Mathematical approach to measurements on embryonic chick lens

Profiles of fresh chick lenses of 5, 8, 11, 14, 17 and 20 days embryological age were photographed and from tracings of these profiles, volumes were calculated using a formula for volume of a solid of revolution. Accurate volumes were also obtained by assuming the lens to be two half oblate spheroids of different minor axis, or, better, two half-solids of profile (x/a)^k + (y/b)^k = 1. In addition, the k in this equation described well the curvature of the lens profiles and thus provided a quantitative measure of developing lens shape. Lens diameter, depth and shift of equatorial diameter upon lens axis as a function of age were also studied.     

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.     

Nuclear ADP-ribosylation in the chick lens during embryonic development

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.     

In vitro reformation of the perichondrium from perichondrial-free Meckel's cartilages of the embryonic chick

Perichondria were removed from Meckel's cartilages of chick embryos of Hamburger and Hamilton stages 34, 38, or 39 (8, 12, or 13 days of incubation) and cultured, either at the air-medium interface or submerged, under standard organ culture conditions, for 7 to 21 days. Meckel's cartilages formed a new fibrous perichondrium by the 10th day of culture. Perichondria both formed earlier and were thicker in those cartilages cultured at the air-medium interface than in those cultured submerged. Histological and ultrastructural analysis indicated that the outermost layer of Meckelian chondrocytes dedifferentiated into fibrous cells to form the new fibrous perichondrium; i.e., the fibrous perichondrium can arise from superficial chondrocytes.     

Anin vitro model for chick embryonic notochords

A two step method to obtain mesenchymal free 3.5 day old chick embryonic notochordsin vitro is presented. 1.) Notochords are isolated by mechanical microdissection from the embryos below the head and above the leg-buds. 2.) The dissected notochords are trypsinized to eliminate contaminating mesenchymal cells, while the perinotochordal sheath (PNS) is retained. After isolation and trypsinization, notochords are cut in standard 8mm lengths, explantedin vitro and incubated at 37°C. Immediately before incubation and after 3 and 6 daysin vitro, notochords are fixed and stained to follow the morphological changes. The total DNA content of notochords is measured before and during maintenancein vitro to evaluate their metabolic activities. Results show that during thein vitro period, the isolated mesenchymal free notochordal fragments can conserve their characteristic architecture. The total DNA content measurements indicate proliferative activity and a high viability of the notochords in ourin vitro system. In the present study, an isolation andin vitro method is offered which might be an effective tool to study the metabolic activities of chick embryonic notochordsin vitro in comparison toin vivo behaviour, in order to study the underlying mechanism of notochord regression.     

Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in vivo and in vitro

Protein synthesis and ultrastructure were studied in the cultured epithelium and in the intact lens of the 6-day chick embryo. Proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Synthesis of delta crystallin from embryonic chick lens messenger ribonucleoprotein complex

A single, major 21 S messenger ribonucleoprotein (mRNP complex) was isolated and purified by sucrose gradient centrifugation after EDTA treatment of high salt washed polysomes from 15 day embryonic chick lenses. A 17 S mRNA was released from the 21 S mRNP. The 21 S mRNP complex coded for a 50 000 molecular weight protein identical to the subunit of delta crystallin. Similar results were obtained with the 17 S mRNA released from the 21 S mRNP complex.     

Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells

Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Sulfate esterifying activity of embryonic chick liver in vitro

A method has been described for the study of tissue sulfate-conjugating systems in vitro. Liver slices from embryonic chicks were maintained in vitro in a medium containing labeled inorganic sulfate and phenol. It was found that more of the sulfate was esterified at 20 °C. than at 37 °C. due to the longer continued activity at the lower temperature. All sulfate-esterifying activity was lost in liver slices maintained at 37 °C. for 30 hr. while those cultures maintained at 20 °C. continued to esterify sulfate for 70 hr.On the basis of our data there would appear to be a change in the thermal stability of the sulfate-esterifying enzyme system of the chick liver upon its transition from the embryonic stage to the stage of the fully developed chick. Data were presented for the chick 4 months ex ovo. We have been unable to detect any analogous temperature effects upon the sulfate-esterifying system in the livers of embryonic and adult rats.