THE ANATOMICAL RECORD 249:389–398 (1997) Developing Human Biliary System in Three Dimensions VIJAYALAXMY VIJAYAN1* AND CAROLYN E.L. TAN2 of Paediatric Surgery, Singapore General Hospital, Singapore 2Department of Paediatric Surgery, Singapore General Hospital, Singapore 1Department ABSTRACT Background: In the development of the human biliary system, although the extrahepatic bile ducts develop from the embryonic hepatic diverticulum, there is increasing evidence to suggest that the intrahepatic bile ducts originate within the liver from the ductal plate. The ductal plate develops as a sheath of primitive biliary epithelium in the mesenchyme along the portal vein branches. Through an orderly process of selection and deletion, the ductal plate is remodelled into the adult system of anastomosing tubular bile ducts. The ductal plate remodelling process occurs at the porta hepatis between 11 and 13 weeks of gestation and progresses towards the periphery of the liver. Methods: In this project, for the first time, we have used computerised three-dimensional reconstruction techniques to visualise the developing human biliary system. Paraffin-embedded tissue from eight human embryos or fetuses between 5.5 and 16 weeks of gestation were serially sectioned, and their images were aligned, digitised, and used for threedimensional reconstruction. Results and Conclusions: Three-dimensional images of the extrahepatic and the intrahepatic biliary systems were obtained, and the following conclusions were drawn. (1) The intrahepatic biliary system, both at the porta hepatis and within the liver, developed from the ductal plate through a consistent pattern of remodelling. (2) Prior to the remodelling process, the ductal plate was of similar morphology irrespective of site and gestation. (3) The extrahepatic biliary system was in direct luminal continuity with the developing intrahepatic biliary system throughout gestation and did not show the presence of a ‘‘solid stage’’ in any of the embryos or fetuses studied. Anat. Rec. 249:389–398, 1997. r 1997 Wiley-Liss, Inc. Key words: ductal plate; bile ducts; biliary development; liver embryology In the development of the human biliary system, the extrahepatic and intrahepatic systems differ in their origin. The extrahepatic bile ducts (EHBD) and the liver arise from the embryonic hepatic diverticulum, which is a ventral projection from the developing foregut (Severn, 1971, 1972). Several theories have been proposed for the development of the intrahepatic bile ducts (IHBD) that link the bile canaliculi and the EHBD. One that is gaining increasing support is that the IHBD arise from the ductal plate, a sheath of primitive biliary epithelium that develops in the mesenchyme along the portal vein branches (Desmet, 1985; Terada and Nakanuma, 1995). Through an orderly process of selection and deletion, the ductal plate is remodelled into the anastomosing system of tubular bile ducts seen in the adults (Fig. 1). This ductal plate remodelling process starts at the porta hepatis, where it is said to occur between 11 and 13 weeks of gestation, and progresses towards the periphery of the liver (Tan and Moscoso, 1994a,b). Thus, the porta hepatis is the crucial region where the EHBD merges into the IHBD. According to some reports, remodelling along the more distal portal vein r 1997 WILEY-LISS, INC. branches may even occur after birth (Van Eyken et al., 1988; Woolf and Vierling, 1993). The incorporation of computers in microscopy has opened the exciting new possibility of visualising developing organs in three dimension. With serial sections, it is now possible to reconstruct the entire organ under study. In manually drawn reconstructions, the observer’s preconceived ideas may affect the results, but computer-generated three-dimensional (3-D) reconstructions can eliminate such errors. In the references cited above, the fetal biliary system and the ductal plate remodelling have been studied using manual reconstructions of serial sections. In the present study, we used computerised 3-D reconstruction techniques to visualise the developing human biliary system. Contract grant sponsor: Department of Clinical Research, Ministry of Health, Singapore; Contract grant sponsor: Department of Paediatric Surgery, Singapore General Hospital, Singapore. *Correspondence to: Vijayalaxmy Vijayan, Department of Paediatric Surgery, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899. Received 11 December 1996; Accepted 10 April 1997 390 V. VIJAYAN AND C.E.L. TAN TABLE 1. Fetuses and embryos used in the present study Fetuses/embryos F25 F16 F46 F4 F7 F28 F47 F45 Gestational age (weeks) Foot length (mm) 5.5 7 8.5 11 11 12 13 16 1 1 4.2 6 7 10.5 11.16 19.7 1These embryos were staged based on external morphology. cies for psychosocial reasons. Approval was obtained from the hospital’s ethical committee. All embryos and fetuses were karyotyped and were normal 46XY or 46XX. The specimens were immediately fixed in 4% buffered paraformaldehyde for 24 hr at room temperature, transferred to 15% sucrose in phosphate buffered saline, and left at 4°C until embedding in paraffin wax. The developmental stages of the embryos were determined according to the method of O’Rahilly and Müller (1987) and the fetuses were staged according to the method of Streeter (1920). 3-D Reconstruction Fig. 1. Diagrammatic representation of the formation of an intralobular bile duct within a portal tract. To simplify the illustration the hepatic artery branch and mesenchymal tissue have not been drawn. A: A single layer of biliary cuboidal epithelium; the ductal plate has developed at the liver parenchyma–mesenchyme margin. B: The ductal plate is separated from the hepatic margin by proliferating mesenchymal tissue. C: Continued mesenchymal proliferation (arrowheads) has pushed the ductal plate farther away from the hepatic parenchyma. The ductal plate has duplicated, and luminal spaces have appeared between the two layers of cuboidal epithelium. D: One portion of the ductal plate is actively remodeled by surrounding mesenchyme into a tubular bile duct (*), while the rest of the redundant ductal plate structures are being deleted. E: The ductal plate remodeling process is complete, and an intralobular bile duct has developed, whereas its biliary precursor, the ductal plate, has disappeared. The mesenchymal cuff around the definitive bile duct is not shown. (Figure reproduced with permission from Tan et al., 1995, Pathol. Int. 45:815–824, Fig. 1.) MATERIALS AND METHODS Embryos and Fetuses Eight normal human embryos and fetuses of 5.5–16 weeks gestation were studied in this project (Table 1). All embryos and fetuses were obtained from healthy mothers undergoing elective termination of pregnanAbbreviations b/bd cbd cd chd dp gb h lhd m ph pv rhd bile duct common bile duct cystic duct common hepatic duct ductal plate gall bladder liver parenchyme left hepatic duct mesenchyme porta hepatis portal vein right hepatic duct Serial sections of 5 µ thickness were cut, and every third section was stained with toluidine blue and used in the 3-D reconstruction. Digitisation was done through a CCD camera (Progressive Research 3012, Carl Zeiss, Germany), which was attached to either a microscope (Axiophot, Carl Zeiss, Germany) or a AF Micro-Nikkor 60 mm f/2.8 D lens (Nikon), depending on the magnification required. Sections were aligned using a RGB monitor connected to the VIOB output of the IBAS 2.5 system (Kontron Elektronik, Germany). To align an image, the preceding image was converted into an overlay, and the current image was aligned according to the overlay. Alignment was done by using clearly visible structures in the images as references. Each section was stored as a separate image of 512 3 512 pixel resolution with 256 (0–255) grey levels. The biliary structures and blood vessels were manually traced in each image and the other structures were erased, and the traced image was scaled to grey levels 0–254. The lumina within the biliary structures were assigned a grey level of 255. The voxel-based 3-D reconstruction technique of distance shading was used to generate 3-D images (Moss, 1992). For each embryo or fetus, the EHBD, consisting of the common bile duct (cbd), the gallbladder (gb), the cystic duct (cd), and the common hepatic duct (chd), was reconstructed. The IHBD, consisting of biliary structures at the porta hepatis, was reconstructed in all embryos and fetuses, and where possible the biliary structures proximal to the porta hepatis also were reconstructed. In all the specimens, reconstructions along the luminal spaces of the biliary structures also were generated to get a 3-D view of the lumen. Wherever possible, the veins were included in the reconstructions. A median filter of matrix size 3–7 was applied to smooth the edges of the final 3-D image. Details of the DEVELOPING HUMAN BILIARY SYSTEM IN 3-D 391 Fig. 2. EHBD and its lumen. A: The 3-D image of a 5.5-week embryo showing the cbd and a funnel-shaped widening of the cbd at the porta hepatis. The cd joins the cbd at the ph with a very short chd in this specimen. The gb points upwards in this specimen, probably due to a fixation artefact (Fig. 4 shows the ph region at higher magnification). B: Photomicrograph of a transverse section through the cbd (arrow) just distal to the ph of a 5.5-week embryo, showing the presence of a clear and patent lumen. C: The 3-D image of a 7-week embryo in which the gb is well formed and the chd ends in fingerlike ductal plate structures at the ph. D: The 3-D image of the lumen corresponding to C. Scale bar 5 50 µ. methodology have been reported elsewhere (Vijayan and Tan, 1995, 1996). through the stages of remodelling until the adult configuration is achieved. RESULTS AND DISCUSSION Extrahepatic Bile Ducts (Figs. 2, 3) Reports about the development of the human biliary system, particularly the ductal plate remodelling, have been based on manual reconstruction of serial sections. The present study is the first computer-generated 3-D reconstruction of the developing human biliary system The EHBD of all eight embryos or fetuses at 5.5–16 weeks of gestation were reconstructed. The EHBD was clearly visible and had a continuous and patent lumen through its entire length in all the specimens studied. This observation confirms the absence of a ‘‘solid stage’’ 392 V. VIJAYAN AND C.E.L. TAN Fig. 3. The 3-D reconstructions of the EHBD and its lumen. A: An 8.5-week fetus showing the EHBD and (B) its lumen. C: An 11-week fetus showing the EHBD and (D) its lumen. In all the gestations, a continuous lumen is present throughout the EHBD. during the development of the cbd, as previously reported (Tan and Moscoso, 1994a). Intrahepatic Bile Ducts At the porta hepatis (Figs. 4–11) There is little controversy regarding the development of the EHBD, which arises from the embryonic hepatic diverticulum (Severn, 1971). However, there are two main theories about the origin of the IHBD. One is that the IHBD originate from the EHBD at the porta hepatis and grow into the liver in an infiltrative manner (Hammar, 1926; Koga, 1971). The other, has accumulated compelling evidence in recent years, is that the IHBD develop from within the liver from the ductal plate, which through a process called ‘‘ductal plate remodelling’’ matures into the tubular anastomosing biliary tree of the adult (Desmet, 1985; Jorgensen, 1977; Terada and Nakanuma, 1995; Van Eyken et al., 1988). DEVELOPING HUMAN BILIARY SYSTEM IN 3-D 393 Fig. 4. A 5.5-week embryo. A: Photomicrograph of a transverse section at the porta hepatis showing the chd (short arrow) and the dp structures (long arrows) attached to the chd. The separation (*) between the chd and the liver parenchyma is artefactual. B: A 3-D image of a 5.5-week embryo at the porta hepatis showing ductal plate structures attached to the chd/cbd. Scale bar 5 50 µ. Fig. 5. A 7-week embryo A: Photomicrograph of a transverse section at the level of the porta hepatis showing the chd (short arrow) and fingerlike dp structures (long arrows) attached to it. Tiny luminal spaces are seen in the dp. B: The 3-D images show early dp structures attached to the chd, projecting into the liver like fingers of a hand. Scale bar 5 µ. All the specimens in this study showed the presence of ductal plate structures at the porta hepatis. In the 5.5-week embryo (embryo F25; Fig. 4), early ductal plate structures were seen at the porta hepatis. The cbd showed a funnel-shaped widening at the porta hepatis, to which ductal plate structures were attached. At 7 weeks of gestation (embryo F16; Fig. 5), early ductal plate structures with luminal spaces were seen at the porta hepatis. In three dimensions, they were seen as fingerlike projections continuous with the chd at the porta hepatis. Proximal to the porta hepatis, within the liver, no ductal plate structures were observed in these 394 V. VIJAYAN AND C.E.L. TAN Fig. 6. A: The 3-D reconstruction at the porta hepatis of an 8.5-week fetus showing the ductal plate structures closely hugging the portal vein branch. The chd is continuous with the ductal plate structures. B: Reconstruction of the corresponding luminal spaces showing that there is luminal continuity between the chd and the ductal plate structures. Biliary structures are shown in yellow, and the portal vein branch is shown in grey. Fig. 7. A 3-D reconstruction at the porta hepatis of two 11-week fetuses: A: Fetus F4 show the chd in continuity with ductal plate structures along the vein. One prominent tubular bile duct is beginning to form (arrow). B: Fetus F7. In this fetus, two tubular branches of the chd can be seen, which are in continuity with the ductal plate structures along the two branches of the vein. Biliary structures are shown in yellow, and the portal vein branches are shown in grey. early embryos. Other investigators have reported the appearance of the ductal plate at the porta hepatis at 6–9 weeks gestation (Van Eyken et al., 1988; Tan and Moscoso, 1994a), which suggests some variability in the onset of ductal plate formation at the porta hepatis. DEVELOPING HUMAN BILIARY SYSTEM IN 3-D Fig. 8. Photomicrograph of a transverse section through the ductal plate in an 11-week fetus: Ductal plate structures (arrows) in the mesenchyme surround a portal vein branch. Discontinuous luminal spaces are seen in the ductal plate (*). Scale bar 5 50 µ. Fig. 9. A 3-D reconstruction at the porta hepatis of a 12-week fetus. The adult pattern of tubular left and right hepatic ducts (arrows) can be seen with some ductal plate remnants still attached to them. Vein and gb are not included in this reconstruction. The ductal plate consisted of a network of abundant, flat biliary structures hugging and closely following the portal vein branch. However, within the ductal plate, the luminal spaces formed a network connected at some 395 Fig. 10. A 3-D reconstruction at the porta hepatis of a 13-week fetus. At this gestation, the adult pattern is seen clearly. The left and right hepatic ducts (arrows) and their branches are seen. Biliary structures are shown in yellow, and the portal vein branches are shown in grey. Gallbladder is not shown. Fig. 11. A 3-D reconstruction at the porta hepatis of a 16-week fetus. Well-defined tubular bile ducts of the adult configuration are seen with first- and second-generation branches of the chd. Biliary structures are shown in yellow, and the portal vein branches are shown in grey. Gallbladder is not shown. places and isolated at others, similar to that described in the mouse embryo (Shiojiri and Mizuno, 1986). At 8.5 weeks gestation (fetus F46; Fig. 6), the curved platelike network encompassed half the circumference of the vein. The 3-D reconstruction along the luminal spaces 396 V. VIJAYAN AND C.E.L. TAN Fig. 12. Several 3-D reconstructions proximal to the porta hepatis. A: An 11-week fetus and B: a 12-week fetus show ductal plate structures along a portal vein branch within the liver. C: A 16-week fetus shows a tubular, well-defined interlobular bile duct along a portal vein branch. Biliary structures are shown in yellow, and the portal vein branches are shown in grey. of the ductal plate and the lumen of the chd showed that they were in direct luminal continuity at the porta hepatis, a feature clearly observed in all stages of gestation. At 8.5 weeks of gestation, the treelike pattern of the adult-type bile ducts was not seen, suggesting that the biliary structures at the porta hepatis had not yet been remodelled from the ductal plate configuration to tubular bile ducts. Two fetuses were studied at 11 weeks gestation (fetuses F4 and F7; Fig. 7). Abundant ductal plate structures were seen that morphologically resembled those of the 8.5-week fetus. In fetus F4, the chd was in direct continuity with the ductal plate structures, among which one tubular bile duct was already beginning to form. Figure 8 shows a photomicrograph of the ductal plate in transverse section. In fetus F7, a more adultlike pattern was seen with tubular left and right branches (first-generation branches) of the chd. The two branches were continuous with ductal plate structures seen poximally along the two branches of the vein. The foot length measurements of the two fetuses show that F7 is older than F4, thus explaining the more adultlike DEVELOPING HUMAN BILIARY SYSTEM IN 3-D pattern at the porta hepatis in fetus F7. Many normal variations in the right and left hepatic duct morphology may be due to the variations in the remodelling process at the porta hepatis (Tan and Moscoso, 1994a). At 12 weeks of gestation (fetus F28; Fig. 9), the ductal plate at the porta hepatis had been remodelled almost fully into adult configuration. Tubular left and right branches (first-generation branches) of the chd were seen clearly; however, remnants of ductal plate were still seen attached to them. Further proximally into the liver, the bile ducts were in continuity with ductal plate structures. By 13 weeks (fetus F47; Fig. 10) and 16 weeks (fetus F45; Fig. 11) of gestation, the remodelling had produced not only left and right hepatic (first generation) ducts but also second-generation ducts that were branches of the hepatic ducts. The adult pattern of bile ducts was seen clearly at the porta hepatis at these gestations. Proximal to these definitive ducts, the biliary system remained in the ductal plate configuration, and the tubular bile ducts were continuous with these ductal plate structures. These results confirm previous reports that the intrahepatic biliary system develops within the liver from the primitive fetal biliary structure, the ductal plate, and not by branching growth from the EHBD into the liver. It also establishes that the process of ductal plate remodelling changes the platelike ductal plate into tubular bile ducts of the adult, starting at the porta hepatis and progressing into the liver with gestation. The exact intrauterine period during which the remodelling occurs at more proximal regions of the intrahepatic biliary system is not entirely clear. In rat embryos, the remodelling continues after birth to achieve the adult configuration (Gall and Bhathal, 1989). Similarly in humans, Woolf and Vierling (1993) suggested that, at the periphery of the liver, biliary remodelling may continue after birth. In our series of fetuses, the remodelling process is complete at the porta hepatis by 13 weeks, which is similar to the 11–13-week crucial period suggested by other reports (Tan et al., 1994). Proximal to the porta hepatis (Figs. 12, 13) Biliary structures proximal to the porta hepatis, well within the liver, were also reconstructed. The primitive intrahepatic biliary structures within the liver were morphologically identical to the ductal plate structures seen at the porta hepatis before the onset of ductal plate remodelling. The biliary structures along the portal vein branches showed ductal plate configuration in the two younger fetuses at 11 and 12 weeks of gestation. As gestation progressed, the ductal plate was remodelled into tubular bile ducts, which could be observed by 16 weeks. Figure 13 shows a transverse section of a definitive intralobular bile duct in a portal tract from the 16-week fetus. Before the start of the remodelling process, the ductal plate structures remain consistent in morphology, irrespective of (a) site within the liver, i.e., at the porta hepatis or more proximally, and (b) gestational age. The mechanism by which the flat lacelike ductal plate is remodelled into a treelike pattern of tubular bile ducts has been the topic of much speculation. A recent report has shown that balanced cell proliferation and apoptosis play a role in the remodelling (Terada 397 Fig. 13. Photomicrograph of a transverse section through a portal tract in a 16-week fetus. A tubular definitive bile duct (arrow) is shown within a portal tract. Scale bar 5 50 µ. and Nakanuma, 1995). Cytokines such as transforming growth factor a1 play a role in the remodelling process (Terada et al., 1994). Reports have shown that the immunolocalisation pattern of transforming growth factor b1 within the biliary epithelium changes as the ductal plates matures into definitive bile ducts (Tan et al., 1995). These reports suggest that molecular and cellular signals are involved in the ductal plate remodelling process. The mesenchyme also plays an important role in the development of the intrahepatic bile ducts (Shiojiri and Nagai, 1992), which has been shown by the transplant experiments of Shiojiri (1984), in which the immature mouse hepatocytes differentiated into IHBD cells only when transplanted into the subcutaneous connective tissue and not into the testes of newborn mice. A timed inductive signal appears to trigger the wave of ductal plate remodelling from the porta hepatis outwards to the rest of the IHBD, the exact nature and origin of the which remains unclear at this point. In summary, the IHBD at the porta hepatis and within the liver arise from the ductal plate, which is the primitive fetal biliary system, through an orderly process of selection and deletion termed ‘‘ductal plate remodelling.’’ Before the start of the remodelling process, the ductal plate is consistent in morphology irrespective of site and gestation. 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