The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

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.     

Expression of mutant genes in mouse aggregation chimeras. 4. The aphakia gene

Analysis of aphakia (ak) gene expression in ak/ak C/C in equilibrium +/+ c/c experimental chimaeras has shown that the ak gene acts in the lens rudiment cells blocking it differentiation. In the lens of 12 day old ak/ak C/C in equilibrium +/+ c/c chimaeric embryos undifferentiated ak/ak cells were present among the normally differentiating fibres. In 14 and 18 day old chimaeric embryos and 20 day old chimaeric mice ak/ak cells are located under the lens epithelium and the capsule of posterior lens half. In the locations of ak/ak cells on the posterior lens surface capsule breaks resulted in the extrusion of lens material into the secondary eye cavity. In all studied chimaeric embryos the lens structure is more similar to that in the normal embryos, than in ak/ak embryos. This suggests that in the developing chimaeric lens ak/ak cells are sorted out as the development proceeds. The proliferation rate of +/+ cells appears to be higher than that of ak/ak cells.     

Immunohistochemical detection of prolactin in the hypophysis of the chick and chick embryo

The appearance and localization of immunoreactive prolactin in the adenohypophysis of chick embryos and chickens was studied by antisera to the bovine prolactin. Immunoreactive prolactin was found in the chick embryos from the 15th day of development on using the methods of indirect immunofluorescence and of unlabelled antibodies with a complex PAP. In the chick embryos and in chickens during the first 10 days of life, the prolactin-containing cells were distributed, mainly, in the cephalic part of adenohypophysis; in the chickens, scarce cells were also found in the caudal part. These results suggest that in the domestic fowl the immunoreactive prolactin, similar by immunochemical specificity with the mammalian prolactin, is a late appearing marker of the adenohypophysis differentiation.     

Immunohistochemical study of the appearance of somatotropic hormone in the adenohypophysis of chick embryos

The differentiation of somatotropocytes was studied in the Leghorn chick embryos during 11 to 18 days of incubation and in chickens during the first week of life using the immunohistochemical method of nonlabelled antibodies with PAP complex and antiserum against human somatotropic hormone (STH). Unlike in humans, the fixation of pituitaries in the Carnoy mixture is optimal for STH to be immunohistochemically estimated in the chickens and chick embryos. STH was found in adenohypophysis from 12-13 days of development. Besides predominant localization of somatotropocytes in the adenohypophysis caudal lobe, individual STH-positive cells are also present in the cephalic lobe of chickens and chick embryos. The results obtained suggest a relatively late appearance of STH during histotypical development of adenohypophysis in chick embryos.     

Transferrin enhances lentoid differentiation in rat egg cylinders cultivated in a chemically defined medium

Rat egg cylinders at the primitive streak stage were grown in modified organ culture for 2 weeks using a chemically-defined medium. The purpose of the experiment was to determine whether the terminal tissue differentiation is modified by human transferrin. The control sets were grown in medium with or without rat serum. In explants treated with transferrin, groups of atypical cells of the ocular lens (lentoids) appeared more frequently than in both control sets; however neuroblasts were observed as often as in the serum-supplemented medium. Bovine serum albumin (BSA) stimulated the differentiation of neuroblasts but did not promote lentoid formation. We conclude that human transferrin does stimulate the differentiation of lentoids in rat embryonic explants, but the mechanism of its action remains unknown.     

Immunohistochemical study of fibronectin localization during the formation of the vascular coat under the influence of the pigment epithelium in chick embryos

The role of fibronectin (FN) in cell interactions of retinal pigment epithelium (RPE) and mesenchyme surrounding the optic cup during choroid formation in chick embryos was studied by indirect immunofluorescence using antibodies against FN. Experimental coloboma of retina and choroid was used as a model. During the initial stages of coloboma the regions structured like retina rudiment appear in the outer layer of the optic cup. Such regions were formed in microphthalmic eyes obtained by excision of lens from the eyes of 3.5 day old chick embryos (stage 21). At stage 21 bright FN-specific immunofluorescence was observed in basal membrane located along the external surface of the normally differentiated RPE. Later on, FN-specific immunofluorescence appeared in mesenchyme condensing along the RPE. The most intensive FN-specific immunofluorescence was observed in chorio-capillary layer of choroid after 5-7 days of incubation. In microphthalmic eyes retina-like regions of RPE and adjacent mesenchyme showed negative reaction, and the choroid was not formed from the adjacent mesenchyme in such zones. The data obtained suggest that the presence of normally differentiated RPE producing FN-containing basal membrane is necessary for the formation of chorio-capillary layer of the choroid in chick embryos.     

Somatotropic cell differentiation in an organ culture of the chick embryo adenohypophysis

Morphogenetic potencies of the adenohypophysis tissue from 4.5 to 11-day old chicken embryos used for the differentiation of somatotropic cells were investigated by methods of organ tissue culture. STG-cells were detected in cultures by immunofluorescence using an antiserum to human STG. In vitro studies of organ cultures revealed differentiation of STG-cells when adenohypophysis tissue was cultured from the 5.5th, 7th, 9th and 11th day of development in the absence of the diencephalon. Differentiation of STG-cells occurred predominantly in embryo caudal lobe transplants after chorion-allantois culturing of Rathke's pocket fragments from 4.5-, 5.0- and 5.5-day old embryos. The data obtained suggest that at late stages of Rathke's pocket development differentiation of STG-cells is preprogrammed and that determined precursors of these cells are located in the caudal lobe of the germ.     

Immunofluorescence study of the formation of evolutionary stable lens proteins in chick embryos]

In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.     

Biochemical investigation of lens induction in vitro. I. Induction properties of the eye cup and ectodermal response

1. Optic cups of 48, 72 and 96 hours old chick embryos were prepared, cultured and recombined with ectoderm. With the optic cups of 48 hours old embryos, lens formation occurred in 16% of the cases. With the optic cups of 72 hours old embryos, lens formation occurred in 28% of the cases. Optic cups of 96 hours old embryos were not able to induce a lens. 2. The optic cup proved to be able to induce a lens more than once. 3. Ectoderm of the head of 72 hours old embryos was still able to form a lens. 4. Using homogenized eye cups of 72 hours old embryos, lens induction occurred only in a few cases. When the optic cups were cut into small pieces, lens induction occurred in 30% of the cases. This suggests that intact cells are necessary to obtain lens induction.     

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.     

Differentiation of lens fibers in explanted embryonic chick lens epithelia

Central regions of explanted lens epithelia from 6-day-old chick embryos were maintained in tissue culture for 4 weeks to determine the extent to which lens fiber differentiation would progress in vitro. Cellular outgrowth from the explants created 3 distinct zones; namely, a thick central zone, a thicker annular zone and a flattened peripheral zone. Cells of the central and annular zones underwent morphological and biochemical changes which correspond to the differentiation of lens fibers in vivo. The mean cell length increased a minimum of 25-fold. The nuclei in the longer cells became pycnotic; DNA remained in the nuclei but accumulated single-strand breaks. The cytoplasm became filled with a homogeneous granular matrix. Organelle density decreased, but microtubules persisted, mostly along surface membranes; free ribosomal clusters were present. There were occasional desmosomes and infoldings of cell membranes. The proportion of ribosomal RNA synthesized decreased relative to the total RNA synthesized, especially in the central zone. Finally, the proportion of delta crystallin synthesized increased to 40–50% of the newly synthesized protein. These data suggest that the transformation of lens epithelial cells into fibers results from a programmed differentiation which can take place in tissue culture.     

A tissue-specific inhibitor of the cell proliferation of the retinal pigment epithelium in chick embryos

The salt extract of the nuclear fraction of a homogenate of the retinal pigment epithelium from 12-15 day old chick embryos inhibits selectively the proliferative activity in the retinal pigment epithelium of 3-5 day old embryos. The inhibiting effect of the nuclear factor is found within 20 h after its introduction into the egg. The nuclear extract from the pigment epithelium does not affect the level of proliferation in retina and lens anterior epithelium.     

The effect of growth rate on differentiation of chick embryo limb bud mesenchyme in organ culture

Small explants of limb bud mesenchyme of day chick embryos which form muscle in organ culture synthesize proportionally less protein than DNA than do large explants which form cartilage. Chondrogenesis occurred in the central area of greatest population density in reaggregating limb bud cells, myotubes in areas of lesser density and fibroblasts in the sparsely populated periphery. Small explants grown in microdrops in plastic dishes undergo less cell division and form cartilage, but not muscle. Small explants on lens paper undergo more cell division and form muscle, but not cartilage.     

Correlations between the processes of cell differentiation and development of blood vessels in the (chick embryo) adenohypohhysis

Utilizing semi-thin and, respectively, thick sections after indian ink injections, we have analyzed morphohistogenesis and vessel development in the adenohypophysis of chick embryos from the 4th to the 19th incubation day. Our preliminary results indicate that a close correlation exists between cellular differentiation and vasculogenesis. Vessels begin to enter the gland only at the 6th day when the first endocellular granules become detectable, and, from the 10th day, they markedly increase in number and size, gradually acquiring a peculiar sinusoidal arrangement around cellular groups, as cell differentiation further progresses.     

The determination of the cytodifferentiation of the adenohypophysis in embryonic development

This is a review of the literature and author's own data on determination of various cell types of adenohypophysis during embryonic development. Recent studies using techniques of organ culture and immunohistochemistry have established the time of determination of glandular cells of adenohypophysis. It has been shown in rat embryos that the direction of differentiation of all major cell types of adenohypophysis is programmed late during the development of the epithelial anlage of this organ. Similar data as concerns somatotropic and prolactin cells have been obtained on chick embryos. Chick embryos possess regional type of determination of prolactin and somatotropin-containing cells in the anlage in correspondence with their location in definitive adenohypophysis.     

Cell differentiation of the head ectoderm in mouse embryos in vitro

Cell differentiation has been studied in the explants of head ectoderm of 8, 9 and 10 day old mouse (CBA) embryos and of head epidermis of 13 day old embryos. Pieces of ectoderm were taken from the temporal region. It was established by indirect immunofluorescence that within 10, 15 and 20 days of cultivation spheroids with keratins or crystallins in some groups of fibres formed in the head ectoderm explants from 9 and 10 day old embryos. When cultivating the regions of head epidermis from 13 day old embryos, spheroids formed with keratin only in their cells. The data obtained suggest that there appear to be two clones of cells determined to the synthesis of keratins or crystallins in the head ectoderm of early mouse embryos. During embryogenesis, the number of cells determined to the synthesis of keratins appears to increase in the regions not related to the eye area. At the same time, the clone of cells determined to the synthesis of crystallins appears to be eliminated.     

Tissue specific antigen of the caudal lobe of the chicken adenohypophysis]

The tissue-specific water-soluble antigen of the chick pituitary gland was revealed by immunochemical analysis. The content of this antigen was found to be predominant in the caudal lobe of the adenohypophysis. The antigen was found from the 13th day of embryogenesis by immunoelectrophoresis and immunofluorescence. Two kinds of the antigen (with high and low relative electrophoretic mobility) were discovered in the chick adenohypophysis. A conclusion was drawn that the adenohypophysis cells differed by the differentiation level.