Development of the human fetal visceral pleura. An ultrastructural study

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Development of the human fetal visceral pleura. An ultrastructural study* Krassimira N. Michailova Department of Anatomy and Histology, Preclinical University Centre, Faculty of Medicine, BG-1431 Sofia, Bulgaria

Summary. The visceral pleura of human fetuses aged from 9 to 36 weeks of gestation was studied by means of transmission electron microscopy. The main components of the visceral pleura (mesothelium, basal lamina and sub mesothelial connective tissue layer) are formed in the fetal period. They develop asynchronously in different pleural areas, and do not reach maturity. Fetal differentiation of the lung pleura can be divided in two stages -- early (until 17 gestation week) and late stage - up to birth. The high mesothelial cells appear later than the flat cells, but the first type predominates in the final covering layer during the period investigated. The most significant developmental phenomena of the mesothelium involve membranous differentiation (the microvillous covering, vesicular system and intercellular contacts). The different transport and secretory potentials of the mesothelial cells during the various prenatal periods are discussed. The mode of development of the basal lamina suggests its mesothelial origin. The elastic membrane appears during the late stage of fetal life. The components of the submesothelial connective tissue layer (fibroblasts, collagen and elastic fibres, blood and lymph vessels) undergo several phases of differentiation. Key words: Serous membranes Visceral pleura Mesothelium - Human - Electron microscopy

Introduction Numerous investigations have been made into the earliest phases of the development of the serous coelom. This group of studies describes the different stages of formation of the pleural cavity, as well as those of the pericardial and peritoneal coverings (Searls 1986; Moore and Persaud 1993; and others). After the 4th gestational week (GW) the

* Dedicated with much gratitude to my SCientific supervisor, Prof. Wassil A. Wassilew, M. D., Ph. D., D. Sci Med. Ann Anat (1996) 178: 91- 99 Gustav Fischer Verlag .lena

splanchnic mesoderm layers are fused in the midline and form the intraembryonic coelom (Sadler 1990). The most important structure dividing the intraembryonic coelomic cavity is formed by the septum transversum, but leaves large openings, the pericardioperitoneal canals and the lung buds expand into them. At 5 GW the lung buds divide into main bronchi. At the 6th GW the mesoderm of the body wall is divided into two components: the thoracic wall and the pleuropericardial membrane. Since the diaphragm is still incomplete, the pleural cavities communicate with the abdominal cavity. At the 7th GW the pleuroperitoneal fold fuses with septum transversum and the pleural cavities are definitely formed. According to O'Rahilly and Muller (1992) the pleuropericardial membranes are closed in stage 17 (41 days), whereas the septum transversum is closed in stage 21 (52 days). During further development the main bronchi divide repeatedly in a dichotomous· fashion and by the end of the 6th month approximately 17 generations of subdivisions have been formed (Sadler 1990). The fetal development of the lung may be divided into pseudoglandular (5 -17 GW), canalicular (17 - 26 GW) and alveolar (24 GW to birth) stages (Burri 1984). Another group of investigators (Hislop et al. 1984; Kitterman 1984; Langston et al. 1984; and others) have described only the process of differentiation of the terminal spaces. There are significant species differences in their rates and mode of formation. The airway ramifications have an endodermal lining which is in direct contact with the surrounding mesenchym and is necessary for lung development (Riso 1983). According to Tournier et al. (1992), the morphogenesis and maturation of the lung depend both on the nature of the extracellular matrix, which facilitates the cell migration, and on the epithelial-mesenchymal interaction, which induces the proliferation and differentiation of epithelial cells. The mesoderm, which covers the outside of the lung, develops into the visceral pleura. The somatic mesoderm layer, covering the body wall from the inside, becomes the parietal pleura (Langman 1975).

Some observers have described the initial changes of the mesothelium. They compare its cellular apparatus, microvillous covering, vesicular system and intercellular contacts with those of the endothelium of the yolk sac (Hoyes 1969; Tiedemann 1976). Using contrast materials, King and Wilson (1983) characterised the transport capabilities of the primary mesothelium and endothelium. Investigations of the human mesothelium have been controversial. It has been variously described as being composed of cubic, prismatic, flat or fibroblast-like cells (Hessedahl and Larsen 1969; Whitaker 1982). Components of both serous sheets have been the subject of individual studies, which describe them as uniform (Ivanova 1975). More often, the developmental changes in the lung epithelium, pleural mesothelium and connective tissue components have been mainly investigated in experimental studies during postnatal life (Emery 1970; Jones and Barson 1971; Krause and Leeson 1975; Hislop et al. 1984; Scheuermann et al. 1988). In contrast to the numerous studies of lung development, investigations on pleural differentiation are rare, and mainly concern the initial changes in the mesothelium. The main

reason for our undertaking this study is the absence of contemporary investigations on the prenatal development of the human visceral pleura as a complex structure (mesothelium, basal lamina and submesothelial connective tissue layer).

Material and methods Our study involved lungs from 28 human stillborn fetuses without lung disease, or from fetuses obtained from abortions, aged 9 to 36 GW, of both sexes. They were divided into the following groups: 4 aged 9 - 10 GW, 6 aged 11 - 13 GW, 5 aged 14 - 20 GW, 5 aged 21 - 24 GW, 5 aged 25 - 32 GW and 3 aged 33 - 36 Gw. Samples from both lungs were taken from different parts of the visceral pleura together with the underlying lung tissue. The strips (1 xl x4 mm), oriented parallel to the pleural surface, underwent initial immersion fixation in 1070 or 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 hour. Subsequent fixation was in 1% OS04 in the same buffer. After dehydration in a graded series of ethanol of increasing concentration, the samples were embedded in Durcupan AMC (Fluka). The semithin sections were stained with

Fig. 1. 9-10 Gw. a) Mesothelial cells with microvillous evaginations of various sizes and shapes in the apical membrane (empty arrows). Undulated course of the basal membrane (curved arrows). Fine collagen fibres (arrow heads) and a cell in the submesothelial layer. x 7 000. b) Electron-lucent (L) and electron-dense (D) mesothelial cells with glycogen accumulations (asterisks). Fine basal lamina (curved arrows). x 8 500; c) Interdigitations (arrows) between the x 35 000. mesothelial cells. d) Mesenchymal cells with large intercellular spaces in the deep zone of the submesotheliallayer. Lumen of a blood capillary (Ca). x6000.

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1% pyronin. The thin sections were comrastcd wit h uranyl acetate and lead citrate, and examined with a Hitachi U-SOO electron microscope.

Results The visceral pleura of the fetuses aged 9 -10 GW was represented by a mesothelium, basal lamina (BL) and submesothelial mesenchymal layer (Fig. 1 a). The mesothelial cells formed a monolayer, with sectors of stratified arrangements of two or three cell rows. The cells were elongated and of two different types of electron density: light and dark (Fig. 1b). The apical plasmalemma ran an unfolded course with occasional short microvillous evaginations. The basal membrane followed an undulating course. The large elongated nucleus with single folds was centrally located and contained a small amount of heterochromatin, attached to the nucleolemma. The cytoplasm was poor in organelles. There were single vesicles of various sizes and content in the cytoplasm. Specific for this stage were

glycogen accumulations, lipid droplets and single electrondense granules. Between the mesothelial cells, simple interdigitations with apical zonula occludence-like contacts were found (Fig. 1c). Mesothelial BL was distinguished by a superficial electron-lucent layer and a deep electron-dense region. The latter was partly broken-up, with thinner regions and a finely granulo-filamentous structure. The thick sub mesothelial layer was composed of mesenchymal cells (Fig. 1 d). Numerous cytoplasmic processes from these cells formed large intercellular spaces with scattered short, thin collagen fibres. The single blood vessels and distal airways were few in number and situated far from the mesothelium. At 11- 13 GW, large regions of the visceral pleura had a mesothelial covering of high (cubical) and flat cells (Fig. 2 a, b). The central (nuclear) portions of the first cell type protruded towards the pleural cavity, or occupied the invaginations of the submesotheliallayer (Fig. 2c). Most of the high cells had long cytoplasmic processes. The remaining cells formed densely packed groups. The nucleolemma of the large, rounded nucleus formed single, shallow invaginations, while the nucleus of the flat cells were elongated. The organelle content of both cell types was poor. Small collagen

Fig. 2. 11 - 13 GW. a) Nuclear portions of the high mesothelial cells (M) protruding towards the pleural cavity. Large nuclei with folded nucleolemma. Vesicles in the apical cytoplasm (arrow). Poor organelle content. Collagen fibres under the basal lamina. x 8 000. b) The nuclear portions of the mesothelial cells (M) occupy the invaginations of the submesothelial layer. x 5 000. c) Flat mesothelial cell with elongated nucleus. The apical membrane forms rare, short microvillous evaginations (empty arrows). x 7 500. d) Larger collagen bundles (asterisks) surround fibroblasts in the sub mesothelial layer. x 7 000.

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and the cytoplasm showed greater electron density in both cell types (Fig. 4 a, b). The electron-dense part of the BL was wider than at the previous stage. The arteries of the submesothelial layer had two or more layers of smooth myocytes in their tunica media. They built a mesh throughout the pleura, accompanied by lymph vessels. The number of superficial blood capillaries in the submesothelial layer had increased, and they were smaller in size (Fig. 4c). They had a thick, continuous endothelium, with numerous microvillous evaginations of the apical membrane, and electron-dense bodies in the cytoplasm. The endothelial cells were located on a thin, interrupted BL. An interesting finding was the large lymph capillaries with lightlystaining luminal contents in the submesothelial layer (Fig.4d). They could be seen in the vicinity of the mesothelial BL. Their endothelium was extremely thin, interrupted and poor in organelles, and formed large evaginations, resembling endothelial valves. The endothelium had no BL. At 25 - 32 GW, the number of microvesicles had increased and they formed complicated groups in the mesothelial cells (Fig. 5 a). Most of the microvesicles were in contact with apical and basal cell membranes. The microvilli were more numerous and longer, as compared to the previous stage, especially in the peripheral zones of the cells (Fig. 5 b). The interdigitations ran a complicated course, with zonulae occludentes located apically, followed by zonulae adhaerentes. In some places large intercellular dilatations were visible. They showed specialised contacts only apically. Cytofilaments forming bundles could be identified. They

bundles formed the fine layer under the BL. Most of the submesothelial cells were elongated and formed a thick layer with few blood vessels (Fig. 2 d). They surrounded smaller intercelullar spaces, compared to those of the previous stage. The number and size of the collagen fibres had increased and they formed bundles. At 14 - 20 GW, the shape and arrangement of the mesothelial cells was still the same. Microvesicles predominated over other vesicular structures. The microvilli were more numerous than at the previous stage, but other forms of apical evaginations occurred still more frequently. Single high cells possessed particularly rich microvillous coverings (Fig. 3 a). Single mesothelial cells had cil ia in the vicinity of the nucleus. The interdigitations ran a more complicated course and short adherent-type contact s wert' scattered along them (Fig. 3 b). The BL was thicker, forming single evaginations in the submesothelial layer. Fine collagen bundles retained the same kind of arrangement under the BL. The submesothelial connective tissue layer in fetuses of over 17 GW could be divided into three zones: I. sparse fibroblasts, large intercellular spaces and fine collagen bundles; 2. densely packed fibroblasts, single extravascular cells, smaller intercellular spaces and larger collagen bundles, and 3. extravascular celis, fibroblasts, small intercellular spaces, fine collagen bundles and numerous blood capillaries (Fig. 3 c). At 21- 24 GW, the high mesothelial cells covered large areas, while the flat cells were single or formed small groups. The content of the ribosomes, polysomes and cysternae of the rough endoplasmic reticulum was increased

Fig. 3. 14-20GW. a) High mesothelial cell with nu-

merous microvilli (arrows). Fine basal lamina (arrow heads). x6000. b) Zonula occludence (small arrows) in the apical part of the intercellular spaces. Adherent types of contacts (large arrows). Group of microvesicles (asterisk). x 40000. c) On the right sidesuperficial zone with large intercellular spaces and small collagen bundles, and on the left side - dense network of fibroblasts and larger collagen bundles. x3500.

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were located free in the cytoplasm or we re II , dose contact with the apical and the intercellular membranes (Fig. 5 c). The elastic-like fibres with a microfibrilla r periphery and an amorphous centre could be seen under I he RL . The thick submesothelial connective tissue layer was composed of fibroblasts, a few macro phages and extravascular cells, large collagen bundles, numerous blood capillaries and a few large lymph vessels (Fig. 5 d). In some places I his layer continued to run directly into the con nective tissue of the lung interstitium. At 33 - 36 OW, the high cells were the predominant cell type in the visceral pleura, while the nat cells were less numerous (Fig.6a, b). The multiple-layered arrangement mesothelium was not visible. Both cell types had more numerous and longer microvilli compared to the previous stage. The organelle content (especiall y the Golgi complexes and cisternae of the granular endopla,mic reticulum), microvillous covering and vesicular system, appeared to be richer in the high cells as compared [ 0 the nat cells. The BL was thicker and for long sections oniy its electron-dense region could be identified. Th e frequentl y interrupted elastic

membrane could be observed under the BL, together with single isolated short thin elastic fibres in the underlying connective tissue. The submesothelial layer generally remained markedly thick, but occasionally it was extremely thin, and in these zones the l'f' ripheral alveoli were in close contact with the mesothelium BL (Fig. 6c).

Discussion The main components (mesothelium, basal lamina and submesothelial connective tissue layer) of the visceral pleura are formed in the fetal period, but they do not attain their final differentiation. It is well known that the lung and the chest remain immature during prenatal life (Davies and Reid 1970; Krause and Leeson 1975; Scheuermann and DeOroodt-Lasseel 1981; Reid 1984). The development processes of the different pleural areas proceed asynchronously, which could be explained with similar changes in the lung. The processes of differentiation during fetal stages advance

Fig. 4. 21-24GW. a) The central part of the high mesothelial cell follows the shape of the nucleus (N). x 7 000. b) Flat mesothelial cell with elongated nucleus (N). x 5 700. c) Endothelial cells (E) of a blood capillary of the submesothelial layer. x 5 000. d) Lumen of a lymph vessel (Lv) with thin endothelium (E) in the vicinity of the mesothelial layer (M). x 5 000.

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Fig. 5. 25-32GW. a) Group of microvesicles with dif-

ferent content (asterisk) in the mesothelium. x24000. b) Microvilli in the vicinity of the intercellular contacts (small arrows). Single cilium (large arrow). x 15000. c) Numerous microvilli. Cytofillamentous bundles (asterisks) in the mesothelium. x 10000. d) Mesothelial cells (M). Large collagen bundles (C) in the thick submesotheliallayer. x 4 000.

Fig. 6. 33 - 36 Gw. mesothelial cell with lobulated nucleus, microvilli, microvesicles in apical and basal cytoplasm (asterisks). Undulated electron-dense basal lamina with thickened parts (arrows). Elastic fibres (EF). x 10000. b) Flat mesothelial cell with rare microvilli and elongated nucleus. Collagen bundles (C) in the submesothelial layer. x 6 500. c) Elastic membrane (arrows). Alveolus (A) and blood capillary (Ca) in the vicinity of the mesothelium. x4500. a) High

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of the apical and basal membranes, microvilli, vesicular system, and intercellular contacts) ensure mesothelial transport. The transcellular pathway appears later than the intercellular. The development of the cytoskeleton at the mesothelial cells in the late fetal stage suggests an additional mechanism of intercellular transport (DiBona and Schafer 1984). Our findings on the glycogen accumulations, lipid droplets and electron-dense granules in the mesothelium from 9 - 10 OW suggest a different manner of formation of some products of these cells in comparison with the adult mesothelial cells. On the other hand, Whitaker et aJ. (1982) have observed large amounts of glycogen in the mesothelium between the 25th and 37th day, and its amount is reduced to only small deposits towards term. According to Whitaker et al. (1982), mesothelial development is unrelated to vasculogenesis. Vessels are practically absent in the submesothelial connective tissue layer during the early stage. Thus, the present data suggest that the mesothelial cells remain the sole source of the secretion that lubricates the organ surfaces at this stage. The further mesothelial changes (marked in the high cells) may be connected with the relatively large nucleus, increase of the cisternae of the granular endoplasmic reticulum, the Golgi apparatus and secretory granules representing the morphological substrate for the secretion potential of the mesothelium for proteoglycans (Simionescu et aJ. 1988), for cytoskeletal proteins (Wu et al. 1982), for fibrinolytic factors (Raftery 1981), for some submesothelial extracellular components (Rennard et al. 1984; Davila and Crouch 1993), and for some products of the basement membranes (Haidar et al. 1990). The secretion capability mentioned above, the results from cell cultures studies (LaRocca and Rheinwald 1984; Rheinwald et al. 1984), the present data on the position of the BL, as well as its changes parallel to the mesothelial cells indicate the mesothelial origin of the BL. Obata (1978) explained the differences in the BL in both pleural layers by variations in the mesothelial covering and the submesothelial connective tissue. Our study (Michailova 1993) on the pleura after experimental haemothorax shows changes in the BL which follow those in the mesothelium. The fibroblasts and collagen fibres at the late stage form a thick submesothelial layer. Jackson et al. (1990) explain the greater rate of collagen synthesis in prenatal life as compared to the adult by faster lung growth during gestation. At the end of fetal life the submesotheliallayer becomes thinner in some areas. The thinning of the connective tissue may be explained by the rapid penetration into the submesothelial layer of respiratory saccules by transitional ducts (Perelman et al. 1981). On the other hand, according to Duncker (1990), in human there is no saccular phase, and from the beginning of the peripheral differentiation there are alveoli, respectively - bronchioli alveolares. The elastic fibres, as well as the elastic membrane under the BL, appear during the late period of fetal life. The amount of elasticity in the pleura increases continuously after 25 OW, parallel to its development. Our findings are in agreement with the electron microscopic study of fetal lung by Collet and Des Biens (1974) showing the appearance of the elastic fibres during

from the centre to the periphery of the lung for the bronchus tree whereas the alveolar differentiation follows a distalproximal sequence and is asynchronous between the lobes (Kotas et al. 1977; Zeltner et al. 1990). Burri's classification of the stages of prenatal lung development. (1984) could be a model for staging the fetal differentiation of the pleura: early stage (until 17 OW), and late stage - up to birth. We have divided the pleural components into two basic groups: first - mesothelium with BL, which undergoes epithelial development, and second submesothelial mesenchyme, which differentiates as a connective tissue layer. The mesothelial changes start early in gestation and continue throughout the entire period investigated. The submesothelial connective tissue layer differentiates predominantly during the second half of fetal development (after 17 OW), while the elastic membrane develops after the 25 OW of prenatal life. The submesothelial connective tissue layer and the subalveolar connective tissue follow a different path of differentiation. The results of the present study show that the differentiation of the mesothelium appears early, whereas the development of the sub mesothelial connective tissue occurs later. On the other hand, Fukuda et al. (1983) demonstrated a relatively late differentiation of the lung epithelium, although the septal subalveolar connective tissue develops early. Our findings of the appearance of the high after the flat mesothelial cells (after 11 OW), a greater number of high cells at the late stage, intimate contact between the two cell types, their different organelle content, microvillous covering, and vesicular system, show that the existence of high and flat cells is already evident at the fetal stage. Fetal "breathing" begins after 20 OW (Perelman er al. 1981), i. e. significantly later than the first appearance of the high mesothelial cells. Thus, it seems unlikely that the smooth contact of the two pleural sheets has some influence in regard to the differentiation of the pleural mesothelium. The high cell type, as a basic type of the visceral pleura, is not an artificial result of the moving lung (Dodson et al. 1983). The primary mesothelial cells withinterdigitations and undulating apical and basal membran es form a complicated layer, which fulfils the initial barrier function of the pleura. Multi-layered sectors were also observed by us (Michailova and Wassilev 1991 a) at the basis of the fetal rat lung. Whitaker et al. (1982) described the double cell layer of the visceral pleura of the 4 112 GW embryo as a rare, interesting finding. Another function of the mesothelial cells is transport. The morphology of the intercellular spaces suggests that permeability starts in them . Suzuki and Nagano (1979) define three types of right junction in the mesothelium of the mouse embryo which are permeable. The vesicular system and the microvilli , as a morphological substrate for transcellular transport , increase rapidly, and changes in their distribution in the m e~othelial cells occur during the late fetal stage. Ukeshima et al. (1986) demonstrated a parallel increase only in the microvilli and microvesicles. Our results show that the furrows and ridges of the pleural surface, the undulations between the cells and the membrane specializations (evagin ation and invagination 97

Burri PH, Weibel ER (1977) Ultrastructure and morphometry of the developing lung. In: Hodson WA (ed) Development of the Lung. Lung Biology in Health and Disease, Vo!' 6. Marcel Dekker Inc., New York, pp215-268 Collet Al, Des Biens G (1974) Fine structure of myogenesis and elastogenesis in the developing rat lung. Anat Rec 179: 343 - 360 Cossar D, Bell 1, Lang M, Heune R (1993) Development of human fetal lung in organ culture compared with in utero ontogeny. Vitro Cell Dev Bioi Anim 29: 319-324 Davies G, Reid L (1970) Growth of the alveoli and pulmonary arteries in childhood. Thorax 25: 669-681 Davila RM, Crouch EC (1993) Role of mesothelial and submesothelial stromal cells in matrix remodeling following pleural injury. Am 1 Pathol 142: 547 - 555 DiBona DR, Schafer lA (1984) Cellular transport phenomena. In: Staub NC, Taylor AE (eds.) Edema. Raven Press, New York, pp61-80 Dodson RF, O'Sullivan MF, Corn Cl, Ford 10, Hurst GA (1983) The influence of inflation levels of the lung on the morphology of the visceral pleura. Cytobios 37: 171-179 Duncker HR (1990) Respirationstrakt. In: Hinrichsen KV (ed) Humanembryologie, Springer, Berlin, pp 571 -606 Emery lL (1970) The postnatal development of the human lung and its implications for lung pathology. Respiration 27: Supp!. 41 -50 Fierer lA (1976) Ultrastructural studies of developing pulmonary alveolar septum. Adv Exp Med Bioi 79: 31-37 Fukuda Y, Ferrans Vl, Crystal RG (1983) The development of alveolar septa in fetal sheep lung. An ultrastructural and immunohistochemical study. Am 1 Anat 167: 405 -439 Gnepp DR (1984) Lymphatics. In: Staub NC, Taylor AE (eds) Edema. Raven Press, New York, pp 263 - 298 Haidar A, Wigglesworth lS, Krausz T (1990) Type IV collagen in developing human lung: a comparison between normal and hypoplastic fetal lungs. Early Human Dev 21: 175 - 180 Hasseldahl H, Larsen lH (1969) Ultrastructure of human yolk sac: Endoderm, mesenchym, tubules and mesothelium. Am 1 Anat 126: 315 - 326 Hislop A, Howard S, Fairweather DVI (1984) Morphometric studies on the structural development of the lung in Macaca fascicularis during fetal and postnatal life. 1 Anat 138: 95 -112 Hoyes AD (1969) The human yolk sac: An ultrastructural study of four specimens. Z Zellforsch 99: 469 -490 I van ova VF (1975) Embryonic and post-embryonic development of the parietal and visceral peritoneum in white mice. Arkh Anat Gistol Embriol 68: 45 - 53 Jackson lC, Clark lG, Standaert TA, Truog WE, Murphy lH, Juul SE, Chi EY, Hodson WA (1990) Collagen synthesis during lung development and during hyaline membrane disease in the nonhuman primate. Am Rev Resp Dis 141: 846-853 lones AW, Barson AS (1971) E1astogenesis in the developing chick lung: a light and electron microscopical study. 1 Anat 110: 1 -15 King BF, Wilson 1M (1983) A fine structural and cytochemical study of the rhesus monkey yolk sac: Endoderm and mesothelium. Anat Rec 205: 143 -158 Kitterman JA (1984) Fetal lung development. 1 Develop Physiol 6: 67-82 Kotas RV, Farrell PM, Ulane RE, Chez RA (1977) Fetal rhesus monkey lung development: Lobar differences and discordance between stability and distensibility. 1 Appl Physiol 43: 92-98 Krause Wl, Leeson CR (1975) Postnatal development of the respiratory system of the opossum. Acta Anat 92: 28 -44 Langman] (1975) Medical Embryology. Human Development -

the saccular stage, or in areas corresponding to the primitive alveolar zones according to a recent immunohistochemical investigation by Cossar et al. (1993). On the other hand, several observers describe the formation of the elastic fibres in earlier glandular (Shibahara et al. 1981) or canalicular (Fierer 1976) stages of the developing lung. Our previous study (Michailova and Wassilev 1991 b) on rat pleura supports the results of Krause and Leeson (1975), in which the elastic membrane differentiates after birtll. These different types of development may be related to a )pecies difference. Recently, special attention has been paid to the mechanical role of the pleura (Oldmixon and Hoppin 1984) played by the subpleural elastic fibres which persist m the pulmonary interstitium. A strong "elastic sac" part icularly important for the breathing lung, is formed (Liggim et aL ! 981). We have not found that the fibroblasts close t(l the mesothelium resemble myofibroblasts or smooth muscle ceUs. The latter become associated with several types of extracellular com· ponents in the connective tissue and are especially important for the process of elastogenesis (Collet an d Des Biens 1974; Fukuda et al. 1983). The process of blood vascularization in 1he visceral pleura follows the same process as in the lung. The invasion of capillaries into the terminal airways of the developing lung starts at the canalicular stage (Burri and Weibel 1977), and according to Perelman et al. (1981), the capillaries penetrate the terminal spaces. Our data show that thc blood vessels enter the submesothelial layer of the pleura from the lung after 24 GW, and appear earlier than the lymph vessels. The present results demonstrate the thick-walled pleural blood vessels, suggesting that the blood supply of the pleura is provided by the systemic (bronchial), rather than by the pulmonary arteries (see also Agostoni 1972; Nagalshi 1972; Albertine et al. 1982). The fetuses invesugated show an extensive lymphatic network in the submesothelial layer of all areas. The lymph vessels are often associated with blood vessels. The ultrastructure of the human pleural lymph vessels appears to be uniform, as has already been found in other body regions and in different species (Leak 1980; Gnepp 1984). The fetal pleura seems to be moderately thick and extends into deeper interlobular connective (jssue septae. The latter feature, as well as I he presence of blood vessels with a thick endothelium and a rIch lymphatic network in the submesothelial layer, slIgge'-fS 1hat (he visceral human pleura is formed as a "thick type" 10 the prenatal period, and is not linked with final lung differentiation.

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