The specificity of the epithelial mesenchyme inductive relations in the morphogenetic synergisms algorithm inside branched tube-alveolar and

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1 Rom J Leg Med [18] [2010] DOI: /rjlm Romanian Society of Legal Medicine The specificity of the epithelial mesenchyme inductive relations in the morphogenetic synergisms algorithm inside branched tube-alveolar and tube-acinar systems Gheorghe S. Drăgoi *, Petru Răzvan Melinte, Mihaela Tudosie, Ileana Dincă Abstract: The authors have made a micro anatomic analysis of the phenotype transformations determined by the epithelial mesenchyme relations at the level of bronchopulmonary and salivary systems in order to evaluate the mechanisms of the architectural genesis of the tube-alveolar and tube-acinar structures during general and forensic ortology and pathology. Key words: Mesenchyme, Epithelial mesenchyme inductive relations, Tube-alveolar and tube-acinar structures T he genesis and 3D evolution of the dichotomic branching structures are of interest for the fundamental as well as for the applicative research. The study of the epithelial type branching models that is characteristic to some organs remains a partially explored field [1]. We selected two anatomic and functional systems for the study of the epithelial mesenchyme inductive interactions: the bronchopulmonary and the salivary glands systems. Two phenomena brought our attention during the ontogenesis of those two systems: first of all, the particularities of the morphogenesis and differentiation processes that involved the tube-like structures, and second, the determinant mechanism that leads to functional specialized structures the blood air barrier inside the pulmonary respiration system and the glandular acinus inside the salivary glands secretion system. During the morphogenesis processes, an important role is played by the mesenchyme that is responsible for the regional specificity of induction inside competent structures [2, 3]. In the algorithm of the pulmonary respiration system morphogenesis, this specificity is spectacular [4]. Structural heterogeneity of organs containing dichotomized branching elements leads to problems in understanding their architectural mechanisms of ontogenesis on one side, as well as of evaluating the evolution status in normal and pathologic conditions on the other side, such as: What are the elements or structures that interact in the algorithm of morphogenetic synergisms? What are the rules to ratify the stages of formation for the branched structures? What are the consequences of the epithelial mesenchyme inductive relations? What are the mechanisms for epithelial mesenchyme inductive relations? How is it possible, the growing and dichotomy branching of the epithelial buds inside the tubealveolar and tube-acinary systems? What are the means of inductive interactions? * Corresponding author, Professor MD, PhD, member of the Romanian Academy of Medical Sciences, Academic Hospital no. 2, Craiova, Romania, dragoigs@yahoo.com 171

2 Dragoi GS et al Morphogenetic synergisms inside branched tube-alveolar and tube-acinar systems What are the determinant factors of the mesenchyme phenotype transformations during the achievement of functional sectors: blood air barrier inside lungs and secreting acinus inside salivary glands? What is the role of the under epithelial basal membrane at the epithelial mesenchyme border? What is the practical value for the knowledge of the specific tissue interrelations during the branched structures morphogenesis? What are the structure patterns for the derived elements from pulmonary and salivary glands mesenchyme? Materials and methods Our research was conducted on tissue fragments obtained from human embryos of 5 months old (7 cases) and 6 months old (8 cases) as well as on serrial sections through four week embryos (3 cases) and 6 weeks embyos (4 cases), from the Pathology Laboratory of the Clinic and City Hospital Filantropia from Craiova during Biological samples were processed by means of classical histological methods and the imagistic findings have been digitally computed. Results We evaluated micro anatomic the phenol type changes determined by the inductive epithelial mesenchyme interrelations during the genesis and evolution of the primordial epithelial buds and of the branched structures derived from those buds inside the respiration pulmonary and salivary glands systems. A. The micro anatomic evaluation of the inductive epithelial mesenchymeinterrelations that determine the genesis and evolution of the primordial epithelial buds 1. In the 4 weeks human embryo pulmonary system, we identified epithelial aggregates formed inside pulmonary mesenchyme. When examining at the x63 objective we noticed those aggregates of epithelial cells with hyper chromatic nuclei that are sometimes mitotic (Fig.1, A and B). On the serried sections through lungs fragments from a 16 weeks fetus, we identified terminal bronchi surrounded by hyper cellular mesenchyme (Fig. 1, C). When examining the wall of the terminal bronchi with the x 20 objective, we observed prismatic cells with hyper chromatic nuclei and caliciform cells. At the periphery of the terminal bronchi one can easily identify cells with a fusiform nucleus, located at the border of the adjacent pulmonary mesenchyme (Fig. 1, D). Newly formed blood vessels exist inside the pulmonary mesenchyme surrounding the terminal bronchi (Fig. 1, E). 2. On the serried sections through the salivary glands system of a 6 weeks embryo we pointed paralingual mesenchyme aggregates containing endoderm epithelial buds that will form the submandibular gland parenchyma. One can easily notice the dichotomized branching of the endoderm buds and their progression to the adjacent mesenchyme (Fig. 3, A). On the Hematoxiline eosin stained sections, the endoderm buds appear as epithelial cells with hyper chromatic and sometimes mitotic nucleus (Fig. 3, B). On the Gomori argentic impregnation sections there is a slight differentiation of the basal membrane at the periphery of the epithelial buds (Fig. 3, C). The gland acini have various shapes depending on the sectioning planes, on the serried sections through a submandibular gland of a 6 months fetus. Some acini form a lobe that is delimited by mesenchyme bands from the neighboring structures (Fig. 3, D). Inside the lobe, the acini are surrounded by highly vascularized mesenchyme (Fig 3, D). When examining at the 20x objective, we identified inside the gland acini some cubic cells that have contiguity relations to the myoepithelial cells at the border with the adjacent mesenchyme (Fig. 5, E). The intermediate and intralobular ducts ensure the continuity of the acini lumen to the extra lobular tube excretory system (Fig. 3, F). The reticulin fibers appears well differentiated at the level of the 172

3 Romanian Journal of Legal Medicine Vol. XVIII, No 3 (2010) basal membrane underneath the intralobular duct epithelium, as seen in the Gomori argentic impregnation sections (Fig. 3, G) A B C D 7 6 E 7 8 Figure 1. Phenotype transformations inside pulmonary mesenchyme during the pseudo glandular stage at a four weeks embryo. 1. Pulmonary mesenchyme; 2. Epithelial aggregate inside pulmonary mesenchyme; 3. Epithelial like cells inside mesenchyme epithelial aggregate; 4. Transverse section through terminal bronchi; 5. Oblique section through terminal bronchiae; 6. Epithelial and caliciforme cells inside terminal bronchiae wall; 7. Epithelial mesenchyme border; 8. Medium artery with nucleated red cells inside the mesenchyme surrounding the terminal bronchiae. Hematoxiline Eosine Stain, x 70 (A, C); x 140 (D), X 280 (E), x 441 (B) 173

4 Dragoi GS et al Morphogenetic synergisms inside branched tube-alveolar and tube-acinar systems Figure 2. The morphogenesis of the bronchial tree and pulmonary parenchyma in a 7 weeks embryo (A) and a 5 month fetus (B-F, H). Capillary network in adult (G). 1. Trachea; 2. Tracheal bifurcation; 3. Right main bronchi; 4. Left main bronchi; 5. Respiratory bronchi; 6. Alveolar canal; 7. Atrium alveolaris; 8. Sacus alveolaris; 9. Mesemchyme crest; 10. The epithelium of the alveolar canal; 11. The epithelium of the sacus alveolaris; 12. Blood vessel inside the sacus alveolaris wall; 13. Vascular network; 14. Lymph node inside pulmonary parenchyma. Cajal Stain (A); Hematoxilin Eosine Stain (B-F); China Ink (G), x 28 (A-C), x 140 (C), x 280 (D-G). 174

5 Romanian Journal of Legal Medicine Vol. XVIII, No 3 (2010) Figure 3. Inductive interactions during the formation and evolution of the submandibular salivary gland in a 6 weeks embryo (A-C) and 6 months fetus (D-G). 1. Epithelial bud; 2. Primordial mesenchyme of the submandibular gland; 3. Reticulin fibers inside epithelial bud; 4. Basal membrane beneath endothelium; 5. Interlobular duct; 6. Glandular acinus; 7. Blood vessel; 8. Acinus; 9. Intermediate duct; 10. Intralobular duct; 11. Basal membrane beneath epithelium. Gömöri Stain (A; C; G); Hematoxilin Eosine Stain (B; D-F), x 28 (A); x 70 (D); x 140 (E-G); x 280 (B,C) 175

6 Dragoi GS et al Morphogenetic synergisms inside branched tube-alveolar and tube-acinar systems Figure 4. The differentiation of the vascular networks inside the salivary gland tube-acinar system at a 5 months fetus. 1. Acinus; 2. Intermediate duct; 3. Intralobular vascular network; 4. Mesenchyme; 5. Blood vessels around salivary ducts; 6 and 7. Vascular network around acinus; 8. Lymph node inside pulmonary parenchyma; 9. Glandular acinus inside lymph node; 10. Interlobular duct; 11. Periphery cellular layer; 12. Central cellular layer. Hematoxilin Eosine Stain, x 70 (A; E); x 140 (B-D); x 280 (F). B. The micro anatomic analysis of the branching structures differentiation by morphogenesis synergisms 1. On frontal section through the cervical thoracic region of a 6 weeks embryo we identified inside the pulmonary respiration system some components of the tracheobronchial tree, such as: tracheae, right and left 176

7 Romanian Journal of Legal Medicine Vol. XVIII, No 3 (2010) main bronchi and lobular bronchi (Fig. 2, A). Pulmonary acinus tributary to terminal bronchi can be observed on the serried sections through the lungs of a 5 months fetus (Fig. 2, B). Structures belonging to the pulmonary parenchyma ductus alveolaris, atrium alveolaris and sacus alveolaris are visible at the 10x objective, and they appear in the immediate neighborhood of the pulmonary adjacent mesenchyme (Fig. 2, C). After examining the hematoxiline eosin stained sections with the x40 objective, we identified the phenol type transformations of the pulmonary mesenchyme around acinus that contribute to the processes of alveolus genesis, angiogenesis and remodeling of the space adjacent to the sacus alveolaris (Fig. 2, E and F). On adult lung injected with China ink one can easily see a luxuriant capillary network around alveolus (Fig. 2, G). In the interlobular stroma there are lymph node formations visible in the lungs of 8 month fetus (Fig. 2, H). 2. In the salivary glands system we noted the differentiation of the acinus structures especially of its terminal section and of the vascular network around acinus groups and at the periphery of the intralobular and intermediate ducts; these findings are specifically seen on submandibular gland fragments harvested from a 5 months fetus (Fig. 4, D). The epithelium has a double layer and there is a differentiation of the peripheral myoepithelial cells at the level of the interlobular ducts wall (Fig. 4, E and F). Discussions The micro-anatomic analysis of the genesis and evolution of the human branching structures allows us to evaluate the epithelium mesenchyme interactions, the morphogen synergisms that play a major role in its differentiation and evolution, as well as the effects of these changes in the ontogenesis of the dichotomized branching systems. The interactions between epithelium and mesenchyme are well known in the genesis of organs containing tubular structures such as kidney, lung, pancreas, salivary and mammal gland; in their genesis is described the processes of reciprocal induction of epithelium by mesenchyme, and of mesenchyme by epithelium [5]. We raised ourselves the question whether itis possible to appear changes of the epithelial cells in lungs due to the interaction of the bronchi buds to pulmonary mesenchyme; after this event, some mesenchyme cells organize themselves as epithelium. This phenomenon is quite obvious in kidney development where two tissues derived from mesoderm interact urethral bud and metaneprogenic mesenchyme. The latter determines the prolonging and branching of the urethral bud. It is well proven that the extremities of the urethral bud induce the mesenchyme cells into producing an epithelial aggregate [6]. On the other hand, the metanephrogenic mesenchyme is capable to respond to the urethral bud induction by forming renal tubes. One has observed that the metanephrogenic mesenchyme induction by other epithelium such as from salivary gland or neural tube will lead to the formation of renal tubes and not other structures [7,8]. Based on that observation, Etheridge [9], hypothesized that induction results after the interaction with the endoderm that is closer on the development scale. During the morphogenesis of all organs, connections are established between the tissues that interact. In the interactions between epithelium and mesenchyme, the mesenchyme influences the epithelium and the modified epithelial tissue secretes factors that can modify the mesenchyme [1]. The identification of those factors that intervene in the inter tissue communication has brought attention to many research laboratories [1]. Many researchers consider that mesenchyme controls the specificity of the epithelial structures branching. Saxen [8] pointed out that inside the kidney there is a single type of mesenchyme that can impose that branching. In salivary glands, the mesenchyme determines the branching pattern while the epithelium differentiation is imposed automatically by the epithelium itself [10,11]. In respiratory and digestive systems the regional mesenchyme determine ramification models and the protein types that intervenes in each region [4, 12-14]. The extra cellular matrix has a major role in the ramification process. Bernfield et al (1984) [15] and Mizuno and Yasugi (1970) [16] hypothesized that the selective degrading of the underneath epithelial basal membrane is important for the branching mechanism. The ramification of the epithelial buds is dependent on the presence of mesenchyme. The mesenchyme can interact to a epithelial tube to provoke its branching by inducing fissures that generate lobules on each side of the fissure. The control of the fissure formation is conditioned by the presence of collagen. It is yet unclear the mechanism by which the collagen intervenes. It has been proven that during the mesenchyme induction that leads to the formation of epithelial branching models, it is TGF-1 protein and activin that intervene. TGF-1 favors protein synthesis inside extra cellular matrix and inhibits metal-proteinases that are capable to digest the extra cellular matrix [18,19]. The activine elaborated by the salivary glands disturbs the normal ramification model [5,20]. In lungs, the mesenchyme is inductive to the endoderm bud growing [21]. The initiation of pulmonary growth is determined by the sysnthesis of the FGF-10 by the mesenchyme cells that soon acts upon III B isoform of the FGF R2 receptor present on the endoderm cells surface. 177

8 Dragoi GS et al Morphogenetic synergisms inside branched tube-alveolar and tube-acinar systems The antepartum development of lungs is particular to other organs. Although the lungs are not necessary as respiratory organs before birth, it must be developed in order to be functional at the time of parturition. There have been described four stages in the genesis and evolution of the lung structural and functional elements. During each stage, elements of the bronchial tree and pulmonary parenchyma are formed. The mesenchyme exerts three major functions in the tube alveolar pulmonary system: it inducts the growing and differentiation of the endoderm branched canals system up to the terminal bronchi; it generates the vascular networks, bio dynamic structures (smooth muscles) and cartilage structures; and finally it is implicated in the genesis of the respiratory changes sector that includes alveolar ducts, sacus alveolaris and pulmonary alveolae. In our opinion, the structural characteristics of the pulmonary parenchyma during its ontogenesis dynamics and the immaturity of the gas exchange structures at the time of parturition, raise the problem of pulmonary mesenchyme competence for the genesis and differentiation of the pulmonary alveoli under the inductive action of endoderm derivates. Conclusions The bronchial tree with a competent aerial transport function is formed after a complex tube genesis process from the endoderm material in the anterior intestine under the inductor action of the pulmonary mesenchyme. The pulmonary parenchyma specialized for aerial exchange derives from the pulmonary mesenchyme that undergoes mesenchyme and epithelial phenotype changes, remaniation of the newly formed structures, vasculogenesis and time and space remodeling. References 1. Gilbert SF, Ritvos O, Vaahtokari A, Eramaa M and Saxen L., Effects of activin and TGF-beta 1 on epithelial branching in developing murine organs. J. Cell.Biol., 1991, 115: Saunders JW Jr, Cairns JM, Gasseling M.T, The rol of the apical ectodermal ridge of ectoderm in the differentiation of the morphological structure of and inductive specificity of limb parts of the chick. J Morphol. 1957, 101: Saunders JW Jr. Developmental biology. Macmillan, 1980, New York. 4. Wessels NK. Mammalian lung development: Interactions in formulation and morphogenesis of tracheal buds. J Exp Zool. 1970, 175: Gilbert SF. Biologie du development. Se Boeck & Larcies, Paris, Bruxelles, Romanoff AL. The Avian Embryo. MacMillan, New York, Sariola H, Ekblom P, Saxen L. Restricted developmental options of the metanephric mesenchyme. In M. Burger and R. Weber (eds.) Embryonic Development Part B: Cellular Aspects. Allan R. Liss New York, 1982, Saxen LM, Sariola H. Early organogenesis of the kidney. Pediat Nephrolog 1987, 1: Etheridge AL. Determination of the mesonephric Kidney. J Exp Zool, 1968, 169: Lawson, KA. Mesenchyme specificity in rodent salivary gland development: the response of salivar epithelium to lung mesenchyme in vitro. J Embryol Exp Morphol. 1974, 32: Sakakura T, Nishizuka Y, Dawe, CJ. Mesenchyme dependent morphognesis and epithelium specific cytodifferentiation in mouse mammary gland. Science, 1976, 194: Cunha GR. Epithelial stromal interactions in the development of the uorgenital tract. Int. Rev. Cytol. 1976, 47, Hilfer SR, Rayner RM, Brown JW. Mesenchymal control of branching pattern in the fetal mouse lung. Tiss Cell. 1985, 17: Haffen K, Kedinger M, Simonassman P. Mesenchyme dependent differentiation of epithelial progenitor cells in the gut. J Pediat Gastroent. 1987, 6: Bernfield M, Banarjee SD, Koda JE, Rapraegar AC. Remodeling of basement membrane as a mechanism of morphogenic tissue interaction. In. R.L. Trelstad (ed), The role of Extracellular Matrix in Development. Alan R Liss. New York, 1984, Mizuno T, Yasugi S. Susceptibility of epithelia to directive influences of mesenchymes during organogenesis: Uncoupling of morphogenesis and cytodiffernetiations. Cell Differ Devel. 1990, 31: Penttinen RP, Kobayashi S, Bornstein P. Transforming growth factor beta increases m RNA for matrix proteins in the presence and in the absence in the changes in mm RNA stability. Proc Nat Acad Sci. 1988, 85: Saxen L. Failure to demonstrate tubule induction in heterologous mesenchyme. Dev Biol. 1970, 23: Nakamura T, Okuda S, Miller D, Ruoslahti E, Border W. Transforming growth factor beta (TGF-beta) regulates production of extracellular matrix (ECM) components by glomerular epithelial cells. Kidney Int (9) Ritvos O, Tuuri T, Eramaa M, Sainio K, Hilden K, Saxen L, Gilbert SF., Activin A disrupts epithelial branching in developing murine organs. Mech Dev. 1995, 50: Dameron F. L influence de divers mesenchymes sur la differenciation de l epithelium pulmonaire de l embryon de poulet en culture in vitro. J Embryol Exp Morph. 1961, 9: