Brn-3.0 expression identifies early post-mitotic CNS neurons and sensory neural precursors.

The mammalian POU-domain factor Brn-3.0 (Brn-3, Brn-3a) is a member of the POU-IV class of transcription factors which resemble the C. elegans factor unc-86 in structure, DNA-binding properties and expression in subsets of sensory neurons. Using specific antisera, we have explored the expression of Brn-3.0 in the early development of the mouse nervous system. Brn-3.0 expression begins at embryonic day 8.5 (E8.5) in a specific set of midbrain tectal neurons whose time and place of appearance are consistent with the earliest CNS neurons previously identified using non-specific markers of neural differentiation. By E9.5, Brn-3.0 immunoreactivity also identifies early CNS neurons in the hindbrain and spinal cord. In the peripheral sensory ganglia, Brn-3.0 expression is first observed at E9.0 in migrating precursors of the trigeminal ganglion, followed by the other sensory cranial and dorsal root ganglia, in a rostral to caudal sequence. Double-label immunofluorescence with Brn-3.0 and the markers of cell division PCNA and BrdU demonstrate that Brn-3.0 is restricted to the post-mitotic phase of CNS development. In the sensory cranial and dorsal root ganglia, however, Brn-3.0 is expressed in dividing neural precursors, suggesting that the nature or timing of developmental events controlled by Brn-3.0 are distinct in the CNS and peripheral neurons. Restriction of Brn-3.0 expression to post-mitotic CNS neurons demonstrates that Brn-3.0 is not required for neurogenesis or patterning of the neuroepithelium in the CNS, but suggests a role in specification of mature neuronal phenotypes.

[The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos]. / Vozmozhnost' linzoobrazovania v éksplantatakh setchatki i pigmentnogo épiteliia glaza kurinykh zarodyshei.

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.

[Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine]. / Induktsiia sinteza al'fa- i beta-kristallinov v organnoi kul'ture zachatka adenogipofiza kur pod vliianiem 5-iod-dezoksiuridina i 5-brom-dezoksiuridina.

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.

[Lenticular and adenohypophyseal differentiation in the oral region ectoderm of chick embryos in tissue culture]. / Khrustalikovye i adenogipofizarnye differentsirovki v éktoderme rotovoi oblasti kurinykh zarodyshei v usloviiakh kul'tury tkanei.

The ectoderm of oral regions from the chick embryos at the stage of 10 to 19 somites was cultivated in vitro and on chorioallantois in the complex with underlying tissues. In all the explants which, besides ectoderm, contained head gut and mesenchyme, lentoids and lenses formed within 6 days of in vitro cultivation. All specific antigens of chicken lens (alpha, beta- and delta-crystallins) were found in them by means of immunofluorescence. In the explants which contained diencephalon, besides single lentoids or lenses, adenohypophyses were found. The possibility of direct lens-inducing effect of the head gut endoderm on the ectoderm of oral region and the participation of diencephalon in this process are discussed.

[The distribution of competence for adenohypophysis development in the ectoderm of chick embryos]. / Raspredelenie kompetentsii k adenogipofizarnomu razvitiiu v éktoderme kurinykh zarodyshei.

The ability of various zones of the cephalic and trunk ectoderm to differentiate into adenohypophysis after the contact with the bottom of the prosencephalon was studied in tissue culture of chick embryos as the stage of 10-13 somites. Stomodeal presumptive lens ectoderm and lateral cephalic ectoderms were shown to be competent for development into adenohypophysis. In all cases adenohypophyseal cords were formed in the zones of ectoderm contact with the brain. The cords contained antigens A-2, A-3 specific for chicken adenohypophysis as well as ACTH and beta-lopotropin. Trunk ectoderm proved to be incapable to differentiate into adenohypophysis.

[The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research]. / Sintez i lokalizatsiia kristallinov v razlichnykh kletochnykh kompartmentakh khrustalika glaza vzroslykh liagushek: immunoavtoradiograficheskoe i immunofluorestsentnoe issledovanie.

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikhailov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.

[Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]. / Konkanavalin-sviazyvaiushchie belki i tsitokeratiny raznykh tkanei rannei gastruly amfibii (Rana temporaria, Xenopus laevis).

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhailov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.

The distribution of lens differentiation capacity in the head ectoderm of chick embryos.

The distribution of lens self-differentiation capacity in the head ectoderm of chick embryos during stages 4-13 (defined by Hamburger and Hamilton) was investigated. Different fragments of head ectoderm were cultivated in organ culture in vitro. Lens cells and lentoids formed in explants were identified by the indirect immunofluorescence technique with monospecific rabbit antisera to chick lens proteins: alpha-, beta-, and delta-crystallins. It was established that besides presumptive lens ectoderm, the lateral head ectoderm and ectoderm of stomodeal area of stages 10-11 chick embryos differentiated into lens cells when separated and cultured alone. The cultivation of presumptive lens ectoderm resulted in lens fiber differentiation in most explants. In the explants of lateral head ectoderm and those of the ectoderm of the stomodeal area, the lentoids and groups of cells containing crystallins were also found in most cases. In some, all cells of the explants taken from these areas differentiated into lens tissue. These results show that in the chick embryos at stages 10-11, capacity for lens self-differentiation is distributed over a large continuous area of the head ectoderm. The potential seems to arise during the process of gastrulation. The head ectoblast of stage 4-5 chick embryos can achieve at least three differentiation in vitro--lens, epidermal-like, and neutral. In the chick head ectoderm, at the beginning of neurulation two areas can be distinguished according to their capacity for self-differentiation. The area situated in front of the head fold develops into lens and epidermal-like tissues in vitro, whereas the area between the head fold and Hensen's node develops into neural tissue. In the course of further development, lens potential was restricted to the presumptive lens ectoderm. Most of the head ectoderm develops in normogenesis in several directions without any traits of lens differentiation. The formation of Rathke's pouch (adanohypaphysis anlage) by cells which have synthesized beta-crystallins is the only known case (besides the lens anlage) in which an expression of lens potential of head ectoderm occurs.

[Analysis of water-soluble antigens of chick iris]. / Analiz vodorastvorimykh antigenov raduzhiny kur

Three serum and twelve tissue antigens were revealed in adult chick iris by immunodiffusion and immunoelectrophoresis. Among the tissue antigens five were characteristic of the eye tissues alone: a specific iris antigen, an antigen revealed in the iris and the retina, and three antigens characteristic of the lens (the alpha-, beta- and delta-crystallines). The rest seven antigens were interorganic differing by their distribution in the tissues and organs investigated; among them one was specific of muscle tissue.

[Synthesis of a lens-specific antigen (delta-crystallin) in rudimentary chicken adenohypophysis]. / Sintez spetsificheskogo antigena krustalika (delta-kristallina) v zachatke adenogipofiza kus.

The synthesis of two lens-specific proteins, delta- and beta-crystallins, by adenohypophyseal anlage of 4-day chick embryos was studied by the immunofluorescence technique in conjunction with autoradiography. Isolated anlages were incubated for 16 hours in a culture medium containing 14c-leucine. The synthesis was determined with the use of an unlabelled carrier, extract of chick lens, as well as of antisera against delta- and beta-crystallins. 14C-Leucine incorporation was found to occur only in delta-crystalline precipitation line rather than in beta-crystallin line. This evidence attests to the synthesis of delta-crystalline by the chick embryo adenohypophyseal anlage. The results are in agreement with the previously obtained immunohistochemical data on delta-crystalline localization in cells of the developing adenohypophysis.

Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord.

Rathke's pouch, the epithelial primordium of the anterior pituitary, differentiates in close topographical and functional association with the ventral diencephalon. It is still not known whether the ventral diencephalon acts as the initial inducer of pituitary development. The roles of the adjacent mesenchyme and notochord, two other tissues located in close proximity to Rathke's pouch, in this process are even less clear. In this report we describe an in vitro experimental system that reproduces the earliest steps of anterior pituitary development. We provide evidence that the ventral diencephalon from 2- to 4-day-old chick embryos is able to function as an inducer of pituitary development and can convert early chick embryonic head ectoderm, which is not involved normally in pituitary development, into typical anterior pituitary tissue. This induction is contact-dependent. In our experimental system, there is a requirement for the supporting action of mesenchyme, which is independent of the mesenchyme source. Transplantation of the notochord into the lateral head region of a six-somite chick embryo induces an epithelial invagination, suggesting that the notochord induces the outpouching of the roof of the stomodeal ectoderm that results in formation of Rathke's pouch and causes the close contact between this ectoderm and the ventral diencephalon. Finally, we demonstrate that the ventral diencephalon from e9.5-e11.5 mouse embryos is also an efficient inducer of anterior pituitary differentiation in chick embryonic lateral head ectoderm, suggesting that the mechanism of anterior pituitary induction is conserved between mammals and birds, using the same, or similar, signaling pathways.