Biology of mammary fat pad in fetal mouse: capacity to support development of various fetal epithelia in vivo

Epithelia from the lobular part of submandibular salivary gland, glandular stomach, intestine and colon of 14-day C3H/HeN fetuses, and from pituitary gland and pancreas of 12-day fetuses were recombined with 14-day mammary fat pad precursor tissue and syngrafted under the kidney capsule. The normal organogenetic development typical of the epithelium occurred. The same epithelia taken from earlier stage fetuses did not develop normally. Thus, 14-day fetal mouse mammary fat pad precursor tissue has the capacity to support normal organogenesis of various fetal epithelia of developmentally advanced stages. This supportive capacity is decreased in the fat pad precursor tissue of 17- to 18-day fetal mice and is entirely lost postnatally.     

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog,Rana catesbeiana

Summary The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).     

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.     

An improved technique for dissociating hepatocytes from fixed mouse liver for cytochemistry

A refined version of a method described previously for dissociating hepatocytes from fixed liver is described. The objective was to develop a procedure that would dispense with the postosmication of previously fixed tissue. In the new procedure fixed liver blocks are wrapped with a quadruple layer of nylon cloth, and, by squeezing them in a buffer solution, individually dissociated cells pass through the cloth without significant damage. The procedure makes it possible to dissociate liver tissue fixed with modified Karnovsky's fixative, Zamboni's fixative or cacodylate buffered glutaraldehyde. The subsequent compatibility of the single cells obtained with many cytochemical procedures has been confirmed.     

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xvii. a Population Carrying Genetic Variability Explicable by the Classical Hypothesis

About 400 second chromosomes were extracted from the Aomori population, a northernmost population of D. melanogaster on Honshu in Japan, and the following experimental results were obtained. (1) The frequency of lethal chromosomes was 0.23. (2) The effective size of the population was estimated to be about 3000, from the allelism rate of lethal chromosomes and their frequency. (3) The detrimental and lethal loads for viability were 0.243 and 0.242, respectively, and the D/L ratio became 1.00. (4) The average degree of dominance for mildly deleterious genes was estimated to be 0.178 ± 0.056. (5) Additive (σ²_A) and dominance (σ²_D) variances of viability were estimated to be 0.00276 ± 0.00090 and 0.00011 ± 0.00014, respectively. (6) There was no significant difference in environmental variances between homozygotes and heterozygotes. Using these estimates, we discuss the maintenance mechanisms of genetic variability of viability in the population. The mutation-selection balance explained these experimental results.     

Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

The Role of c-kit Proto-oncogene during Melanocyte Development in Mouse. In vivo Approach by the In utero Microinjection of Anti-c-kit Antibody

In order to investigate the role of the c- kit oncogene in the melanoblast development, a rat monoclonal antibody (ACK2) against the mouse c-kit protein was used to localize cells expressing c-kit during fetal development. ACK2 was also injected directly into the amniotic cavity of mouse fetuses at successive developmental stages. After birth, the offspring were examined to determine the resulting coat color patterns. c-kit positive melanoblasts first appeared in dermis of fetuses at 11.5 days postcoitum (dpc). Subsequently, these cells increased in number and migrated dorsolaterally to the ventral region, and by 12.5 dpc some of them began to invade the epidermis. Treatment of fetuses by ACK2 microinjection appeared to affect the pigmentation in the coat, inducing a variety of spotting patterns in offspring, and the location of the spots was closely correlated with gestational stage. ACK2 injection of early fetuses produced major changes in coat color even though few c-kit positive cells were detectable in the dermal mesenchyme at the time of injection. Large spots were also induced when mid-stage fetuses with a only few c-kit positive cells in the dorsal region were injected. By contrast, except for spot formation in the center of ventral region, ACK2 injection did not appear to affect melanogenesis in late stage fetuses that had many c-kit positive cells.