Morphogenesis of intestinal villi. I. Scanning electron microscopy of the duodenal epithelium of the developing chick embryo

Development of villi in the duodenum of the chick was studied in stages ranging from 11 days of incubation to one week after hatching. Formation of definitive villi is preceded by development of a set of previllous ridges that run lengthwise along the duodenum. The first set of 16 previllous ridges (Set I) is complete by about 13 days of incubation; all ridges in the set are fairly uniform and proceed through their subsequent development in synchrony. Previllous ridges in Set I fold into a highly regular zigzag pattern between 14 and 16 days of incubation. Definitive villi develop from Set I ridges beginning at about 17 days when populations of distinct cells appear on the crests of the ridges between angles in the zigzag folds. Cells in these populations lack the rounded appearance of cells seen in earlier stages; their apical surfaces are densely covered with microvilli. A second set of villi (Set II) develops at about 16 days of incubation when about 16 rows of tongue-like flaps erupt between the previllous ridges of Set I. At hatching, Set II villi are still smaller than villi of Set I; this distinction disappears by about the fourth day after hatching. The significance of the morphological changes in epithelial cells is discussed in terms of several hypotheses bearing on the mechanisms of villus formation.     

The effects of exogenous cortisone acetate on development (especially skeletal development) and on circulating levels of corticosteroids in chick embryos.

Chick embryos were treated with cortisone acetate on day 8 of incubation and the subsequent growth of the whole embryo and the development of the tibia studied to day 18 of incubation. Cortisone, at 10 ng to 2 mg/embryo decreased general body growth; above 0.5 mg/embryo it also retarded morphogenesis by as much as 3 Hamilton-Hamburger stages; and above 1 mg/embryo gross abnormalities were produced. The growth and differentiation of the tibia were affected to a greater extent than was the whole body. The reductions in tibial and total body weight were not linearly related to dose of cortisone injected. The exogenous cortisone acetate resulted in drastic alterations in the circulating levels of cortisone, cortisol, corticosterone, and progesterone, but plasma progesterone level was most readily correlated with the growth retardation. This study emphasizes that avian embryos can readily compensate for exogenous corticosteroids and that caution must be exercised when attributing causality to the substance administered.     

Distributions of Actin, Vinculin and Fibronectin in the Duodenum of Developing Chick Embryos: Immunohistochemical Studies at the Light Microscope Level

Microscopic studies were made on the localizations of three different cytoskeletal proteins, actin, vinculin and fibronectin, in the duodenum of developing chick embryos and chicks by an indirect immuno-fluorescent staining method with specific antibodies.
Topographical changes in the distributions of these three proteins seemed to be related to stages in morphogenesis of the duodenum. In early stages of embryonic development, findings suggested interaction between actin and vinculin in the apical region of epithelial cells and between actin and fibronectin in the basal region of these cells. From this stage, vinculin and fibronectin seemed to be of importance in determination and continuity of the polarity of the duodenal epithelium, and in control of the intracellular arrangement of actin. This relation between actin and vinculin seemed to continue throughout embryogenesis.
The main role of actin in epithelial cells seemed to change on day 12 from that of forming constricting bundles for morphogenesis of previllous ridges to that of microfilaments in the microvilli and the terminal web.     

Studies on the histogenesis of the tunica mucosa of the small and large intestines of the camel (Camelus dromedarius). I. Histogenesis of the small intestinal mucosa

The histogenesis of the small intestinal mucosa passes through a previllous stage, comprising development until the formation of villi (at a C RL of about 15 cm), and a villous stage, comprising the further development. In the previllous stage, the mucosa consists of a Lamina epithelialis made up of undifferentiated cells with stratified nuclei and a Lamina propria/submucosa made up of homogeneous mesenchyme. The villi appear as luminal epithelial outgrowths associated with vascularized mesenchymal stalks and their growth advances in a cranio-caudal direction. The intestinal glands were firstly observed at the 26 cm-stage as downgrowths of the intervillous epithelium into the underlying mesenchyme.     

Various aspects of organogenesis of the colon in Gallus domesticus: morphologic observation

The authors have investigated the morphological aspects of the wall components of the developing colon in the chick embryo (Gallus domesticus) from the 7th to th 15th day of incubation. Particular attention has been given to the lumen recanalization, phenomenon which occurs also in other animal species. The most significant results can be summarized as follows: 1) the lumen is recanalized at the 7th day only at the proximal part of the colon (Fig. 1, Tav. 1), while at the distal tract it is still completely filled by an epithelial plug (Fig. 2, Tav. 1). Therefore the recanalization of the lumen takes place cranio-caudad. 2) At the 8th day the process of recanalization of the lumen shows, in the distal part of the colon, well defined modalities. Radially oriented intraepithelial spaces within the epithelium filling the lumen join other semilunar intercellular spaces, which are placed near the central part of the occluded lumen (Fig. 3). By the junction of a couple of radially oriented spaces with one semilunar space, an U-shaped intercellular space derives, which delimits an incoming epithelial fold (Tav. 3). Such a phenomenon is continued also during the 9th and 10th day of incubation (Fig. 6, Tav. 2). 3) At the 11th day the colonic lumen is completely open and, in its distal part, the appearance of the primordial previllous ridges can be observed (Fig. 7). In the proximal tract the previllous ridges develop one day later (Fig. 8). 4) At the 13th day, in the distal part of the colon, the first appearance of crypts occurs (Fig. 10). So, while the process of recanalization of the lumen is cranio-caudad, the formation of previllous ridges and crypts proceeds caudo-cranially. 5) From the 11th day onwards the lamina propria is actively involved in the process of formation of the previllous ridges. Only at the 14th day, in the distal part of the colon anlage, the appearance of the muscularis mucosae is observed (Fig. 11). 6) The muscle layer and the subserous stratum do not show appreciable morphological changes in the course the considered period of incubation.     

Regulation of goblet cell differentiation by calcium in embryonic chick intestine

The potential role of calcium as a regulator of goblet cell differentiation was tested by culturing duodenal explants from 14-day chicken embryos in media containing a range of calcium concentrations. Extracellular calcium in vitro approximated the range of plasma calcium concentration that was found to rise by 16% during the third week of embryonic development. As ionic calcium was increased from 0.9 to 2.0 mM in 48-h cultures, the number of goblet cells per 100 ridges increased progressively from 29 +/- 2.2 to 158 +/- 6.9 while attaining a distribution on the previllous ridges similar to that in ovo. The rate of goblet cell differentiation in vitro exceeded that in ovo when extracellular calcium was increased to 1.1 mM and was maximal at 1.6-2.0 mM calcium. Neither the growth of previllous ridges nor gross morphology of the tissue was altered as extracellular calcium was increased. Goblet cell differentiation in 1.3 mM calcium was stimulated by 0.1 microM calcium ionophore and inhibited by the calmodulin antagonist trifluoperazine, indicating an intracellular site for calcium action. These results indicate that calcium plays a primary role in regulating goblet cell differentiation in embryonic intestine and suggest that rising levels of plasma calcium influence the rate of differentiation in ovo.     

Preliminary observations on the effects of selenate on the development of the embryonic skate, Raja eglanteria

Morphogenesis of the clearnose skate, Raja eglanteria, was not significantly inhibited as a result of 7 days of exposure to 1-2 mM selenate in the sea water during Days 59-69 of embryonic development (hatching would normally have occurred at 82 +/- 4 days of incubation). Although corneal transparency appeared normal in the eye, preliminary measurements of the thickness of Bowman's layer of the cornea suggested that it was significantly thinner in the corneas of embryos exposed to 1-2 mM selenate. Selenate is an ion reported to inhibit sulfation of glycosaminoglycans in connective tissue.     

The distribution and ontogeny of gastrin/CCK-, somatostatin- and neurotensin-immunoreactive cells in the gastrointestinal tract of the chicken

The distribution and time of appearance of cells with gastrin/CCK-, neurotensin- and somatostatin-like immunoreactivity were studied in samples from eight regions of the gastrointestinal tract of chick embryos from 11 days of incubation to hatching. No immunoreactive cells were found in any region at 11 days of incubation. Somatostatin- and neurotensin-immunoreactive cells appeared for the first time in the proventriculus, pyloric region and duodenum at 12 days of incubation with cells immunoreactive for neurotensin occurring in the rectum at the same stage. Gastrin/CCK-immunoreactive cells were detected in the small intestine first at 14 days and in the pyloric region two days later. Cells immunoreactive for somatostatin and neurotensin appeared in the upper and lower ileum at 14 days of incubation for the first time; neurotensin-immunoreactive cells, present in the caecum at 14 and 16 days, were rare. Cells of all three types were plentiful in the pyloric region by 17 1/2 days of incubation. No immunoreactive cells were detected in the gizzard at any stage studied. Endocrine cells were present in the relatively undifferentiated surface epithelium which occurs throughout the gastrointestinal tract of chick embryos at 12 days of incubation. Thereafter cells of all three types were detected in the glandular epithelium at or very soon after morphogenesis and differentiation of the latter had occurred.     

Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.     

Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.     

The anatomicohistological characteristics of the formation of the mucosa of the human small intestine in the prenatal period of ontogeny]

The time, when the first villi appear, is defined. Quantitative composition, percent content, height of the villi of the mucous membrane in all parts of the small intestine during various periods of the intrauterine development are presented. In 17-week-old embryos at the level of the duodenum the first villi are noted and their formation in the middle part of the intestinal tube is revealed. By the 8th-9th weeks of development they appear at the level of the caudal part of the intestine. This demonstrates asynchronism of their formation in the cranio-caudal direction. The periods of their intensive growth in height in the first part of the intestine correspond to 8-10, 12-16, 24 weeks of the fetus age, in the second part--to 8-10, 18-20 weeks, in the third--to 10-12, 16-18, 26-28 weeks. These periods alternate with an abatement in growth intensity, which is connected with an increasing width of the villi themselves, with amount of newly formed villi and with changes of the intestinal tube diameter. The form of the villi is rather variable.     

Transcriptional diversity in myogenesis.

Duodenal morphogenesis in the chick embryo has been studied to see if changes in organization of extracellular fibers are associated with villus formation. Three major processes occur during morphogenesis; longitudinal ridges form, then these ridges become zigzag in shape, and finally a double row of villi form from each zigzag ridge. Tissues of different developmental stages were progressively trypsinized to remove cellular material and were prepared for scanning microscopy to show the organization of extracellular fibers. Results show that fiber systems of increasing complexity form as the dudoenum develops, and suggest that some cellular events such as initial ridge formation precede these changes. Tissues with longitudinal ridges contain only randomly organized fibers. In tissues with zigzag ridges, oriented fibers appear along the ridges and some extend laterally from flexures of each zigzag ridge, but interridge fibers are still randomly organized. When villi form, fibers in the body of the villus are random but fibers at the base of villi and between villi are highly oriented. Transmission microscopy and collagenase treatment of partially trypsinized tissue indicate that most, if not all extracellular fibers viewed by scanning microscopy are collagenous. Therefore, since collagen fiber organization is so closely related to morphogenetic changes, we presume it plays an important role.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Pomegranate peel extracts effects to reduce mono sodium glutamate toxic effects on chicken embryos: Morphological studies

BackgroundMonosodium glutamate (MSG) is a flavoring agent added to various foods. This experimental study investigated MSG effects on chicken embryos morphology and the possible ameliorative effects of pomegranate peel extracts (PPE) at different incubation periods.MethodsSeven hundred and twenty fertilized chicken eggs were used and divided into six groups: control, PPE, MSG, PPE + MSG, preventive (PPE–MSG) and therapeutic (MSG–PPE) groups. Fertile chicken eggs were injected with MSG (0.1 ml) and/or PPE (0.3 ml) twice before incubation at days 0, 1. Embryos were extracted at days 7, 10, 12, 14 and 16. Effects of MSG and/ or PPE on embryo development during different incubation periods were studied.ResultsMSG injected into embryos led to congenital anomalies that appeared mainly in MSG and MSG + PPE groups. These anomalies included growth retardation, absent eye, abdominal swelling and hernia. Mortality rate was the highest in MSG, then in MSG + PPE and MSG–PPE groups. PPE treatment reduced MSG toxic effects and these results were better in MSG–PPE and PPE–MSG groups than MSG + PPE group.ConclusionsMSG injection affected chicken embryonic development causing growth retardation and decline in total body length, break length, and total body weight in all the treated groups. These harmful actions can be ameliorated with PPE treatment depending on embryo age.     

N-cadherin is crucial for heart formation in the chick embryo

The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5–12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.     

Cysteamine-induced duodenal ulcer: effects on calcium absorption across rat duodenum

The effect of cysteamine-induced duodenal ulcers on calcium transport across rat duodenum was investigated. Intracellular calcium accumulation measured after 24 hr and 3 days of cysteamine injection showed a significant increase (P less than 0.001) in the duodenal strips isolated after 3 days with no change noticed in those isolated after 24 hr, although the morphological changes in both were very similar. The relationship between increasing calcium concentration in the incubation medium and intracellular calcium concentration is a saturable process that conforms to the Michaelis-Menten type of kinetics. The average maximal flux (Vmax) increased from 8.93 nmole/hr-gdw in normal to 12.5 nmole/hr-gdw in 3-day-ulcerated rats, with no apparent change in the Michaelis constant (Kt) (0.8 mM). Unidirectional influx of calcium across the mucosal membrane was significantly increased (P less than 0.001) in 3-day-ulcerated duodenum suggesting that the increase in calcium transport could be due to the activation of the active step at the mucosal border.     

Effect of clofibrate on the small intestine of fetal mice

Pregnant Swiss ICR mice were injected with clofibrate at different dosages and time intervals, and embryos were removed either at 17 or 18 days of gestation. In embryos sacrificed at 17 days the level of intestinal catalase activity of the proximal and distal halves in the treated groups is identical in any case to that of the controls. In embryos sacrificed at 18 days, the rise in the level of catalase activity in the proximal half of the small intestine in treated groups is dose dependent up to a certain limit: with repeated injections the increase reaches a plateau. The distal halves of treated groups are much less responsive and an increase in catalase activity was noted only with repeated injections. In untreated embryos circular DAB-positive microperoxisomes (200 nm in diameter) and tubular structures (100 nm in thickness) are seen in the duodenum at 18 days of gestation. At the same stage, only circular microperoxisomes are identified in the ileum. After clofibrate treatment circular and tubular microperoxisomes are observed in the ileum also. It is concluded that clofibrate induces a rise in catalase activity in the embryo, only after 17 days of gestation. These observations are discussed in relation to the biogenesis of microperoxisome.     

The initial expression and patterned appearance of tenascin in scutate scales is absent from the dermis of the scaleless (sc/sc) chicken.

Morphogenesis of the anterior metatarsal skin (scutate scale region), from 9.5 to 12 days of development, results in the formation of orderly patterned scale ridges. It is after the initial formation of the Definitive Scale Ridge that the characteristic outer and inner epidermal surfaces differentiate. The hard, plate-like beta stratum, with its unique beta keratins, characterizes the epidermis of the outer surface, while the epidermis of the inner surface elaborates an alpha stratum. The anterior metatarsal region of the scaleless mutant does not undergo scale morphogenesis. Therefore, scale ridges do not form nor do the outer and inner epidermal surfaces with their characteristic beta and alpha strata. We have found that the extracellular matrix molecule, tenascin, first appears in the scutate scale dermis at 12 days of development when the scale ridge is established. Tenascin is found in the dermis only under the scale ridge and is not associated with the dermal-epidermal junction. Tenascin is not found in scaleless anterior metatarsal dermis at this time. As outgrowth of the Definitive Scale Ridge takes place, tenascin distribution correlates closely with the formation of the outer epidermal surface of each scale ridge. By 16 days of development tenascin is also found in close association with the dermal-epidermal junction. Tenascin does not appear in scaleless anterior metatarsal dermis until 16 days of development and then it is randomly and sparsely distributed at the dermal-epidermal junction. Tenascin's initial appearance and pattern of distribution in the scutate scale dermis and its abnormal expression in the scaleless dermis suggest that morphogenesis plays a significant role in regulation of its expression.     

The occluding junctions of mouse duodenal enterocytes during development

Summary The architecture of occluding junctions during the differentiation of the mouse duodenum was studied in freeze-fractured material. Irregular zonulae occludentes (ZO) (Type I) are numerous during fetal life, and are characterized by their irregular width, and by the presence of basal open-ended extensions fused with the discontinuous basal strand of the ZO. Regular ZOs (Type II), typical of the adult villous epithelium, appear after Type I junctions by day 16 of gestation. Two patterns are distinguishable: in the first, parallel strands of ridges and furrows are found without crossing branches; in the second pattern, the junction zone is organized like a network of short branches forming various types of polygons. In fetal and adult mice fasciae occludentes (FO) (Type III) are present on the lateral cell membranes; in unfixed specimens particles are found in the furrows of the E-face and pits on the ridges of the P-face. In fixed tissues, the particles are aligned on the ridges of the P-face. These results indicate that fixation with glutaraldehyde modifies considerably the affinity of junctional particles toward the P-face during the fracture process. Moreover, the presence of numerous large FOs on the lateral cell membranes of enterocytes during late fetal life and in the adult, is possibly related to cell movement along the intestinal villi.