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Depletion of definitive gut endoderm in Sox17-null mutant mice

¹ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
² The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan
³ Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia

View larger version (108K): [in a new window]   Fig. 1. Expression of Sox17 and Sox7 during gastrulation and early organogenesis. (A-C) Pre-streak (A,B; 6 and 6.25 dpc, respectively) and early-streak (C; 6.5 dpc) embryos showing the progressive expansion of Sox17 expression (arrow) to the whole extra-embryonic region of the visceral endoderm. (D) Transverse section of the extra-embryonic region of an early-streak embryo showing expression of Sox17 in the visceral endoderm (arrows). (E) Mid-streak (7.0 dpc) stage embryo showing strong expression of Sox17 in the extra-embryonic visceral endoderm (unbroken arrow) and the endoderm at the anterior end of the primitive streak (broken arrow). (F,G) Lateral (F) and anterior (G) views of early-bud stage (7.5 dpc) embryo revealing Sox17 expression in the extra-embryonic visceral endoderm and the anterior-most endoderm (broken arrows). (H,H') Early- to late-bud stage (7.5 dpc) embryos demonstrate the increasing number of Sox17-expressing cells in the anterior definitive endoderm (H; broken arrow), but no expression of Sox7 in the definitive endoderm (H'). (I-L) Embryos at early-bud stage (7.5 dpc): whole embryo (I) and histological section (J) showing strong Sox17 expression in the visceral endoderm (arrows). Section in situ hybridisation by radioactive riboprobes display strong expression of Sox17 (K) and Sox7 (K') in the parietal (arrowheads) and extra-embryonic visceral endoderm (arrows). Strong Sox17 expression in the definitive endoderm (L; broken arrows). Planes of sections are indicated by arrowheads in the embryo (I) of a similar developmental stage. (M,M') Early neural plate stage (7.75 dpc) embryo showing the expansion of Sox17-expressing cells (M; broken arrow) and the absence of Sox7-expressing cells in the prospective foregut and midgut endoderm (M'). (N-Q) Early headfold stage (8.0 dpc) embryo (arrowheads indicating the plane of the histological sections) showing the descending gradient of Sox17 expression from the ectoplacental cone (O) to the allantoic region of the visceral endoderm (P), and enhanced expression in the definitive endoderm of the prospective midgut and hindgut (Q). (R,S) Section in situ hybridisation by a DIG-labelled riboprobe showing the regionalised Sox17 expression (broken arrows) in the posterior definitive endoderm of the 8.25 dpc embryo (four to five somite stage). The broken rectangle in R demarcates the region magnified in S. (T-W) Sox17 expression in the posterior endoderm of the mid- and hindgut (V,W), but not in the foregut (U) of seven- to eight-somite stage (8.5 dpc) embryo (arrowheads indicating the plane of the histological sections). al, allantois; epc, ectoplacental cone; dc, decidua; fg, foregut; hg, hindgut; mg, midgut. Scale bars: 100 µm.

View larger version (30K): [in a new window]   Fig. 2. Targeting a null mutation to the murine Sox17 gene. (A-C) The Sox17 targeting strategy. Restriction sites: B, BamHI; E, EcoRI; C, ClaI. The green bold bar indicates the position of the probe for genomic Southern blot hybridisation using the BamHI-digested samples (mutated allele, 9.0 kb; wild-type allele, 5.0 kb) (genotyping results of ES cells and adult mice are shown in B), while the red arrowheads show the primer positions for genome typing by PCR (results of 8.5 dpc embryos are shown in C). (D) Lack of Sox17 synthesis in mutant 7.5 dpc mouse embryos revealed by immunoblot analysis using anti-Sox17 C17-antibody (Kanai et al., 1996).

View larger version (84K): [in a new window]   Fig. 3. Defective gut development of Sox17–/– embryos. (A-F) Lateral views of phenotypically normal (+/+,+/–) and mutant (–/–) embryos at 7.5 (late-bud stage; A), 8.0 (headfold stage; B), 8.25 (five- to six-somite stage; C), 8.5 (10- to 12-somite stage; D), 9.5 (E) and 10.5 (F) dpc. The homozygous Sox17 embryos show no axis turning and poor posterior development from 8.5 dpc onwards. Arrows indicate the plane of histological sections (G-S); unbroken arrow for transverse section and broken arrow for sagittal section. (G) Sagittal and (H,I) parasagittal sections of 8.0 dpc embryos. In the mutant embryo, the visceral endoderm extends further into the prospective gut on the lateral aspects (H,I) than the medial aspects (G) in the posterior region. Arrowheads and arrows mark the frontier of the visceral endodermal cells at the anterior and posterior aspects of the embryo, respectively. (H,I) Insets (right) also show a magnified view of the posterior region. (J,K) Transverse sections at the midgut level of 8.25 dpc embryos. In the mutant (–/–) embryo, the definitive endoderm in the lateral region of the prospective gut is replaced by the cuboidal and vacuolated cells that resemble the visceral endoderm. (L-O) Transverse sections at the fore- and hindgut (L,M) and midgut (N,O) levels of 8.5 dpc embryos. The sections of the heterozygous (+/–) embryo at a similar developmental stage (before axis turning) are shown. In the foregut and hindgut portal of the 8.5 dpc mutant embryos, cells that resemble the visceral endoderm are found in the lateral and ventral regions of the gut tube (L,M). In the lateral region of the open midgut, the endoderm cells are juxtaposed to the mesothelium of the splanchnopleure. By contrast, the mutant (–/–) embryo lacks the layer of splanchnic mesenchyme (red arrows; O) that separates the endoderm from the mesothelium. (P,Q) Sagittal sections of the 9.5 dpc mutant (–/–) embryo, showing normal development of (P) the head, the heart and (Q) the thyroid bud. (R) Transverse sections at the caudal foregut level of 9.5 dpc mutant (–/–) embryos reveals a small foregut, but proper liver bud formation. (S) Transverse sections of the hindgut of 9.5 dpc embryos. In the mutant (–/–) embryo, the hindgut has regressed to a cord-like structure containing significantly fewer endodermal cells. The broken rectangle (J,L,N,P) marks the area shown in the magnified view of the histological section. Arrowheads (J,K,N,O) indicate the border between the definitive endoderm and the presumptive visceral endoderm. ce, coelomic epithelium; da, dorsal aorta; fg, foregut; hg, hindgut; ht, heart; lb, liver bud; mes, mesothelium; sm, splanchnic mesenchyme; thy, thyroid bud. Scale bars: 100 µm.

View larger version (112K): [in a new window]   Fig. 4. Depletion of the definitive gut endoderm in Sox17–/– embryos. (A-D) Hnf3 expression in the definitive gut endoderm of the 8.5 (A,C) and 9.5 (B,D) dpc normal and mutant embryos [A includes both anterior (left) and posterior (right) views of the same embryos]. Hnf3 expressing cells are confined to a much narrower medial and paraxial domain in the embryonic gut of the mutant embryo. (E) Transverse sections of 9.5 dpc embryos demonstrate normal Hnf3ß expression pattern in the reduced hindgut endoderm and the floor plate of the heterozygous (+/–) and null mutant (–/–) embryos. (F,G) 9.5 dpc mutant (–/–) embryos showing normal Cdx2 expression in the endoderm of the gut posterior to the level marked by the red arrow (F, lateral view; G, transverse section at the plane indicated by the broken line in F). (H,I) Hex expression in the foregut endoderm of 8.5 dpc embryos (H; anterior view) and in the thyroid (small arrows) and liver (large arrows) buds of 9.5 dpc embryos (I; lateral view). (J) Pdx1 expression in dorsal (small arrow) and ventral (large arrow) pancreatic regions of the gut endoderm in 9.5 dpc heterozygous embryos (left-most in the figure). Pdx1 expression is completely absent (broken arrows) in the prospective pancreatic tissues of the homozygous (–/–) mutant embryos (two on the right hand side). (K-P) Shh expression in the definitive gut endoderm (open arrows for foregut, solid arrows for mid to hindgut) and notochord of 8.5 (K, posterior view; L,M, transverse sections) and 9.5 dpc embryos (N, lateral view; O,P, transverse sections). In plate P, the broken arrow shows the Shh-negative posterior gut in the mutant (–/–) embryo. (Q,R) Ihh expression in the visceral and definitive endoderm of 8.5 (Q; ventral view) and 9.5 (R; lateral view) dpc embryos. The unbroken arrows indicate the Ihh-positive endoderm in the heterozygous embryo and the broken arrows indicate the absence of Ihh expression in the equivalent endodermal tissues in the homozygous mutant embryo. (S,T) Ptch expression in the ventral structures of the 8.5 dpc mutant embryos (S, ventral view; T, transverse section). The unbroken arrows indicate the Ptch-positive splanchnic mesothelium in the heterozygous embryo and the broken arrows indicate the absence of Ptch expression in the equivalent tissues in the homozygous mutant embryo. Broken lines on the whole-mount figures show the plane of histological sections. Arrowheads in C,H,M,T indicate the border between the presumptive visceral endoderm and the definitive endoderm. fg, foregut; fp, floor plate; hg, hindgut; nc, notochord; mes, mesothelium; ve, visceral endoderm; asterisk, nonspecific signals that are due to antibody trapping in lumen. Scale bars: 100 µm.

View larger version (119K): [in a new window]   Fig. 5. Loss of Sox17 function elevates apoptosis but does not affect the initial formation of anterior definitive endoderm. (A-I) Whole-mount TUNEL staining (brown) of the mutant embryos at early neural plate (A, anterior view), late neural-plate to early head-fold [anterolateral (B), posterior (C), sagittal section (D) and transverse sections (E,F)] and early somite stages [anterior (G), posteroventral (H) and transverse section (I)]. Mutant embryos do not show any more TUNEL-positive cells than do the normal (+/+ and +/–) embryos prior to the neural plate stage (A). However, at the late neural plate to early somite stages, more TUNEL-positive cells are found in the prospective foregut endoderm (B,E-G; arrows in D,I). By contrast, no increase in apoptosis is found in the posterior endoderm (C,D,H). In H, the broken red lines indicate the border between anterior and posterior gut segments. The broken arrows in B,G show the plane of sectioning (D,E,I); the broken rectangle (E) marks the area shown in the magnified view (F). (J,K) Cer1 expression in the anterior definitive endoderm of the normal and mutant embryos at early- to late-bud stage (J, lateral view) and at late neural-plate stage (K, lateral view). Proper expression of Cer1 was found in the prospective foregut endoderm of the normal (+/+ and +/–) and null mutant embryos. Scale bars: 10 µm.

View larger version (90K): [in a new window]   Fig. 6. Visceral endoderm replaces the definitive endoderm in the lateral region of the embryonic gut. (A-C) The 8.0 dpc [late headfold stage; anterior (A) and posterior (B) views of the same embryos] and 8.5 dpc (C, ventral view) mutant embryos showing the endocytotic activity of visceral endoderm revealed by the uptake of horseradish peroxidase (HRP; 2 mg/ml, brown staining reaction) for 30 minutes. The visceral endoderm in the yolk sac of the mutant embryos demonstrate normal HRP-uptake activity, but the endodermal cells displaying similar uptake activity are also found in the prospective gut region of the embryo (A,B, broken arrows; C, arrowheads). (D,E) Expression of Hnf4 in the visceral endoderm of the 8.5 dpc mutant embryos (D, lateral view; E, transverse section), reveals the expansion of the Hnf4 expression domain into the lateral region of both anterior and posterior gut in the mutant embryo (arrowheads). (F,G) Expression of Gata4 in the cardiogenic mesoderm (ht) and visceral endoderm (F, anterior view; G, transverse section) of the 8.5 dpc mutant embryos. Gata4 expression, which is normally restricted to the visceral endoderm, extends into the lateral regions of the posterior gut in the mutant embryos (G, arrowheads). (H-J) Afp expression in the visceral endoderm of the normal and mutant embryos at early neural plate (H, lateral view) and headfold stages [anterolateral (I) and posterior (J) views of the same embryos]. In the mutant embryos, Afp expression is associated with the yolk sac endoderm until the early neural plate stage (H), but by the headfold stage, expression extends into the posterior and lateral regions of the embryonic gut (I,J, broken arrows). In A,B,H-J, the anterior, lateral and posterior border between visceral and definitive endoderm is indicated by arrowheads, broken arrows and arrows, respectively. In C-E,G, arrowheads mark the border between the visceral endoderm and the definitive endoderm. The broken lines in D and F mark the plane of sectioning. fg, foregut; hg, hindgut; ht, heart. Scale bar: 100 µm.

View larger version (81K): [in a new window]   Fig. 7. The lack of contribution of Sox17–/– ES cells in the gut endoderm in the chimera. ROSA26-derived cells are positive for X-gal staining (blue staining), while the ES clone (Sox17–/–)-derived cells are negative. (A,E) Whole mount view of chimaeric embryos with the high contribution of Sox17–/– ES cells and non-chimaeric littermates (right) at 8.25 (A) and 9.5 (E) dpc. (E) Three chimeras with various degrees of mutant ES cell contribution: one chimera with high mutant ES cell contribution displays no axis rotation (upper left), while two other chimeras containing moderately high mutant ES cell contribution are slightly retarded in posterior trunk development [one shows delayed axis rotation (lower left)]. The broken arrows show the plane of the sectioning image in the designated plate. In E, red arrowheads show the posterior gut endoderm, which was composed mostly of the ROSA26 (Sox17+/+) host-derived cells. (B-D,F-K) Transverse sections of chimaeric embryos at 8.25 (B-D) and 9.5 (F-K) dpc. Sox17–/– ES cells were excluded from the posterior or mid- and hindgut endoderm, which is mainly composed of ROSA26 host-derived cells (B-D,F,G,J,K). Sox17–/– mutant cells can contribute to the anterior or foregut endoderm (open arrows in B,C,H,I). The chimeras with the high mutant ES cell contribution display a reduction in cell population of the posterior definitive endoderm (B,C) or embryonic gut (F). Arrowheads show the border between visceral endoderm and definitive endoderm. The broken rectangle encompasses the area magnified in the designated plate. fg, foregut; hg, hindgut; mg, midgut; ve, visceral endoderm. Scale bars: 100 µm.