Spatial and temporal changes in the pattern of glycosylation of the developing chick limb tissue components as revealed by fluorescent conjugated lectin probes.

The changing pattern of expression of glycoconjugates during the differentiation of the chick leg bud between stages 17 to 34 (days 3 to 8 of incubation) was studied using fluorochrome-labelled plant lectins. Limb buds were fixed in cold acetic-alcohol and wax-embedded. Agglutinins of peanut (PNA), soybean (SBA) and succinylated wheat germ (WGAs) revealed a specific binding pattern in the apical ectodermal ridge (AER) between Hamburger and Hamilton stages 19-32. These stages coincide with the period of elevation of the AER. This specific binding pattern was absent from the adjacent dorsal and ventral ectoderm. Prechondrogenic cells were positive for WGA and for PNA, and the PNA-binding capacity was intensified after neuraminidase treatment. Premyogenic cells at stage 23 can be identified as negative to PNA after neuraminidase, while the blood vessels became positive. PNA, SBA, WGA, WGAs and, in addition, Ricinus communis (RCA-I) lectins stained the basal membrane. Strands of extracellular matrix which connect with the basal membrane and cross the limb transversely between dorsal and ventral ectoderm were stained by RCA-I, SBA and PNA after neuraminidase.

Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud.

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.

Diaminobenzidine histochemistry in the rat limb bud.

Diaminobenzidine (DAB) histochemistry was used to determine enzymatic or non enzymatic peroxidase activity in the rat limb bud on days 12 and 13 of gestation. The optimal technical conditions were determined. Light microscopy revealed the presence of positive intracellular granules in the apical ectodermal ridge (A.E.R.) and in the condensed mesoderm. Their distribution in the A.E.R. shows a caudo-cephalic gradient. Electron microscopy, red oil red, and supravital Nile blue staining suggest the lipidic nature of these granules. The hypothesis of an endogenous pseudo- or peroxidase activity are discussed.

Acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) activity distribution pattern in early developing chick limbs.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) activities was studied by histochemical, quantitative and electrophoretical methods during the early development of chick limbs, from stage 16 to stage 32 H.H. (Hamburger & Hamilton, 1951). By quantitative methods, true AChE activity was found, and increased about threefold during the developmental period, together with a smaller amount of BuChE which increased more rapidly in comparison with the AChE activity from stage 25 to 32 H.H. Cholinesterase activity was histochemically localized mainly in interacting tissues, such as the ectoderm (including the apical ectodermal ridge) and the underlying mesenchyme. True AChE was histochemically localized around the nuclei and on the plasma membrane of ectodermal (including AER) and mesenchymal cells, and at the plasma membrane of mesenchymal cell processes reaching the basal lamina between the ectoderm and the mesenchyme. AChE together with BuChE activity was found in the basal lamina between the ectoderm and the mesenchyme, in underlying mesenchymal cells and in deeper mesenchymal cells, especially during their transformation into unexpressed chondrocytes. During limb morphogenesis, the cellular and regional localization of the enzyme activities showed variations depending on the stage of development and on the occurrence of interactions. The possibility of morphogenetic functions of the enzyme id discussed.

Limb development in chick embryos: cyclic AMP-dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis.

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20-23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25-28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP-dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80-85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane-enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP-dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP-protein phosphorylation system in the differentiation of limb tissues.

Estimation of creatine kinase isoenzymes: the effects of caffeine and retinoic acid on CK levels in fetal mouse limbs.

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

The brachymorphic mutation of mice and altered developmental patterns of limb bud 3':5' cyclic adenosine monophosphate.

Concentrations of cyclic AMP (cAMP) were determined in paired fore and hind limbs from day 12-16 of development in murine fetuses homozygous for the brachymorphic (bm) mutation and normal controls. A developmental rise in cAMP occurred 1 day earlier in bm/bm than in +/+ hind limbs and cAMP was higher in day-13 bm/bm than in +/+ fore limbs. Since cAMP is well documented to stimulate chondrogenic differentiation, premature cartilage determination secondary to altered levels of cAMP could play a role in bm/bm short-limbed dwarfism.

Characterization of embryonic cholinesterase in chick limb bud by colorimetry and disk electrophoresis.

In the chondrogenic blastema of the chick limb bud an histochemical cholinesterase activity related to aggregation of the chondroblasts was described by Drews and Drews (1972). This cholinesterase activity was termed "embryonic cholinesterase" (Drews 1975). In the present study embryonic cholinesterase from the limb bud has been characterized by colorimetry and disk electrophoresis and compared to cholinesterase from the myotomes of the same embryos. Embryonic cholinesterase comprises both acetylcholinesterase and butyrylcholinesterase activity. The banding patterns of embryonic cholinesterase from limb bud and of cholinesterase from myotomes are identical. These findings support the hypothesis that the cholinergic system is involved in the regulation of embryonic development.

The development of alkaline phosphatase activity in limb buds of mouse embryos in vitro and its relation to chondrogenesis.

The development of alkaline phosphatase (aPh) activity and chondrogenesis were studied in the limb buds of mouse embryos (day 11 p.c.) that had been grown in an organ culture. During a 12-day culture period an increase in aPh activity to more than 40 mU/limb bud was measured from day 2 in vitro onward. Depending on the time of application, aPh formation can be inhibited by certain substances. Cytosine-arabinoside inhibits aPh activity when the substance is added on day 2, 3, or 4. Chondrogenesis, on the other hand, is affected on days 1, 2, and 3 and to a lesser degree on day 4. Actinomycin D interferes with aPh activity after its addition on day 1, 2, 3, or 4. Chondrogenesis is only inhibited when the drug is applied on the 1st, 2nd, or to a lesser degree on the 3rd day. Cycloheximide inhibits aPh formation on all days of treatment, but to a lesser degree on days 5 and 6; chondrogenesis is most influenced on days 2, 3, and 4. On day 6 of the culture period, aPh activity can be demonstrated histochemically only in the region of humerus and proximal parts of radius and ulna. Alterations in the distal cartilage skeleton, therefore, do not influence the activity data. A prerequisite for an increase in aPh activity is cartilage growth in the proximal part of the limb buds and subsequent induction of a perichondral cell population to proliferation and differentiation.

Alpha-Glycerophosphate dehydrogenase (insoluble) and lactic dehydrogenase activities in the skeletal muscles of two insects.

The flight muscle preparations of the dragonfly Pantala flavescens and the aquatic beetle Cybister confusus showed extremely low levels of lactic dehydrogenase activity and high levels of alpha-glycerophosphate dehydrogenase (insoluble) activity. The activities of these two enzymes in the leg muscle of the beetle were approximately the same (1:1), but lactic dehydrogenase activity was several times higher than that in the flight muscles of both Insects. These results have been interpreted as indicating the high energy-yielding demands of the flight muscles during continuous sustained activity, while the leg muscles of the beetle which are involved in swimming activity derive their energy predominantly through anaerobic glycolysis.