Induction of tubules in rat metanephrogenic mesenchyme in the absence of an inductive tissue

Differentiation of metanephrogenic mesenchyme to renal tubular epithelium requires induction by the ureteric bud in vivo or any of several embryonic tissues in vitro. In an effort to eliminate the tissue requirement in embryonic induction, extracellular matrices and soluble factors were analyzed individually or in combination for their ability to stimulate tubulogenesis in uninduced metanephrogenic mesenchyme from 13-gestation-day rat embryos. These evaluations have established that pituitary extract and epidermal growth factor (EGF) in concert with a matrix can promote morphogenesis of mesenchymal rudiments in culture. While type I collagen, laminin, or fibronectin matrices all promoted tubulogenesis in the presence of pituitary extract and EGF, type IV collagen proved the most effective. Under these conditions, tubules were induced in 23/24 mesenchymal rudiments by 9 days in culture. Mesenchyme was not induced prior to explanation since it formed no tubules when cultured in a medium that allowed tubulogenesis in intact embryonic kidneys. Preliminary characterization of the undefined factor in pituitary extract was consistent with a protein of molecular weight greater than 100,000 but less than 300,000. When uninduced metanephrogenic mesenchyme from mouse was used instead of rat tissue, a similar pattern of morphogenesis was not observed, suggesting that the described medium is inappropriate for promoting differentiation in mouse or, less likely, that different mechanisms mediate differentiation in rat and mouse. These studies show that embryonic induction can occur in explanted rat renal mesenchyme in an appropriate environment and does not require the presence of an inductive tissue.     

Immunocytological study on the differentiation of chick and quail adenohypophysis epithelial rudiments, grafted or cultivated in vitro: evidence for polypeptidic hormones

Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11--13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio-allantoic membrane exhibited cells reacting with the following immune sera: anti-beta-(1--24)ACTH, anti-alpha-(17--39)-ACTH, anti-alpha-endorphin, anti-beta-endorphin and anti-beta-LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to beta-(1--24)-ACTH, alpha-(17--39)-ACTH, and beta-LPH, as did adenohypophysis from quail embryos of corresponding age (9--10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder-cells and/or secrete some factors promoting differentiation.     

Epithelia are interchangeable between facial primordia of chick embryos and morphogenesis is controlled by the mesenchyme

The embryonic chick face is composed of a series of facial primordia, epithelium-covered buds of mesenchyme, which surround the presumptive mouth. The protruding adult upper beak containing the prenasal cartilage is formed from the frontonasal mass, the paired maxillary primordia form the sides of the face, while the lower beak is derived from the paired mandibular primordia which contain the two Meckel's cartilages. When grafted to a host wing bud, the frontonasal mass and the mandibular primordia both form elongated outgrowths, whereas the maxillary primordium forms a ball of tissue. Facial epithelium is required for growth and morphogenesis of all primordia. Recombinations between epithelium and mesenchyme from different primordia show that the epithelia are interchangeable and appear to be equivalent. Even the epithelium from the maxillary primordium that does not grow out in a polarized fashion can support outgrowth of the frontonasal mass and mandibular mesenchyme. The form of the recombined graft is determined by the mesenchymal component.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.     

Mouse urogenital development: a practical approach

A detailed knowledge of the developmental anatomy of the embryonic mouse urogenital tract is required to recognize mutant urogenital phenotypes in transgenic and knock-out mice. Accordingly, the purpose of this article is to review urogenital development in the mouse embryo and to give an illustrated methodological protocol for the dissection of urogenital organ rudiments at 12-13 days of gestation (E12-13) to isolate the urogenital ridge and at E16 to isolate the seminal vesicle, Müllerian duct, Wolffian duct, and prostatic rudiment, the urogenital sinus (UGS). The UGS can be cultured and, in the presence of testosterone, prostatic buds form in vitro. Because of the importance of mesenchymal-epithelial interactions in urogenital development, methods for the isolation of epithelium and mesenchyme from the embryonic urogenital sinus are also described. Urogenital sinus mesenchyme (UGM) and urogenital sinus epithelium (UGE) can be used to construct tissue recombinants that can either be grown in vitro or grafted in vivo for the study of epithelial-mesenchymal interactions in prostatic development.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.     

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.     

INDUCTIVE TISSUE INTERACTIONS IN THE BEAK OF A CHICK EMBRYO *

Series of homologous and heterologous recombination experiments were made between the beak and other skin derivatives, by means of a modified chorioallantoic membrane grafting, to investigate inductive tissue interactions involved in the upper beak of a developing embryo and a hatched chick. 6-day beak epidermis, as well as 6-day cephalic skin epidermis, differentiated into typically normal epidermis of the beak, when they were associated with the mesenchyme taken from 6-day upper beak region. These epithelia, when grafted without association with beak mesenchyme, failed to differentiate into the beak epidermis. 6-day beak epidermis differentiated into typical down feathers when combined with 7-day back skin dermis, prospective feathered area. The inductive potency of the beak mesenchyme was not limited in embryonic life, but persisted even after hatching. These findings were discussed in relation to the role of inductive tissue interactions involved in the expression and stabilization of the differentiated characters of the epidermis in both embryos and adults.     

The genesis of cartilage size and shape during development and evolution

How do cartilaginous elements attain their characteristic size and shape? Two intimately coupled processes underlie the patterned growth of cartilage. The first is histogenesis, which entails the production of cartilage as a discrete tissue; the second is morphogenesis, which pertains to the origins of three-dimensional form. Histogenesis relies on cues that promote the chondrogenic differentiation of mesenchymal cells, whereas morphogenesis requires information that imbues cartilage with stage-specific (e.g. embryonic versus adult), region-specific (e.g. cranial versus appendicular) and species-specific size and shape. Previous experiments indicate that early programmatic events and subsequent signaling interactions enable chondrogenic mesenchyme to undergo histogenesis and morphogenesis, but precise molecular and cellular mechanisms that generate cartilage size and shape remain unclear. In the face and jaws, neural crest-derived mesenchyme clearly plays an important role, given that this embryonic population serves as the source of chondrocytes and of species-specific patterning information. To elucidate mechanisms through which neural crest-derived mesenchyme affects cartilage size and shape, we made chimeras using quail and duck embryos, which differ markedly in their craniofacial anatomy and rates of maturation. Transplanting neural crest cells from quail to duck demonstrates that mesenchyme imparts both stage-specific and species-specific size and shape to cartilage by controlling the timing of preceding and requisite molecular and histogenic events. In particular, we find that mesenchyme regulates FGF signaling and the expression of downstream effectors such as sox9 and col2a1. The capacity of neural crest-derived mesenchyme to orchestrate spatiotemporal programs for chondrogenesis autonomously, and to implement cartilage size and shape across embryonic stages and between species simultaneously, provides a novel mechanism linking ontogeny and phylogeny.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem- or progenitor- cells for islet transplantation.     

Cell proliferation in the developing rat pineal gland. A bromodeoxyuridine immunohistochemical study

The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the cell proliferation in the developing rat pineal gland, from the appearance of pineal primordium in the embryonic day 15 (E15) until 30 days after birth. The results showed three different proliferative phases. From E15 to E21, the pineal gland shows a phase of rapid proliferation. The second phase corresponds to the first postnatal week, in which the number of labeled cells per surface unit decreases suddenly to values between 20% to 10% of those of embryonic period. From the second postnatal week onwards, the number of BrdU-positive cells progressively decreases.     

The effect of neuronal cells on kidney differentiation

During embryonic growth, tissue interactions between dissimilar cells are the driving forces of morphogenesis. Although their importance has been well known for over the past 50 years, the molecular background of these interactions has remained unelucidated. The unrecognized heterogeneity of those mesenchymal cells that are involved in the epithelio-mesenchymal tissue interactions may be one reason for this. For example, studies of kidney differentiation show that the metanephric organ rudiment contains more cell-lines than previously thought. Identification of both neural crest- and mesoderm-derived cells in the nephrogenic mesenchyme helps in re-evaluating the biology of the tubule induction. The neural crest-derived cells of the nephric rudiment differentiate into neuronal cells, and later during differentiation some of them are found in the stroma. There is also experimental evidence for the role of these neuronal cells in the morphogenetic tissue interaction.     

Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.Key Words: islets, stem-cells, development, epithelium, mesenchyme, pancreas, stomach, chick-quail, 3-dimensional, endocrine     

Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development

Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.     

An embryonic pineal body as a multipotent system in cell differentiation

The differentiating potency of pineal cells from 8-day quail embryos was studied with cell culture. It was found that the differentiation of striated muscle fibres occurred abundantly in the pineal cells cultured in hypertonic culture conditions. Muscle nature of these fibres was confirmed by utilizing the antiserum against the striated muscle type creatine kinase (MM-CK). When CO2, NAHCO3, NaCl, KCl and MgCl2 were added in hypertonic concentrations, extensive myogenesis occurred in cultured pineal cells. Myogenesis in pineal cultures began as early as 2 days and, after 3 days in the medium with 75 mM additional NaCl, reached 100-fold when compared with that in the isotonic medium. Muscle fibres from pineal cells in culture were similar in morphology to the skeletal muscle fibres of mesodermal origin in situ. Myogenesis of pineal cells under hypertonic conditions was accompanied by the synthesis of a unique 56 x 10(3) Mr protein, which was not found in the intrinsic muscle cells. Clonal cell culture revealed that about 80% of clonable pineal cells were myogenic precursors. Pineal cells of 8-day quail embryos were not only myogenic but oculopotent (melanogenic and lentoidogenic) in cultures. This study examined whether multipotential progenitor cells with both potentials are present in the pineal or not. The results showed that at least 16% of all clonable pineal cells were multipotent precursors. The embryonic pineal is considered to be a typical multipotent system in parallel with the pigmented and neural retina, the neural crest and the teratocarcinoma.