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.     

The collagen of osteogenic cartilage in the embryonic chick

The diaphyseal region of tibiae, and vertebral bodies from 8-day chick embryos were cultured in the presence of tritiated proline and the radioactive proteins were extracted and co-purified with carrier collagen. Chromatography on carboxymethylcellulose indicated that the radioactively labelled proteins eluted as a single peak which coincided with the carrier α1 chains. On DEAE-cellulose, the radioactively labelled α1 chains eluted with authentic cartilage α1 (II) carrier. The transitory chondrogenic regions of the embryo thus produce a collagen molecule similar, if not identical, to the principal collagen molecule found in cartilaginous structures in the adult.     

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.     

Developmental changes in the ability to express embryonic pepsinogen in the stomach epithelia of chick embryos

Summary The avian stomach is subdivided into two parts, the proventriculus and the gizzard. It has been shown that the gizzard epithelium can express embryonic chick pepsinogen (ECPg) antigen, a marker protein of the proventricular epithelium, as well as normal proventricular epithelium, under the appropriate experimental conditions. To study the possible mechanisms involved in the suppression of ECPg synthesis in the gizzard epithelium during normal development, we carried out heterotypic and heterochronic recombination experiments of the epithelium and mesenchyme of these two organ rudiments. When recombined and cultured with 6-day proventricular mesenchyme, gizzard epithelium of 3.5- to 12-day embryos expressed pepsinogen at all stages tested. However, the ratio of ECPg-positive cells to total epithelial cells in the gizzard epithelium decreased rapidly when epithelium older than 7 days was cultured with proventricular mesenchyme. In contrast to proventricular mesenchyme, 6-day gizzard mesenchyme did not allow ECPg expression in associated proventricular epithelium of 3.5- to 7-day embryos. These results indicate that gizzard epithelium does not express pepsinogen in normal development because of both a decrease in ability to express the enzyme in itself in the course of development and a repressive influence of gizzard mesenchyme.     

Effects of dideoxyforskolin on proteoglycan synthesis and structure in embryonic chick chondrocyte cultures

1,9-Dideoxyforskolin inhibits proteoglycan synthesis and xyloside-initiated glycosaminoglycan (GAG) synthesis in chick embryo chondrocytes. Dideoxyforskolin does not affect the length of xyloside-initiated GAG chains secreted into the medium but chains from the dense proteoglycan secreted into the medium appear slightly longer. Incorporation of labeled serine into the dense proteoglycan and subsequent digestion with Pronase revealed a dramatic decrease in percent of total radioactivity associated with GAG chains in the proteoglyean from cultures treated with forskolin or dideoxyforskolin. These observations suggest that these diterpenes have a specific inhibitory effect on chain initiation reactions and thus may be useful tools in the study of proteoglycan synthesis and processing.     

The occurrence of low buoyant density proteoglycans in embryonic chick cartilage

Two proteoglycan fractions, PCS-H and PCS-L, have previously been isolated from 4 M guanidine HCl extract of embryonic chick cartilages. This communication reports further studies with PCS-L indicating that this fraction contains several different forms, of which one differs from hitherto known cartilage proteoglycans in 1) markedly lower buoyant density, 2) susceptibility to reduction with 2-mercaptoethanol, 3) aggregate-forming ability in 4 M guanidine HCl, and 4) presence of dermatan sulfate-chondroitin sulfate copolymer chains. Also isolated from the PCS-L fraction is a keratan sulfate-rich proteoglycan which represents the smallest molecular size species in cartilage proteoglycan populations.     

Phenotypic characteristics of adipocytes generated from Meckel's chondrocytes in response to chick serum in vitro

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.     

Development of glucose active transport in embryonic chick intestine. Influence of thyroxine and hydrocortisone

1. Glucose active transport is detectable in 12-day-old embryonic chick duodenum and increases at least 11-fold after 4 days of postnatal life. 2. Glucose active transport develops at the in vivo rate in 72-hr cultures of 14-day embryonic duodenum. 3. In the presence of either 1 nM thyroxine or 1 microM hydrocortisone in vitro, glucose active transport reaches levels approximately 200% of control values (equivalent to 18-19 day levels in vivo). 4. Thyroxine and hydrocortisone act by different mechanisms based on their antagonistic interaction and differences in time course of action, requirement for protein synthesis and modulation by extracellular calcium.