lateral membrane expression (arrows). Intestinal development is normal in control MO-injected embryos (J,J′,N), whereas microinjection of AChE MO results in short/malrotated intestines (K,K′,N). Intestinal malformations are rescued by co-injection of RNA encoding wt AChE (L,L′,N) or mutAChE that lacks catalytic activity (M,M′,N). AChE activity assays (O) confirm that AChE MO knocks down AChE, that wt AChE mRNA increases activity, and that mutAChE mRNA has no effect on AChE activity, relative to controls (uninjected, control MO, GFP mRNA). Higher magnification views of the boxed regions in A-D,H-M are shown in A′-D′,H′-M′, respectively. The number of tadpoles with the phenotype shown among the total number of tadpoles in that experimental group is indicated (A-D,J-M). Bar charts show mean±s.e.m. Significant differences between the percentage of tadpoles with abnormal gut phenotypes or between AChE activities from n=3-16 independent experiments (16-30 embryos per condition per experiment) are indicated by lowercase letters (P<0.05). Scale bars: 1000 μm in A-D′,J-M′; 100 μm in H,I; 25 μm in H′-I′.
	Fig. 2. AChE is required for endoderm cell rearrangement and polarization. In NF 46 intestine sections of embryos injected with control MO (A), AChE MO (B), AChE MO plus wt AChE mRNA (C), or AChE MO plus mutAChE mRNA (D), β-catenin (red) outlines membranes of injected (GFP labeled, green) and uninjected cells. Serial sections from embryos in A-D were immunostained for integrin (green) and aPKC (red) (E-H); the boxed regions are shown at higher magnification in I-L. A single-layer columnar epithelium of polarized cells forms in control MO-injected intestines (I). AChE MO-injected cells (J) are rounder (asterisks), unpolarized [absence of aPKC (red), arrowhead] and fail to form a single layer. Defects are rescued by co-injection with wt AChE mRNA (K) or mutAChE mRNA (L). Scale bars: 100 μm in A-H; 25 μm in I-L.
	Fig. 3. AChE is required for microtubule organization and endoderm differentiation. In NF 46 intestine sections of embryos injected with control MO (A), AChE MO (B), AChE MO plus wt AChE mRNA (C), or AChE MO plus mutAChE mRNA (D), β-catenin (red) outlines cell membranes of injected (GFP labeled, green) and uninjected cells. Serial sections from embryos in A-D were immunostained to visualize microtubules (α-tubulin, green) (E-H); boxed regions are shown at higher magnification in I-L. Microtubules are apically enriched and oriented along the apical-basal axis of columnar epithelial cells (I). Microtubule organization is disrupted when AChE is knocked down (J), but is rescued by co-injection of the AChE MO with wt AChE mRNA (K) or mutAChE mRNA (L). Serial sections from embryos in A-H were immunostained for E-cadherin (green) and IFABP (red) (M-P); boxed regions are shown at higher magnification in Q-T. IFABP is expressed in control MO-injected intestine (Q). AChE knockdown (R) prevents differentiation of the endoderm, as indicated by the absence of IFABP in AChE MO-injected cells. Differentiation is restored by co-injection of the AChE MO with wt AChE mRNA (S) or mutAChE mRNA (T). Nuclei, blue (TO-PRO-3). Scale bars: 100 μm in A-H,M-P; 25 μm in I-L,Q-T.
	Fig. 4. AChE is not required for cell-cell adhesion, but is necessary for cell-substrate adhesion to fibronectin. Dissociated intestinal endoderm cells from control MO-injected (A) or AChE MO-injected (B) embryos reaggregated 30 min (30′) after introduction of Ca2+ ions into the medium (0′; see supplementary Materials and Methods). Brightfield/fluorescent images show that both injected and uninjected cells from each injection group reaggregated. Assays were performed using at least three different embryos per condition. Transverse sections through wild-type guts (NF 41) were immunostained for laminin (LM; C,C′) or fibronectin (FN; D,D′). LM (red) is found at basement membranes (arrow) surrounding the gut tube. FN (green) is found at the basement membrane (arrow), but is also enriched at endoderm cell basal poles (arrowheads). Endoderm cells from control MO-injected or AChE MO-injected intestines were plated on LM (E) or FN (F). There is no difference in cell adhesion on LM (E), but cells from AChE MO-injected embryos are less adherent than control cells on FN (F). Mean±s.e.m. for the percentage of adherent cells for six to eight independent embryos. *P<0.05. Scale bars: 1000 μm in A,B; 100 μm in C,D; 25 μm in C′,D′.
	Figure S1) Morpholino-mediated knockdown of AChE protein expression. In sections through the intestine of NF 46 embryos injected with control MO (A), AChE MO (B), AChE MO + wt AChE mRNA (C), or AChE MO + mutAChE mRNA (D), β-catenin (red) outlines membranes of injected (i.e., GFP labeled; green) and uninjected cells. Serial sections from embryos in A-D were immunostained for AChE (red) and images of the boxed regions are shown at higher magnification (E-H). Apical localization of AChE protein (indicated by arrowheads in E-H) is observed in control MO injected cells (E), but is missing in AChE MO injected epithelium (F). AChE staining is rescued by co-injection with either wt AChE mRNA (G) or mutAChE mRNA (H). Scale bars = 100 μm (A-D); 25 μm (E-H).
	Figure S2) CRISPR-Cas9 mediated editing of ache gene perturbs gut elongation. Xenopus embryos were injected with Cas9 mRNA/protein (A), ache gRNA (B), or Cas9 plus ache gRNA (C), and allowed to develop until stage 46. Relative to injection of Cas9 or ache gRNA alone (A, B), injection of Cas9 plus ache gRNA (C) disrupts intestinal lengthening. Intestine phenotypes resulted from injection of Cas9 plus ache gRNA at both the 1- and 8-cell stages; craniofacial and spinal deformities, similar to those observed with AChE-inhibitor exposure, were also observed in embryos injected at the 1-cell stage (not shown), though absent in 8-cell endoderm targeted injections. The graph (D) indicates the mean (± S.E.M) percentage of embryos in which the intestine is shortened (n=3 different experiments, both 1- and 8-cell injections). E) Genomic sequencing validates the efficacy of CRISPR-Cas9 mediated editing, revealing the presence of deleterious mutations in representative Cas9 plus ache gRNA-injected embryos. Scale bars = 1000 um.
	Figure S3) Modulation of cholinergic signaling via acetylcholine receptors (AChR) does not affect gut morphogenesis. Exposure of X. laevis tadpoles to the AChR agonist carbachol (B) from NF 33-46 does not affect elongation or rotation of the gut relative to water solvent controls (A). Similarly, exposure to the AChR antagonist atropine (D) has no impact on gut morphogenesis relative to DMSO/ethanol treated solvent controls (C). Gut elongation defects observed in AChE-inhibited embryos (e.g., Malathion; E) are not rescued by simultaneously blocking cholinergic signaling with atropine (F). Higher concentrations of carbachol do not affect intestinal development (G), but do result in a significant increase in embryonic lethality (H). Atropine does not rescue intestinal development in AChE inhibited embryos (I), but does reduce Chlorpyrifos-methyl (10mg/L) induced embryonic lethality (J). Neither atropine nor carbachol significantly alter AChE enzymatic activity (K). Scale bars = 1000 um.
	Figure S4) Quantification of the effect of AChE on endoderm cell shape. Length and width measurements were taken for at least 5 endoderm cells in each Control (Con) MO or AChE MO-injected embryo (+/- wt AChE or mutAChE mRNA) and the average L:W ratio calculated. Graphs represent mean L:W ratio for at least three embryos in each injection group ±S.E.M (n = number of individual embryos used to measure L:W ratios). Significant differences among injected cells are indicated by * (p<0.05). Significant differences between injected and un-injected cells within microinjection groups are indicated by a bar (p<0.05).
	Figure S5) Chemical AChE inhibitors alter endoderm polarity, rearrangement and microtubule architecture. Transverse cross-sections through the gut of NF 46 tadpoles exposed to DMSO (A, A’, D, D’), Malathion (B, B’, E, E’), or Chlorpyrifos-methyl (C, C’, F, F’) from NF 33-46 were stained with integrin (A-C; green) to outline endoderm cell membranes, aPKC (A-C’; red) to highlight the apical surface of the epithelium, and alpha-tubulin (D-F’; green) to visualize microtubules. Compared with the columnar epithelium in DMSO exposed controls (A), the organization is disrupted in AChE inhibitor treated embryos (B, C). Arrowheads highlight robust apical expression of aPKC in DMSO controls (A’), versus patchy aPKC expression in chemically inhibited embryos (B’, C’). Likewise, in DMSO controls (D, D’), α-tubulin bundles (green) are normally aligned along the apicobasal axis of the endoderm cells, with slight apical enrichment (D,D’); however, exposure to Malathion (E,E’) or Chlorpyrifos-methyl (F,F’) disrupts this architecture. Higher magnification images of the boxed regions in A-F are shown in A’-F’. Scale bars in A-C, D-F= 100 um. Scale bars in D’-F’= 50 um. Scale bars in A’-C’ = 25 um.
	Figure S6) AChE activity is not required for endoderm cell-cell adhesion. A-E’) Endoderm cells were dissociated from four NF 41 tadpoles (A-E) previously exposed in vivo from NF 33-41 to DMSO (A,A’), Malathion (B,B’), Chlorpyrifos-methyl (C,C’), Carbachol (D,D’) or Atropine (E,E’). Dissociated cells were allowed to re-aggregate for 60 minutes (60’) after reintroduction of Ca2+ ions into the media (0’). Organophosphate inhibition of AChE (B-C’), chemical stimulation of cholinergic receptors (D,D’), or antagonism of cholinergic receptors (E,E’) had no effect on re-aggregation (A’-E’), indicating cell-cell adhesion is independent of AChE or cholinergic signaling. At least three separate assays were performed per chemical. F-J’) Endoderm cells were dissociated (F-J) from four NF 41 tadpoles and subsequently exposed ex vivo to DMSO (F,F’), Malathion (G,G’), Chlorpyrifos-methyl (H,H’), Carbachol (I,I’), or Atropine (J,J’) during a 60 minute re-aggregation assay. Organophosphate inhibition of AChE (G-H’), chemical stimulation of cholinergic receptors (I, I’), or antagonism of cholinergic receptors (J, J’) had no effect on re-aggregation (F’-J’), indicating cell-cell adhesion is not mediated by AChE or cholinergic signaling.

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