Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.     

FORMATION AND CHARACTERIZATION OF THREE TYPES OF YEAST INVERTASE

Three types of invertase (invertase I, II and III) are separatedfrom the soluble and insoluble fractions (4,500xg, 10 min supernatantand pellets of the homogenate, respectively) of baker's yeastby a DEAE cellulose column chromatography. The invertases Iand II are eluted with 0.1 M sodium acetate buffer (pH 3.9)and with 0.1 M sodium acetate buffer (pH 6.2) containing 0.1M NaCl from DEAE cellulose respectively, whereas the invertase-IIIremains adsorbed on the cellulose under these conditions. Theyare present in proportions of 2.5: 1 : 0.06 in the soluble fractionand 1.4: 1 : 0.12 in the insoluble fraction of the fresh baker'syeast cells. While in-vertase-II remains at a constant level,invertases I and III in the soluble fraction increase upon incubationof cells for the formation of invertase under the continuoussupply of sucrose. Invertases I and II differ from each other considerably in theoptimum pH and slightly in the response to (activation and inactivationby) crude papain and are identical with respect to the heatstability and probably to the affinity for sucrose. ¹Present address: Chemical Laboratory, Nippon Medical School,Konodai, Ichikawa-shi, Chiba-ken.     

Development of Melanocyte Progenitors in Murine Steel Mutant Neural Crest Explants Cultured With Stem Cell Factor,Endothelin-3, or TPA

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sf¹) have no pig-mented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using SI/SI mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.     

Deficiency of the Gene B Impairs Differentiation of Melanophores in the Medaka Fish,Oryzias latipes: Fine Structure Studies

In an orange-colored variant of the medaka fish, Oryzias latipes, which is homozygous for b allele, the melanophores represent a tissue-specific differentiation, manifesting an amelanotic appearance in the skin, an incomplete melanogenesis in the choroid and the peritoneum, and mosaic phenotype-like melano-iridophores in the peritoneum. In a wild-type strain of this species carrying the B gene, all melanophores are terminally differentiated irrespective of the tissues in which they are located. This indicates that the deficiency of B gene impairs the differentiation of melanophores in the medaka. Electron microscopy disclosed that the deficiency of B gene causes deterioration of melanogenesis to occur inside the melanosomes and that the manner of deterioration in the melanophores in the skin, the choroid and the peritoneum is different. The ubiquitous occurrence of reflecting platelet-laden melanophores in the peritoneum of this variant and the total absence of a mosaicism in pigment cells of the wild-type strain indicate that the deficiency of B gene predestines melanoblasts distributed in this tissue to an ambiguous state with regard to their differentiation. Little difference is observed between melanosomes maturation in pigment epithelial cells of the orange-colored variant and the wild-type strain, indicating an innocent role of the B gene in their differentiation.     

Ecological paradoxes*

Two controversial areas of ecology are examined critically to ascertain the limits of some current theories. In the field of population ecology it is argued that no general theory can explain the regulation of animal numbers. Existing theories are considered inadequate because either (1) evidence points to multiple causes rather than a single cause, (2) propositions lose scientific appeal by accumulating qualifiers, or (3) conflicting theories are irrefutable in scientific inquiry. Different research strategies are required to discover generalities in population regulation. In the field of community organization it is pointed out that too much emphasis on the competitive exclusion principle has blinded ecologists from the clustering of resources and of species populations. Conditions of co-existence as well as segregation are considered important in the organization of communities, which allow elements of chance in small scale distribution. The ecological world is likened to the physical world in which small scale disorder leads to order on a large scale. For the development of a global ecology the acceleration of studies in the tropics and the southern hemisphere is urged.     

HORMONAL REGULATION ON THE INDUCTION OF {alpha}-AMYLASE ISOZYMES IN THE EMBRYO-LESS ENDOSPERM OF BARLEY

The -amylase induced by helminthosporol and gibberellic acidin the embryo-less endosperm of barley was separated into thethree fractions, 1, 2 and 3, by DEAE-cellulose column chromatography.A maximum amount of the 2. was induced by gibberellic acid andthat of the as by helminthosporol. After rechromatography, the2 induced by gibberellic acid and the as induced by helminthosporolshowed their respective single bands in an electrophoresis agargel zymogram. On the other hand, the ai induced by helminthosporoland gibberellic acid showed three bands. Dihydrohelminthosporic acid, a derivative of helminthosporol,induced the same isozymes as helminthosporol did. (Received May 8, 1967; )     

Fractal Analysis of Plant Root Systems

The morphology of root systems of crop plants was analysed byfractal geometry using an image processing system. Results indicatethat these root systems have a fractal structure (D; 1.48     

Ecological association of birds and vegetation structure in wet tropical forests of Australia

Birds and environments of tropical rainforests were studied in 18 selected areas from sea level to 1520 m within the humid tropical region of northeastern Australia. A total of 122 sites (20 m × 20 m each) were established within the 18 areas, and bird data (observation and mist-netting) and environmental data (pro forma survey and foliage measurements) were collected at each site. The bird data were analysed numerically in two ways: abundance of 146 species in 18 areas and presence and absence of 108 species at 122 sites. Both classification and ordination analyses produced a major discontinuity between the tableland sites and the lowland sites, each divisible further into various types of vine forest and non-vine forest vegetation. The few rare species did not contribute to the general pattern. The environmental data were reduced to a set of those attributes that showed variation among the sites. They included topographic and edaphic features, physiognomic and structural features of vegetation, fruiting and defoliating habits of plants, foliage height diversity and evenness, and canopy cover. The primary pattern in the data set of 135 attributes by 122 sites was vine forest versus nonvine forest, and altitudinal divisions appeared as a secondary pattern, in both classification and ordination. Canonical correlation coefficients between bird vectors and environmental vectors were reasonably high for the first four canonical vectors, which correlated well with major Gower vectors of birds and environments. The back-correlation to the raw data produced bird species and matching environmental attributes with sites sharing them for both positive and negative ends of each canonical vector. In general, the analysis extracted elements of both fauna and environment that characterized the vine forest as opposed to the non-vine forest and the tableland sites as opposed to the lowland sites. Canonical correlation analysis is limited by the unverifiable nature of derived correlations. Such correlations may suggest certain associations to be tested by independent means (hypothesis-generating function), but may also formalize self-evident or fortuitous associations.