BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

CHANGES IN CELL SURFACE MORPHOLOGY DURING DIFFERENTIATION OF MICROMERE- AND MESOMERE-DERIVED CELLS OF THE SEA URCHIN EMBRYO

Micromeres and mesomeres isolated from 16-cell embryos of the sea urchin, Strongylocentrotus intermedius , were cultured in vitro , and changes in the cells surface architecture during the differentiation of the micromere- and mesomere-derived cells were observed using scanning electron microscopy. Two types of the distribution of the surface microvilli were observed in both blastomere-derived cell masses. One type showed a uniform distribution of the microvilli and the other type showed an uneven one. Though many microvilli were observed in most of both mesomere and micromere-derived cells at the 64-cell stage and the early blastula stage (16 hr after the 16-cell stage at 6°C) respectively, the microvilli decreased in number at the later stages in both blastomere-derived cell masses as compared with the 64-cell stage and the early blastula stage respectively. Rapid disappearance of the surface microvilli was observed in the micromere-derived cells in contrast with the mesomere-derived cells which still had many microvilli even at the midmesenchyme stage.     

人胎儿真皮乳头和皮纹发生的研究

本文用扫描电镜法研究了入胎儿的皮纹发生过程,包括初级真皮嵴、次级真皮嵴、真皮乳头和表皮隆线的发生。为研究人皮纹的发生和皮肤的异常提供了皮肤正常发育的形态学依据。共观察111例从第6周到第9个月胎儿的皮纹区皮肤,表明第3个月末胎儿开始形成初级真皮嵴,以后逐渐加深,至第16周嵴的顶端中央产生纵沟形成两条平行的次级真皮嵴;自19周后,次级真皮嵴局部隆起,由波浪形逐渐形成乳头。至30周乳头呈犬牙状。表皮隆线于第4—5月形成,随真皮乳头的增高而渐趋明显。至第6个月,全部皮纹图样已可辨认。本文还讨论了真皮乳头发生的过程。     

Early development of the digestive tract of cod larvae, Gadus morhua L., during start-feeding and starvation

Cod larvae, Gadus morhua L., were reared in the laboratory and released to a large marine enclosure 4 to 5 days after hatching (6–8° C). The development of the digestive system was studied until day 24 after hatching. Morphological investigations of the jaw apparatus and the digestive tract showed that the larvae are able to absorb ingested food well before exhaustion of the yolk sac. The foregut, and especially the midgut, were particularly active in lipid absorption, and the hindgut was characterized by pinocytotic activity. Duhng the first days of feeding, no distinct prey organisms were observed in the gut, and signs of food absorption in the epithelial cells of the gut were sparse.A distinct red fluorescence, restricted to the hindgut, was observed from day 11 to day 19. On the basis of changes in absorptive pattern in the gut we suggest that changes in digestive and absorptive abilities, as well as in nutritional needs, take place around days 15–17 after hatching.
In starved larvae, signs of degeneration of the gut tissue were first visible in the foregut. By day 9 after hatching, microvilli was degenerated to such an extent that the ability to absorb food must have been severely restricted. If larvae are starved longer than this, they will probably not survive.     

ENZYMATIC ACTIVITIES RELATING TO THE RESPIRATION AND THE ONSET OF METAMORPHOSIS OF THE ASCIDIAN TADPOLE

The oxygen consumption of a single ascidian larva was measured. After hatching the consumption increases gradually. During the period of tail resorption it also increases gradually, but after the completion of tail resorption the consumption decreases conspicuously.
With the development of the larva after hatching, the activities of cytochrome oxidase and succinic dehydrogenase and of Janus green-reduction become detectable in the adhesive papillae, the proximal region of the tail, and the tail muscle. After the completion of tail resorption, these activities become indistinct.
These tissues underwent most profound morphological changes at the onset of metamorphosis. Soon after hatching, Janus green has no effect to induce metamorphosis. In larvae 4 hr after hatching, the shrinkage of adhesive papillae can be induced by Janus green-treatment. In 12 hr larvae, both the shrinkage of adhesive papillae and the tail resorption can be induced by Janus green. The enhancement of respiratory activities in the larvae after hatching may be related to the changes in the adhesive papillae and later to changes in the proximal region of the tail. Only when both of these changes occur can metamorphosis be induced.     

FACTORS CONTROLLING THE REASSEMBLY OF THE MICROVILLOUS BORDER OF THE SMALL INTESTINE OF THE SALAMANDER

Hydrostatic pressure, when applied to segments of the small intestine of the salamander, causes a tremendous reduction in number of microvilli and a loss of the terminal web. The intestinal epithelium strips off from its deeper layers at the level of the basement membrane. When the pressure is released and this epithelial sheet is allowed to recover, the microvilli and its terminal web reappear. Stages in the reformation of microvilli are described. In the earliest stages, foci of dense material seem to associate with the cytoplasmic surface of the apical plasma membrane. From this material, filaments appear and their regrowth is correlated with the extension of the microvilli. We suggest that the dense material nucleates the assembly of the filaments which, in turn, appear instrumental in the redevelopment of microvilli. This concept is supported by the existing literature. Further, since neither the microvilli nor the terminal web reappear on any surface but the apical surface, even though the apical and basal surfaces are bathed with the same medium, we suggest that information in the membrane itself or directly associated with the membrane dictates the distribution of the dense material which leads to the formation of the microvilli and ultimately to the polarity of the cell.     

Morphometrical changes in the apical surface of the colonic absorptive cells in perinatal rats with special reference to the effect of fetal oral administration of milk in utero

Perinatal changes in the apical surface of the colonic absorptive cells in the rat were studied morphometrically. Cell microvilli length increased from day 20 through neonatal day 3, during which a maximum incremental growth rate was noted between fetal day 22 and neonatal day 1. Microvilli width remained almost constant throughout the period. Enlargement of the apical surface of the microvilli showed a similar developmental pattern as was seen from the measurement of length and surface area of any one of the microvilli. Fetal oral administration of milk in utero caused incremental growth in length and surface area, as well as an associated apical surface enlargement. The present study indicates that the function of the colonic absorptive cells, which is acquired later on in utero, is activated by ingestion of maternal milk after birth.     

THE ULTRASTRUCTURE OF THE QUIESCENT CENTER IN THE APEX OF CULTURED ROOTS OF CONVOLVULUS ARVENSIS L.

Cultured roots of the common bindweed, Convolvulus arvensis L. growing at the rate of 15–30 mm/day in sterile nutrient medium were fixed for electron microscopic analysis. The ultrastructure of the quiescent center, the initials of the ground meristem, and the initials of the procambium were studied in order to determine whether sequential structural changes could be correlated with models for specifying the mechanisms by which cell differentiation and cell division might be controlled. The differentiation of cells in the root proper occurs very gradually in linear files from the site of the quiescent center proximally into the different tissue regions. Major structural changes, such as the orientation and subsequent elongation of cells along the longitudinal axis of the root and cell wall changes, indicate that the control of differentiation and perhaps cell division occurs in radial gradients outwardly from the quiescent center.     

Regulation of intestinal brush border microvillus length during development by the G- to F-actin ratio

We examined ultrastructural changes in developing chicken intestinal microvilli and correlated these with changes in the G- to F-actin ratio and the amount of actin per milligram cell protein. Three discrete morphological and temporal changes occur during late microvillus morphogenesis: an increase in microvillus number associated with microvilli becoming hexagonally packed on the cell surface; an increase in core actin filament number; and an increase in the length of microvilli. Dramatic rises in the amount of cell actin occur at the time of the first two morphological changes. Changes in the G- to F-actin ratio suggest that increases in the level of monomeric actin drive the elongation phase of microvillus growth since immediately prior to growth the G- to F-actin ratio shifts from its embryonic and adult 3:7 ratio to a 1:1. Our results also indicate, but do not prove, that an increase in the amount of G-actin precedes the rise in level of F-actin and growth of microvilli by 1 day, implying that an increase in the content of G-actin stimulates actin polymerization. Our findings also suggest that the G- to F-actin ratio and their absolute amounts, perhaps in combination with cytoskeletal protein turnover and/or the pool size of actin binding proteins, plays a role in restricting the mature constant length of microvilli.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

The regulation of saccharase and phosphofructokinase activity in the small intestine of chicks of different ages]

Studies have been made on the onset and regulation of the activity of sucrase and phosphofructokinase from different parts of the small intestine of chicks after hatching up to the 30th day of postnatal life. It was shown that within this period the activity of both enzymes undergoes synchronous changes. Carbohydrate loading increases the activity of sucrase only in 30-day chicks, its effect being similar to that of a stimulator from the duodenum. Changes in the activity of phosphofructokinase exhibit a different pattern being significantly dependent on the region of the small intestine.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

GROWTH, DEVELOPMENT AND RESPIRATION IN THE OVULES OF ZEPHYRANTHES LANCASTERI AT DIFFERENT STAGES OF MATURATION

Changes in the rate of respiration and activity of succinicdehydrogenase in the ovules, embryo and endosperm have beeninvestigated at different stages of seed development in Z. lancasteri.An attempt has been made to find any possible relation betweenphysiological changes and the growth pattern of ovule. The fertilizedovules mature into seeds in 16–19 days from the time ofpollination. The respiratory course and the growth of ovulesfollow identical trends. The curve for oxygen uptake is characterizedby at least two peaks; the first on the sixth or seventh dayis related to the laying down of walls in the coenocytic micropylarchamber of the endosperm and the second on the 11th day precedesthe elongation of cotyledon. In excised endosperm the activityof succinic dehydrogenase is highest during its transformationfrom freenuclear to cellular state, while in the embryo it isso after the attainment of mature size. ¹Present address: AEC Plant Research Laboratory, Michigan StateUniversity, East Lansing, Mich., U.S.A.     

Morphogenesis of intestinal villi. I. Scanning electron microscopy of the duodenal epithelium of the developing chick embryo

Development of villi in the duodenum of the chick was studied in stages ranging from 11 days of incubation to one week after hatching. Formation of definitive villi is preceded by development of a set of previllous ridges that run lengthwise along the duodenum. The first set of 16 previllous ridges (Set I) is complete by about 13 days of incubation; all ridges in the set are fairly uniform and proceed through their subsequent development in synchrony. Previllous ridges in Set I fold into a highly regular zigzag pattern between 14 and 16 days of incubation. Definitive villi develop from Set I ridges beginning at about 17 days when populations of distinct cells appear on the crests of the ridges between angles in the zigzag folds. Cells in these populations lack the rounded appearance of cells seen in earlier stages; their apical surfaces are densely covered with microvilli. A second set of villi (Set II) develops at about 16 days of incubation when about 16 rows of tongue-like flaps erupt between the previllous ridges of Set I. At hatching, Set II villi are still smaller than villi of Set I; this distinction disappears by about the fourth day after hatching. The significance of the morphological changes in epithelial cells is discussed in terms of several hypotheses bearing on the mechanisms of villus formation.     

DEPENDENCY OF THE TENSILE MODULUS ON TRANSVERSE DIMENSIONS,WATER POTENTIAL,AND CELL NUMBER OF PITH PARENCHYMA

Uniaxial tensile tests of solid and hollow cylindrical plugs of pith parenchyma from potato tubers indicate the tensile modulus of elasticity, E, can vary significantly as a function of tissue transverse area and water potential. E increases from 1.2 to 19 MPa as ψ_w changes from -1.4 to -0.4 MPa. E increases from 5 to 19 MPa as transverse area of solid tissue sample increases from 0.2 to 2.5 cm². Variations in E accompanying changes in transverse area appear to be related to cell number along the radii of plugs. Hollow cylindrical plugs for which wall thickness is maintained but total tissue area is changed show constant values of E. It is suggested that shear stresses within tissue samples influence E and are dependent upon cell number and tissue water content. Material with these properties would be a “poor choice” for constructing plant organs experiencing repeated stress and periodic dehydration. However, ground tissue may act as a buffer against localized ovaling of stem and leaf cross sections under loading.     

九连小檗悬浮培养细胞生理生化特性的研究——Ⅲ.药根碱累积与细胞生长的关系

本文叙述了九连小檗植物细胞悬浮培养过程中,药根碱的累积和细胞生长与培养液中可溶性糖转化的关系。实验表明细胞培养过程中培养液的可溶性糖逐渐消耗,细胞生物量和药根碱量都逐渐增加,且细胞生长与药根碱累积的曲线几乎是平行的。然细胞生长速度较快,其生长速率曲线的峰形较尖陡。药根碱累积速度较慢,延续时间较长,其累积速率曲线的峰形较平缓。根据糖的消耗与细胞生物量增长和药根碱累积的关系,计算出蔗糖——细胞转化率为59%,蔗糖——药根碱转化率为3.3%。     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

Summary In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square m rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.Supported by MRC of Canada research grant MA-6069Mr. D. Malka was supported by a studentship from the F.C.A.C.Dr. D. Ménard is a chercheur boursier du Conseil de la Recherche en Santé du Québec     

Organization of impulse flows and selective neuronal death in the hyperstriatum of chick embryos and chicks

Studies have been made on changes in the number of neurones per volume unit of the tissue of hyperstriatum tissue in chick embryos from the 18th day of incubation to the 3rd day after hatching. Significant interhemispheric asymmetry in migration rate and cell death was revealed in the dorsal hyperstriatum from the 19th day of embryogenesis, which may be due to asymmetry in the intensity of visual afferentation which depends on the stationary position of the embryo in the egg. In the activity of neuronal populations within the dorsal hyperstriatum at a stage which immediately precedes mass death of cells (up to 40%), a structure of impulse volley was found which is characterised by the onset of impulse discharges following each other in a form of short series. Sometimes it is possible to reveal correlation between the duration of dominating interdischarge intervals and the extremes in the recovery of excitability of neuronal pools, which indirectly indicates putative reverberative origin of impulse cyclic phenomenon.