Crucial Transcription Factors in Endoderm and Embryonic Gut Development Are Expr - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 25 下一页

返回列表

查看: 405270\|回复: 240	go Crucial Transcription Factors in Endoderm and Embryonic Gut Development Are Expr [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 00:09 |只看该作者 |倒序浏览 |打印

作者：Rie Matsuuraa, Hiroshi Kogoa, Takunori Ogaeria, Takashi Miwaa, Masaki Kuwaharaa, Yoshiakira Kanaic, Takumi Nakagawab, Atsushi Kuroiwab, Toyoshi Fujimotoa, Shigeko Torihashia作者单位：a Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, andb Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan;c Department of Anatomy, Graduate School of Agriculture and Life Science, University of Tokyo, Tokyo, Japan

   2 |; ?2 B! l# q: ~3 J$ a

                     $ k8 _+ |& E' m! t6 X
         - i6 R9 @5 O+ Z) ?# X

         ( c2 o6 G) `4 U5 c
            e1 P' H; g& e7 |  G1 a. z$ v
                  & h8 @9 K9 p; w5 u

      4 H* X8 q. b5 Q4 l2 w# g& o
      ) n7 o3 n3 n( B" X
      【摘要】5 f6 V% m. ]- w; d3 i# M9 l4 \& L
   Mouse embryonic stem (ES) cells are pluripotent and retain the potential to form an organ similar to the gut showing spontaneous contractions in vitro. The morphological features of these structures and their formation, as assessed using the hanging drop method to produce embryoid bodies (EBs), seem to be similar to those in vivo. To determine whether the same molecular mechanisms are involved in the formation process, the expression pattern of transcription factors regulating endoderm and gut development in the mouse embryo was examined by in situ hybridization and compared with in vivo expression. Expression of gene products was also examined by immunohistochemistry, and expression colocalization was analyzed with double staining. The results showed that all factors examined, that is, Sox17, Id2, HNF3&szlig;/Foxa2, and GATA4, were expressed in both EBs and gut-like structures. Moreover, their expression patterns were similar to those in the mouse embryo. EBs after the hanging drop period and before outgrowth already expressed all factors that were colocalized with each other in EB epithelial structures. These findings suggest that the origin of the gut-like structure is determined during the hanging drop period and that the gut-like structure is formed as the epithelial structure in EBs during the hanging drop period. They also indicate that the in vitro system using mouse ES cells mimics in vivo development and should prove useful in the study of molecular mechanisms for endoderm and gut development.
      【关键词】 Embryonic stem cell Embryoid body Transcription factors Development Gastrointestinal motility
   INTRODUCTION: _, Y2 c! s- e! G/ M) d

Mouse embryonic stem (ES) cells, which are derived from the inner cell mass of blastocysts, are pluripotent. They differentiate into endoderm, mesoderm, and ectoderm, and the derivatives of these three germ layers after spontaneously entering a program of differentiation in vitro , and thus appeared to be a good model system to study the differentiation of endoderm and gut organogenesis.
# h0 T1 U6 U2 M1 ?8 K, t7 r1 {
In the present study, to characterize the formation process of the gut-like structure in vitro, we investigated, using in situ hybridization, the expressions of several transcription factors crucial to endoderm formation and gut organogenesis, and compared them with patterns in the embryonic gut. The Sry-related HMG box factor Sox17 is an early endodermal marker that is located downstream of the Nodal signaling pathway in zebrafish and plays a crucial role in the differentiation of the definitive endoderm . None of the transcription factors to date is specific to the definitive endoderm or GI tract. Therefore, an examination of the expression pattern of these factors in the ES cell system and their colocalization may provide an answer to the question of whether this system would be an in vitro model of gut development at the molecular level.2 y7 a! A: v3 t5 U
5 @, Q: F% |* t
As a result of the present study, we found that Sox17, Id2, HNF3ß/Foxa2, and GATA4 were expressed in EBs and gut-like structures, and that their expression pattern was similar to that in the mouse embryo. Our findings indicate that the in vitro system we used reflects normal development in vivo and is useful for the study of molecular mechanisms of endoderm and gut development.
" m! f% a8 A! c) z" R
MATERIALS AND METHODS1 W' R, Q) e: K; s

The feeder-independent ES cell line G4¨C2 (a gift from Dr. H. Niwa, RIKEN, Kobe, Japan) derived from the EB3 cell line (a subline originated from E14tg2a) was cultured in Dulbecco¡¯s modified Eagle¡¯s medium supplemented with 1,000 U/ml leukemia inhibitory factor (LIF), 10% fetal calf serum, 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 0.1 mM nonessential amino acids (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), and 1 mM sodium pyruvate (Sigma). Cultures were then maintained in hanging drops without LIF for 6 days to develop EBs. EBs were transferred to gelatin-coated dishes attached to the substrate and began to outgrow. EBs and developing gut-like structures were used on day 2, 5, 7, 10, 14 and 21 of the outgrowth and termed EB2, EB5, EB7, EB10, EB14, and EB21, respectively. EBs just before plating were also used and termed EB0. Whole mouse embryos at E8.0 and their guts at E13.0 were obtained by mating BALB/c mice. Samples were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline and subjected to whole-mount in situ hybridization, as previously described .2 b4 @. P7 U% ~
! B2 c8 G. _" Q+ z; f7 H; Y7 `
Samples were incubated with digoxigenin (DIG)-labeled antisense riboprobes prepared using the following linearized plasmid templates and RNA polymerases: (a) GATA4, BamHI-digested pBluescript SK phagemid G14a , T3 polymerase. Sense riboprobes were used as a negative control in all cases. After hybridization, the DIG-labeled molecules were detected using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (NBT/BCIP; Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) as a substrate for the anti-DIG antibody¨Ccoupled alkaline phosphatase. After overnight treatment with 4% PFA, some samples were processed for cryosectioning.1 M6 g$ L+ q6 f- S% Y

Colocalization of factors was examined by immunohistochemistry. EBs at EB0 were fixed with 4% PFA and then embedded in OCT compound (Sakura Finetechnical Japan Co., Ltd., Toyko, http://www.sakura-finetek.com) to make frozen sections (6 µm thickness). Double staining was carried out using the following antibodies: goat anti-Sox17 (1:200, R&D Systems Inc., Minneapolis, http://www.rndsystems.com), goat anti-GATA4 (sc-1237, 1:1,000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit anti-GATA4 (sc-9053, 1:200, Santa Cruz), goat anti-HNF3ß/Foxa2 (sc-9187, 1:100, Santa Cruz), rabbit anti-HNF3ß/Foxa2 (1:500, Upstate Biotechnology, Inc., Lake Placid NY, http://www.upstatebiotech.com), and rabbit anti-Id2 (sc-489, 1:1,000, Santa Cruz). These were coupled to second antibodies conjugated with either Alexa Fluor 594 or Alexa Fluor 488 (1:400, Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Confocal images were taken using a laser confocal microscope (LEM 5 Pascal; Carl Zeiss, Inc., Jena, Germany, http://www.zeiss.com ).
8 v4 I6 e& p. R. _( }
RESULTS
8 g4 M# `( K8 W3 i  z- W% K: b
Sox174 ]2 G- w% g- Q& K0 z$ }7 |
' s$ }- m5 c+ e) M$ D. {& F
Although the expression of Sox17 was not observed clearly in the embryonic gut at E13.0 (Fig. 1A), it was detected in the definitive endoderm in the early head-folding stage of the mouse embryo at E8.0 (Fig. 1A, inset). On the other hand, EBs and the gut-like structures from ES cells showed Sox17 expression from EB0 (Fig. 2). EBs at EB0 probably correspond to mouse embryos from E5.0 to E7.5 (the egg-cylinder stage), as has been suggested . Prospective gut-like structures at EB5 showed strong Sox17 expression (Fig. 2C); however, after EB7, its intensity decreased (Fig. 2D, 2F) and almost disappeared at EB14 when organogenesis was complete (data not shown). The expression pattern is summarized in Figure 4.

Figure 1. Expression of transcription factors in the digestive tract at embryonic day (E)13.0. (A): Sox17 is not obviously expressed in the digestive tract at E13.0, but it is in the embryo at the head-folding stage. Sox17 was detected in definitive endoderm. Inset: Cross section at E8.0 showing Sox17 expression located in hindgut (Hg) rather than neural plate (Np). (B): Id2 is expressed throughout the gut. Inset: Cross section of small intestine showing expression located in epithelium. (C): HNF3ß/Foxa2 is strongly expressed in the foregut, that is the esophagus, stomach, and proximal region of the small intestine, whereas the cecum is weakly positive. (D): GATA4 shows a unique expression pattern. The corpus to pylorus of the stomach and anterior region of the small intestine express GATA4, whereas the terminal ileum, cecum, and colon do not. (E): Heterogeneous expression of GATA4 in the epithelium (Ep) is confirmed by a cross section of stomach cut at the position indicated by the broken line in (D). The small arrow, large arrow, white arrowhead, and black arrowhead indicate the small intestine, colon, cardia, and cecum, respectively. Scale bars = 500 µm (in A applies to B¨CD, E), 100 µm (insets).
8 [% w3 ^# [# Y3 q0 V0 M
Figure 2. Expression of Sox17 in embryoid bodies (EBs) and gut-like structures. (A): Sox17 is expressed in EBs just prior to plating (EB0), and is detected in the small head region of the EB (star). (B): At EB2, the area corresponding to the small head region at EB0 retains Sox17 expression (star). (C): Prospective gut-like structures at EB5 (arrows) have definitive Sox17 expression. (D): At EB10, expression in a developing gut-like structure weakens. (E): Cross section of EB at EB0 showing small head region, including an epithelial structure (arrow) expressing Sox17. (F): Cross section at EB10 as shown in (D) indicates that epithelia (arrows) in gut-like structure express Sox17, although expression weakens at EB10. Scale bars = 500 µm (A¨CD), 100 µm (E, F).
5 ^# y7 o2 v2 E6 N% g
Figure 3. Double staining of transcription factors in embryoid bodies (EBs) using immunohistochemistry just prior to plating (EB0). (A): Immunohistochemistry for Sox17. (B): GATA4 immunoreactivity on same section as in (A). (C): Merged image of (A) and (B). Although the two factors exhibit distinct distributions, epithelial structures (arrowheads) express both factors. (D): Immunohistochemistry for Sox 17. (E): Id2 immunoreactivity on same section as in (D). (F): Merged image of (D) and (E). Both factors are colocalized on epithelial structures (arrowheads). Sox17 immunoreactivity is located in nucleus, whereas Id2 is distributed in both cytoplasm and nucleus. (G): Immunohistochemistry for GATA4. (H): HNF3ß/Foxa2 immunoreactivity on same section as in (G). (I): Merged image of (G) and (H). Epithelial structures (arrowheads) show colocalization of both factors. Their immunoreactivities are located mainly in nuclei with some distributed in cytoplasms. (J): Immunohistochemistry for HNF3ß/Foxa2. (K): Id2 immunoreactivity on same section as in (J). (L): Merged image of (J) and (K) revealing that epithelial structures (arrowheads) express both factors. Scale bar = 20 µm (A¨CL).
+ g' c+ t2 M1 r9 a6 S
Figure 4. Time course of transcription factor expression in embryoid bodies (EBs) and gut-like structures in vitro, and in the digestive tract of mouse embryos. All factors are expressed in EBs just prior to plating (EB0), but after EB5, each factor shows a different pattern. In vivo expression in the foregut, midgut, and hindgut is indicated by the lines labeled F, M, and H, respectively. Before differentiation of these three regions, expression in endoderm is shown as dotted lines. Each factor is expressed in the endoderm and presents a characteristic expression pattern in the foregut, midgut, and hindgut. The gradient of each line indicates the intensity of expression.0 q$ J  W8 Z$ M3 `; m% B, {

Id26 o+ M' J9 {- v8 ~4 C; u
: b! Q; f: G4 J1 @( M, e
In the embryo, Id2 was observed in the epithelium throughout the GI tract at E13.0, and intense expression was observed in the gut epithelium (Fig. 1B, inset). Id2 protein was also detected in neonates using immunohistochemistry (data not shown). Therefore, Id2 is expressed in the epithelium throughout the gut from the early embryonic stage to the neonate. As for the EBs and gut-like structures, Id2 was expressed in the epithelium from EB0 to EB14 (Fig. 5) and was first detected in the small head region and/or in the region between the small head and the large body of EBs at EB0 (Fig. 5A), where the epithelial structure was formed and showed Id2 expression (Fig. 5E). Immunohistochemistry for Id2 also demonstrated its expression in the same region of EBs at EB0 (Fig. 3E, 3K). Subsequently, the positive area expanded; the labeling intensity was heterogeneous, but future gut-like structures after EB4 showed definitive expression (Fig. 5B¨C5D). Most gut-like structures, including those in a premature state, were Id2 positive in all stages examined, and Id2 expression was detected in the epithelium (Fig. 5F). In Figure 4, the expression pattern is summarized and compared with that of the embryonic gut.- `$ T" I( ^; i* M* j9 y
, ~8 K! ?; g" s& e" T4 H7 q$ v1 |
Figure 5. Expression of Id2 in embryoid bodies (EBs) and gut-like structures. (A): Id2 is expressed in EBs (arrowhead) just prior to plating (EB0). (B): Strong expression of Id2 is seen as spots in the outgrowing portion of EBs at EB2. (C): At EB5, prospective gut-like structures (arrows) show clear Id2 expression in the center, suggesting the formation of epithelium. (D): At EB10, almost all premature gut-like structures express Id2 in their epithelium (arrows). (E): Expression in EBs at EB0 is demonstrated using a section. Clear expression of Id2 is shown in the epithelium (arrows). (F): Strong expression of Id2 in developing gut-like structures at EB10 is confirmed in their epithelium (arrows) using a cross section. Scale bars = 500 µm (A, B), 100 µm (in B applied to C; D¨CF).
, D# D) @: \* a, b! Q/ {* s  n
HNF3ß/Foxa24 y% @' l$ Y- |& G" z
3 t/ s+ A5 m; v
In the mouse GI tract at E13.0, HNF3ß/Foxa2 was observed in the anterior region of the GI tract, that is, the esophagus, stomach, and anterior part of the small intestine (Fig. 1C). It was also detected in the cecum, and its expression was seen in the epithelium in the sections (data not shown). We observed the expression of HNF3ß/Foxa2 in EBs from EB0 in the epithelial structure of the small head region (Fig. 6A, 6E). HNF3ß/Foxa2 protein was also detected in the same area of EBs by immunohistochemistry (Fig. 3H, 3J). After outgrowth, HNF3ß/Foxa2 mRNA appeared clearly in the epithelium of the developing gut-like structures (Fig. 6C, 6F), but the expression became heterogeneous after EB7 (Fig. 6D) and was lost in most gut-like structures at EB14. The expression pattern of HNF3ß/Foxa2 in EBs and gut-like structures are summarized in Figure 4.

Figure 6. Expression of HNF3ß/Foxa2 in embryoid bodies (EBs) and gut-like structures. (A): HNF3ß/Foxa2 is expressed in the small head regions (stars) of EBs just prior to plating (EB0). (B): Expression in the head region is also seen at EB2, with some forming small spot-like expression patterns at this stage. (C): At EB5, prospective gut-like structures clearly show HNF3ß/Foxa2 mRNA (arrows). (D): Some premature gut-like structures express HNF3ß/Foxa2 strongly (large arrow), but others express it only weakly (small arrows) at EB10. (E): Cross section of an EB at EB0 shows that epithelial structures (arrows) express HNF3ß/Foxa2. (F): Cross section of the prospective gut-like structure at EB5 in (C) demonstrating that HNF3ß/Foxa2 is expressed in epithelium (arrow). Scale bars = 500 µm (in A applied to B¨CD), 100 µm (E, F).) q& z; v' d# q% t& J  f* B
4 b8 f. C% S6 N1 ~7 L) g7 ?3 }
GATA4

GATA4 was expressed in the stomach of the mouse embryo at E13.0. Its expression was restricted to the epithelium of the distal part corresponding to the corps and pylorus (Fig. 1D, 1E). The proximal region of the gut also expressed GATA4, the expression of which decreased toward the colon. GATA4 was also expressed in the small head region of EBs at EB0 (Fig. 7A, 7E); immunohistochemistry for GATA4 confirmed its expression in EBs at that stage (Fig. 3B, 3G). After outgrowth, beating cardiac muscle showed strong GATA4 expression, in addition to developing gut-like structures (Fig. 7B, 7F). We sometimes observed that EBs with well differentiated cardiac muscles formed more gut-like structures. After EB5, the expression became heterogeneous (Fig. 7C, 7D). Some premature structures showed intense expression, whereas others had weak or negligible expression. This heterogeneity of expression continued to EB14, as summarized in figure 4.
( t8 g- Y0 ~" @; x. n7 s- t" `
Figure 7. Expression of GATA4 in embryoid bodies (EBs) and gut-like structures. (A): GATA4 mRNA is detected in the small head region (star) of EBs just prior to plating (EB0). (B): Expression of GATA4 is demonstrated in many regions, including beating cardiac muscles differentiated at EB2. The star indicates strong expression originating from the small head region of EBs at EB0. (C): Prospective gut-like structures (arrow) at EB5 show GATA4 expression. (D): Expression becomes heterogeneous, and at EB10, some developing gut-like structures show intense expression (large arrow), whereas others show none (small arrow). (E): Cross section of an EB at EB0 confirms that GATA4 is expressed in the epithelial structure (arrows) in the small head region. (F): Cross section of GATA4-positive area at EB2 in (B) showing strong expression (star) in epithelial structure. Scale bars = 500 µm (A¨CD), 100 µm (E, F).2 G2 n; w) F& x& g

Double Labeling of Factors in EBs with Immunohistochemistry4 Q9 |! ]1 H: Y. Z6 ~7 o7 j

All factors examined were expressed from EB0. In EBs, transcription factors were prominent in the small head regions where immunoreactivity of the same probes was detected (Fig. 3). Thus, analysis by double staining suggested the origin of the gut-like structures in EBs. Although each factor showed its own particular expression pattern (as shown in Fig. 3), double-positive epithelial structures in EBs were identified in all cases. These results demonstrate that transcription factors were colocalized in EB epithelial structures.
+ }. j1 W7 w( W! @  [; }
DISCUSSION5 I9 }2 |9 X! T  A
8 ?7 x0 @. h1 x, F
Sox174 l' x3 w! N1 Z  S' U
5 B6 r7 o& I) J) I% _, K$ i. Z
Sox17 was originally identified as a stage-specific transcription factor during spermatogenesis . In the present study, we demonstrated Sox17 expression in definitive endoderm at E8.0 and its disappearance in the GI tract at E13.0. The EBs and developing gut-like structures showed similar transient expression patterns, that is, strong Sox17 expression at EB5, a decrease after EB7, and a disappearance at EB14. This suggests that formation of the gut-like structure may follow a process parallel to gut organogensis in vivo (Fig. 4).
& U; Z" M' A" {9 e0 ~0 T
Id2" B6 w' M0 l, P, w( v7 i( Z0 t

The expression pattern of Id2 was investigated in the digestive tract of mouse embryos .4 e: a. D2 b, S* `2 S, U3 Z

In the present study, Id2 was expressed in EBs and developing and mature gut-like structures throughout all stages examined, and the pattern was similar to that in mouse embryos (Figs. 1B, 5). These results demonstrate that Id2 is an appropriate marker of the developing gut and suggest that the gut-like structure in vitro shows the same characteristics as the embryonic gut in vivo.
! B# U0 C% @2 h8 r0 ?8 {
HNF3ß/Foxa2

The fork head family members, including HNF3ß/Foxa2, are evolutionarily conserved throughout metazoans and play critical roles in gut endoderm differentiation .$ d  I6 z9 _0 J" b2 o2 ^& E$ Q; u3 s

In the present study, HNF3ß/Foxa2 was expressed in the anterior region of the embryonic GI tract at E13.0. At this stage, expression reportedly begins to decrease and even become heterogeneous in the GI tract in vivo. Similar expression patterns were observed in the EBs and gut-like structures in vitro. In the early stages, HNF3ß/Foxa2 mRNA clearly appeared in the epithelium of prospective gut-like structures (Fig. 6C), but expression became heterogeneous after EB7 (Fig. 6D). The regional variations in HNF3ß/Foxa2 expression indicate that the gut-like structure in vitro contains heterogeneous portions corresponding to those in the embryonic gut (Figs. 1C, 4).

GATA4
, _, x! ?' _5 P" V. Q; a% _
Both GATA4 and HNF3ß/Foxa2 are expressed in the endoderm, and in vitro footprinting studies have suggested that they function in combination, and that GATA4 dominantly regulates HNF3ß/Foxa2 expression .

In the embryonic gut at E13.0, GATA4 was expressed in the stomach and the proximal intestine, as previously reported. In developing gut-like structures after EB5, GATA4 was heterogeneously expressed (Fig. 7C, D). This heterogeneity is similar to that of the embryonic gut and again indicates that the gut-like structure reflects normal development in vivo (Figs. 1D, 4).

SUMMARY AND CONCLUSION) H' f8 e1 p+ E+ N/ |. o! f
6 ?3 R+ R$ [0 Z; ?# P" Q) f
The expression patterns of transcription factors in EBs and gut-like structures, and those in the mouse embryo reported previously, are summarized in Figure 4. All factors examined are crucial for formation of definitive endoderm and/or gut and are expressed in the epithelium of EBs at EB0 and in gut-like structures, including those in a premature state (Figs. 3, 4). Moreover, such expression patterns are similar to those in the embryonic gut. Our results suggest that the molecular mechanisms underlying gut development in vivo and the formation of gut-like structures in vitro are identical. Each transcription factor is expressed in a characteristic pattern at different segments of the GI tract. Therefore, their heterogeneous expression in the gut-like structure in vitro indicates that segments with regional specificities are reproduced (Fig. 4). Studying the onset of expression allowed us to determine the time course of differentiation during the formation of gut-like structures. EBs at EB0 are equivalent to the egg-cylinder stage (E5.0¨CE7.5), as previously described for the case of suspension cultures .  d% }5 u; y, N

Although the transcription factors examined are not specific to the definitive endoderm and/or gut, all factors were already expressed at EB0 in the small head region of EBs. Further colocalization of these factors was confirmed by immunohistochemical double staining. Because they overlapped with each other in the epithelial structures in EBs, the future gut-like structures were determined and formed in a particular area (the epithelial structure in the small head region of EBs) during the hanging drop period (Fig. 3). This assumption is supported by previous reports showing that HNF3ß/Foxa2 expression was quite low in undifferentiated ES cells but increased in EBs after 3 days of suspension culture preceding the rise in transthyretin, -fetoprotein, and other endoderm gene expression markers . These results suggest that development of the endoderm occurs in EBs even during the hanging drop period.0 E0 K* g4 P' L9 `
3 J4 w+ S% i2 }- x4 U+ X4 z
Our results demonstrate that the gut-like structures in vitro are a valid model system for the study of the mechanisms of early endoderm determination and gut organogenesis, and should provide important information needed to identify essential factors.
+ \0 h  O/ A; ?# S
DISCLOSURES# n: @! C  G& r$ S9 Z+ G% p
$ R4 O) ?+ K, Q8 g2 t! {
The authors indicate no potential conflicts of interest.- E! ?6 i+ \6 A0 ?2 a1 Z
4 S7 `2 ^  k: u. c2 e4 `
ACKNOWLEDGMENTS+ z  ]5 ^' ]+ b, [% h8 C4 r
2 i' d0 U1 i6 ~. O( d" o2 \
We thank Drs. D. Wilson (Washington University), H. Sasaki (RIKEN, Kobe, Japan), and Y. Yokota (Fukui University) for their kind gifts of plasmid templates¡ªGATA4, HNFß/Foxa2, and Id2, respectively. We are also grateful to Dr. H. Niwa (RIKEN, Kobe, Japan) for the gift of the ES cell line G4¨C2 and to Dr. M. Kanai-Azuma (Kyorin University) for valuable technical advice. This work was supported by research grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi Memorial Foundation for Female Natural Scientists.
      【参考文献】  ^7 `% b3 w* @) N
, }# U1 W! Q0 }+ X

O¡¯Shea KS. Embryonic stem cell models of development. Anat Rec 1999;257:32¨C41.! E4 V: d0 n# \5 l' m
+ ?% W; ]; W$ \, M
O¡¯Shea KS. Self-renewal vs. differentiation of mouse embryonic stem cells. Biol Reprod 2004;71:1755¨C1765.
, V2 p6 }8 d4 @
Yamada T, Yoshikawa M, Takaki M et al. In vitro functional gut-like organ formation from mouse embryonic stem cells. STEM CELLS 2002; 20:41¨C49.# N% V. |' g; l  ~, U

Kuwahara M, Ogaeri T, Matsuura R et al. In vitro organogenesis of gut-like structures from mouse embryonic stem cells. Neurogastroenterol Motil 2004;16 (suppl 1):14¨C18.
# h6 Z. ^0 i3 S( v. v- ~" @8 F
Ishikawa T, Nakayama S, Nakagawa T et al. Characterization of in vitro gutlike organ formed from mouse embryonic stem cells. Am J Physiol Cell Physiol 2004;286:C1344¨CC1352.( G5 g" g6 K- E4 F! X) U
/ `! V4 l1 m2 k, |! s7 H
Kikuchi Y, Agathon A, Alexander J et al. casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 2001;15:1493¨C1505.

Sakaguchi T, Kuroiwa A, Takeda H. A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish. Mech Dev 2001;107:25¨C38.9 Y- x" u6 w6 c0 S6 ]

Jen Y, Manova K, Benezra R. Expression patterns of Id1, Id2, and Id3 are highly related but distinct from that of Id4 during mouse embryogenesis. Dev Dyn 1996;207:235¨C252./ {8 r2 y8 W2 h) k; H; w
7 q6 J. z2 d( o
Yokota Y, Mansouri A, Mori S et al. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 1999;397:702¨C706." _" c8 c* A* F) \* H' n

Russell RG, Lasorella A, Dettin LE et al. Id2 drives differentiation and suppresses tumor formation in the intestinal epithelium. Cancer Res 2004;64:7220¨C7225.0 q# x. Y( b% a! ^' t7 D3 B9 Z

Lai E, Clark KL, Burley SK et al. Hepatocyte nuclear factor 3/fork head or "winged helix" proteins: A family of transcription factors of diverse biologic function. Proc Natl Acad Sci U S A 1993;90:10421¨C10423.

Dufort D, Schwartz L, Harpal K et al. The transcription factor HNF3&szlig; is required in visceral endoderm for normal primitive streak morphogenesis. Development 1998;125:3015¨C3025.& E/ ^6 u% O4 Y: I  n) N
5 _) h9 h5 [, k* P; K
Hallonet M, Kaestner KH, Martin-Parras L et al. Maintenance of the specification of the anterior definitive endoderm and forebrain depends on the axial mesendoderm: A study using HNF3&szlig;/Foxa2 conditional mutants. Dev Biol 2002;243:20¨C33.

Arceci RJ, King AA, Simon MC et al. Mouse GATA-4: A retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart. Mol Cell Biol 1993;13:2235¨C2246.3 A% q1 ]' O9 ?

Morrisey EE, Ip HS, Lu MM et al. GATA-6: A zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm. Dev Biol 1996;177:309¨C322.7 |# m$ O0 L8 ?( i" M
" t/ Y# y2 M/ C* R: G$ A
Jacobsen CM, Narita N, Bielinska M et al. Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium. Dev Biol 2002;241:34¨C46.
6 [, E( b- o. @. o% c, q% L
Kuo CT, Morrisey EE, Anandappa R et al. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev 1997;11:1048¨C1060.' T! ~# O9 r/ m: c* d9 Q+ W
4 M8 Q1 ]$ B2 j) k
Molkentin JD, Lin Q, Duncan SA et al. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev 1997;11:1061¨C1072.4 O2 A& F0 E" ~. @/ |& v

Soudais C, Bielinska M, Heikinheimo M et al. Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development 1995; 121:3877¨C3888./ \' w6 S8 q( L

Sakiyama J, Yamagishi A, Kuroiwa A. Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Development 2003;130:1225¨C1234.) U& g% _+ M! u* O$ _# @6 ~9 Y

Kanai Y, Kanai-Azuma M, Noce T et al. Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. J Cell Biol 1996;133:667¨C681.3 O1 n3 r, X; W. v7 B' p4 n
6 i* t8 |* V2 J9 c) X5 f: ?5 ^) p" O
Sasaki H, Hogan BL. Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 1993;118:47¨C59.

Leahy A, Xiong JW, Kuhnert F et al. Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J Exp Zool 1999;284:67¨C81.

Kanai-Azuma M, Kanai Y, Gad JM et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 2002;129:2367¨C2379.: ~) y% s, ^/ B) ?4 u8 X( b

Hollnagel A, Oehlmann V, Heymer J et al. Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem 1999;274:19838¨C19845.( P% ]: c# y/ [* ~2 V( a+ o0 z
/ }  \7 ?9 `2 P, Z" ?  a7 x
Brennan J, Lu CC, Norris DP et al. Nodal signalling in the epiblast patterns the early mouse embryo. Nature 2001;411:965¨C969.
4 t" U3 i9 S& Y0 D7 ^- k0 v
Vincent SD, Dunn NR, Hayashi S et al. Cell fate decisions within the mouse organizer are governed by graded Nodal signals. Genes Dev 2003;17:1646¨C1662.

Zaret K. Developmental competence of the gut endoderm: Genetic potentiation by GATA and HNF3/fork head proteins. Dev Biol 1999; 209:1¨C10.

Ang SL, Wierda A, Wong D et al. The formation and maintenance of the definitive endoderm lineage in the mouse: Involvement of HNF3/fork-head proteins. Development 1993;119:1301¨C1315.* ?9 K: p; _" r2 E$ w$ M! i

Kaestner KH, Hiemisch H, Luckow B et al. The HNF-3 gene family of transcription factors in mice: Gene structure, cDNA sequence, and mRNA distribution. Genomics 1994;20:377¨C385.

Fujikura J, Yamato E, Yonemura S et al. Differentiation of embryonic stem cells is induced by GATA factors Genes Dev 2002;16:784¨C789.

Abe K, Niwa H, Iwase K et al. Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 1996;229:27¨C34.0 z; \1 C: R- X6 e

Levinson-Dushnik M, Benvenisty N. Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 1997;17:3817¨C3822.

收藏0 分享0 顶0 踩0