Effects of retinoic acid on tissue differentiation
Retinoic acid has a posteriorization/lateralization activity on the central nervous system and mesodermal tissue ( Durston et al. 1989 ; Ruiz i Altaba & Jessell 1991a, b; Lopez & Carrasco 1992; Moriya et al. 1993 , 1998). Treatment of the isolated dorsal lip of Xenopus early gastrula with retinoic acid induces pancreas differentiation (Moriya et al. 2000). Activin at high concentrations induces isolated presumptive ectoderm of Xenopus blastula to differentiate into dorsal lip-like cells that also possess inductive activity similar to genuine dorsal lip ( Ninomiya et al. 1999 ). Thus, this suggests that treatment with activin and retinoic acid may induce presumptive ectoderm to differentiate into pancreas.
Ectoderm treated with activin alone became axial mesoderm and anterior endoderm, such as notochord, muscle and pharynx ( Table 1; Fig. 2B). These tissues are normally derived from the dorsal lip region in vivo, suggesting that activin at the concentration used here induces dorsal lip-like cells in ectoderm explants. Treatment with activin at this concentration and retinoic acid at various concentrations for 1 h, however, did not induce pancreas formation. Retinoic acid had a dose-dependent effect on the pattern of differentiation of mesodermal tissue ( Table 1). Notochord formation was first repressed and muscle formation was then repressed, while the formation of pronephros was induced as the concentration of retinoic acid increased. Retinoic acid in combination with activin had a small effect on endodermal tissue differentiation. In normal development, endoderm undergoes morphogenesis, and therefore probably determination, later than mesoderm ( Nieuwkoop & Faber 1956; Chalmers & Slack 1998). Short-term treatment with differentiating factors immediately after isolation of ectoderm may therefore only correspond to the timing of determination of mesodermal tissues, but not endodermal tissues. To avoid this effect, we cultured ectoderm continuously in the presence of activin and retinoic acid, but obtained similar results to short-term treatment. However, with continuous treatment, ectoderm explants were exposed to retinoic acid during the determination of endodermal tissues, which did not alter the pattern of endodermal differentiation ( Table 2). In contrast, when isolated dorsal lips were treated with retinoic acid for 1–3 h immediately after isolation they differentiated into pancreas (Moriya et al. 2000), but dorsal lips had already begun to differentiate into endomesoderm. Therefore, when undifferentiated cells are exposed to activin and retinoic acid at the same time, they may be induced to become lateral mesoderm, but not endomesoderm. In addition, retinoic acid may only be able to induce pancreas in cells that have already differentiated into endomesoderm.
Effects of the time lag between activin and retinoic acid treatments
To examine the effect of retinoic acid on endomesoderm, presumptive ectoderm was first exposed to activin (100 ng/mL) for 1 h and was then exposed to retinoic acid (10–4M) for 1 h at various times after the activin treatment. A time lag of 3–5 h between activin and retinoic acid treatment induced frequent pancreas formation in more than 80% of explants ( Table 3). These findings indicate that once ectoderm has been induced to differentiate by activin, there is a window of 3–5 h in which retinoic acid can induce a very high rate of differentiation of pancreas-like structures. However, beyond this window retinoic acid has little effect and ectoderm differentiation is driven by activin alone, leading to the development of axial mesoderm and pharynx. Therefore, retinoic acid may only be able to drive endomesoderm to form pancreatic tissues and once ectoderm has differentiated into endodermal tissue, which occurs at approximately 15 h after initiation of activin treatment, retinoic acid can no longer affect these cells.
It has been reported that activin alone induces Xenopus ectoderm to express XlHbox8, a marker of anterior endoderm including the pancreas anlage ( Gamer & Wright 1995). XlHbox8 is generally expressed in the posterior foregut region at stages 33–41 ( Wright et al. 1989 ). In the present study, morphological analysis showed that explants treated with activin alone differentiated into pharynx, an anterior endodermal tissue, and did not develop pancreas-like structures ( Tables 1–3). Thus, these findings suggest that activin alone may induce anterior endoderm, but not pancreas.
Acinus-like structures were observed using light microscopy in ectodermal explants treated with activin and then 5 h later with retinoic acid. Cells resembling exocrine or endocrine cells containing secretory granules of normal embryonic pancreas ( Leone et al. 1976 ) were seen under electron microscopy. Production of the islet hormones, insulin and glucagon, has already begun in larva at stage 41 ( Shuldiner et al. 1991 ; Maake et al. 1998 ). In the present study, ectoderm treated with activin and retinoic acid with a 5 h time lag produced both insulin and glucagon ( Fig. 5), suggesting that these explants had differentiated into endocrine pancreas, which was functionally similar to normal pancreas in vivo.
The pancreatic tissues that developed in the explants were covered by intestinal epithelium-like tissue in most cases. Endodermal epithelium forms a duct from mouth to anus and its morphology gradually varies along this duct in vivo. In the present study, epithelia observed in explants were classified into two types, pharyngeal epithelium-like structures and intestinal epithelium-like structures. Intestinal epithelium is very thick, while pharynx is thin ( Chalmers & Slack 1998). These two types of epithelium were distinguished according to the height/width ratio of epithelial cells. A layer with a ratio lower than 3 was classified as a pharyngeal epithelium-like structure and a layer with a ratio higher than 3 was scored as an intestinal epithelium-like structure. Ectoderm treated with activin alone formed a pharyngeal epithelium-like structure, while cells treated with activin and retinoic acid differentiated into pancreas and intestinal epithelium-like structures. Pharynx locates anteriorly, while pancreas and intestine locate posterior to the pharynx in vivo.
It has been reported that Xenopus blastulae treated with retinoic acid lose head structures ( Durston et al. 1989 ). Retinoic acid can repress anterior markers and induce posterior markers ( Ruiz i Altaba & Jessell 1991a, b; Lopez & Carrasco 1992; Kolm et al. 1997 ). Retinoic acid and its receptor are localized to the posterior of the embryo ( Ellinger-Ziegelbauer & Dreyer 1991; Chen et al. 1994 ). Thus, it is possible that ectoderm is first initiated into anterior endomesoderm by activin and then the cells are modified posteriorly by retinoic acid and differentiate into pancreas. Pancreatic structures actually appeared to be accompanied by intestinal epithelium under microscopy ( Fig. 2D).
The effect of retinoic acid on endoderm in Xenopus embryos has been reported previously ( Zeynali & Dixon 1998). Xenopus embryos at stages 22–33 were exposed to retinoic acid, which led to abnormal morphogenesis in the digestive tract, while pancreas developed normally. We treated Xenopus blastula with retinoic acid and looked for pancreas induction in vivo. The embryo lacked a head structure, but all endodermal organs developed normally. There were no organ-specific defects or enlargement with retinoic acid treatment. The location of endodermal organs was, however, abnormal; the organs were located close to each other along the anteroposterior axis (data not shown). These findings suggest that retinoic acid, which drives posteriorization, can only induce correct pancreas formation in ectoderm that has begun to differentiate into anterior endoderm, via, for example, treatment with activin.
Influence of mesoderm on pancreas development
Moriya et al. (2000) have induced pancreas formation in dorsal lip explants by retinoic acid treatment and have also demonstrated that the frequency of pancreas formation increases as the concentration of retinoic acid increases and that the pancreas is covered by intestinal epithelium. Pancreas was accompanied by notochord in these dorsal lip explants, whereas notochord formation was repressed under the conditions that frequently induced pancreas in ectodermal explants ( Table 3). Notochord induces pancreas in the prepancreatic endoderm chick embryo ( Kim et al. 1997 ). Thus, it is possible that the pancreas that developed in dorsal lip was induced by notochord present in the same explant. In the present study, however, pancreas was induced without notochord. Therefore, notochord formation is not necessary for pancreas differentiation.
In the chick embryo, notochord has been shown to repress Shh in prepancreatic endoderm and permit pancreas development; treatment with an Shh inhibitor causes a presumptive stomach region to differentiate into pancreas ( Kim et al. 1997 ; Hebrok et al. 1998 ; Kim & Melton 1998). Candidate notochord-derived inhibitors of Shh expression in prepancreatic endoderm include Shh, activin B and fibroblast growth factor (FGF)2 ( Kim & Melton 1998). It is possible that activin A in the present study may also have inhibited the expression of Shh. Activin alone, however, did not induce pancreas in ectoderm ( Tables 1–3); pancreas differentiation in ectoderm requires both activin and retinoic acid in this system.
Some studies of the effect of retinoic acid on development have suggested that retinoic acid affects Shh expression in the chick limb bud and mammal craniofacial morphogenesis ( Helms et al. 1994 ; Ogura et al. 1996 ; Helms et al. 1997 ). In Xenopus ectoderm, retinoic acid may also have an effect on Shh expression and pancreas differentiation.
In summary, pancreas may be frequently induced by activin and retinoic acid treatment of Xenopus presumptive ectoderm. This is a simple and effective in vitro system for analysis of the mechanisms of pancreas differentiation and endodermal patterning.