Robert L. Trelstad

Cell contact during early morphogenesis in the chick embryo.

Abstract The primitive epithelial layers of the chick blastoderm, the epiblast and hypoblast, are underlain by discontinuous basement laminae. Primary mesenchymal cells, which comprise the mesoblast that migrates laterally from the primitive streak, make close junctions (intercellular space, 25–100 A) and tight junctions (no apparent extracellular space) with the basal surfaces of the epiblast and hypoblast. Close and tight junctions are also prominent between mesenchymal cells, especially along their trailing edges. Filopodia are most numerous on the leading edges of the migrating cells as would be expected if the close and tight junctions played a role in contact inhibition, perhaps by mediating electrical coupling between the cells. The tight junctions within mesoblast, epiblast, and hypoblast are at first focal in nature (maculae occludentes), but subsequently those between cells in the same tissue become more extensive while those between cells in unlike tissues are disrupted. When the primary mesenchyme organizes itself into an epithelium (somite, lateral, and intermediate mesoderm), the trailing ends of the cells, which contain the Golgi organelles, are directed toward the middle of the mass where zonulae occludentes appear next to the newly created free surface. Subsequently, secondary mesenchyme derives from the mesodermal epithelium. The migrating cells are connected by broad tight junctions (fasciae occludentes) which seem to persist as maculae occludentes after the presumptive connective tissue cells differentiate. Within the epithelia, maculae adhaerentes (desmosomes) are particularly well developed at later stages and probably contribute to tissue-specific intercellular adhesion.

Hyaluronate production and removal during corneal development in the chick

Abstract Glycosaminoglycan (acid mucopolysaccharide) synthesis was studied during development of the embryonic chick cornea by the introduction of isotopically labeled precursors both in ovo and to excised corneas in culture. Results obtained for both types of preparations were similar. Hyaluronate is the major glycosaminoglycan synthesized by the embryonic cornea between stages 24 and 35, a period during which the primary corneal stroma swells and is invaded by mesenchymal cells. Subsequent to the completion of invasion a rapid decline in incorporation of isotopic precursors into hyaluronate occurs concomitant with an increasing hyaluronidase activity. The hyaluronidase is present primarily between stages 35 and 38, when the cornea begins to dehydrate prior to becoming transparent.

Tendon collagen fibrillogenesis: Intracellular subassemblies and cell surface changes associated with fibril growth

The fine structure of tendon fibroblasts in developing limbs of chick and mouse embryos have been examined to define the cellular features of collagen fibrillogenesis in vivo . Two structural features of the fibroblast which may relate to fibril assembly have been observed: intracellular aggregates resembling SLS (segment-long-spacing)-like aggregates of monomeric and dimeric lengths and deep recesses of the cell surface containing striated fibrils. A model is proposed which suggests that assembly of a collagen fibril in vivo begins by formation of subassemblies inside the cell which then are excreted by exocytosis and added to a unique region at the fibril end. This end is closely associated with the cell surface in a manner which allows for cellular regulation of some parameters of fibril morphology.

Temporal and spatial transitions in collagen types during embryonic chick limb development.

Abstract To determine whether transitions occur in the types of collagen synthesized during embryonic chick limb development, the α chain composition of the collagens produced by whole limbs and various anatomical regions of limbs was analyzed at different stages (23–24 to 40). The tissues were incubated in the presence of 3 H-proline and 3 H-lysine and the α chain distribution of the purified, labeled collagens was determined by chromatography on carboxymethyl cellulose columns. We found that the stage 23–24 leg mesenchyme is producing predominantly, if not solely, an (α1) 2 α2 type collagen (chain type as yet undetermined). At about stage 25–26 the limb core begins synthesizing detectable amounts of (α1) 3 collagen, which we presume to be cartilage type collagen, [α1 (II)] 3 , while the outer portion of the limb largely continues to produce (α1) 2 α2. The production of (α1) 3 collagen in the core progressively increases until, by stage 33 it is the only species detectable in the tibial diaphysis. Shortly thereafter (by stage 35 + –36) (α1) 2 α2 type collagen reappears in the tibial diaphysis signifying the production of bone collagen, [α1 (I)] 2 α2. During the next several days of incubation, the relative proportion of (α1) 2 α2 increases in the bony diaphysis while (α1) 3 remains the predominant species synthesized in the cartilaginous epiphysis.

Collagen fractionation: Separation of native types I, II and III by differential prectipitation☆

Abstract Collagen types I, II and III can be purified in their native state from heterogeneous collagen solutions by fractional precipitation at neutral pH using ammonium sulfate, sodium chloride and ethanol as precipitants. This method of collagen separation is useful as a preparative procedure and should also serve as an analytical tool for identification of unknown radioactively labeled collagens.

Human aorta collagens: Evidence for three distinct species☆

Abstract Three different molecular species of collagen and a soluble form of elastin were obtained by digestion of human aortas with pepsin. Two of the three collagens contain 1 2 cystine, present in interchain disulfide crosslinkages, and appear to represent type IV collagen previously described in basement membranes and type III collagen, recently found in fetal skin. The third collagen species is type I, the molecule found in a wide variety of connective tissues including skin, bone, tendon and ligaments.

A Collagen Defect in Homocystinuria

The biochemical mechanism accounting for the connective tissue abnormalities in homocystinuria was explored by examining the effects of various amino acids known to accumulate in the plasma of patients with this disease on cross-link formation in collagen. Neutral salt solutions of purified, rat skin collagen, rich in cross-link precursor aldehydes, were polymerized to native type fibrils by incubating at 37 degrees C in the presence of homocysteine, homocystine, or methionine. After the polymerization was completed, each sample was examined for the formation of covalent intermolecular cross-links, assessed indirectly by solubility tests and directly by measuring the cross-link compounds after reduction with tritiated sodium borohydride and hydrolysis. Collagen solutions containing homocysteine (0.01 M-0.1 M) failed to form insoluble fibrils. Furthermore, much less of the reducible cross-links, Delta(6,7) dehydrohydroxylysinonorleucine, Delta(6,7) dehydrohydroxylysinohydroxynorleucine, and histidino-dehydrohydroxymerodesmosine were formed in the preparations containing homocysteine as compared with the control and the samples containing methionine or homocystine. The content of the precursor aldehydes, alpha-aminoadipic-delta-semialdehyde (allysine) and the aldol condensation product, was also markedly diminished in tropocollagen incubated with homocysteine. It is concluded that homocysteine interferes with the formation of intermolecular cross-links that help stabilize the collagen macromolecular network via its reversible binding to the aldehydic functional groups. Analysis of the collagen cross-links in skin biopsy samples obtained from three patients with documented homocystinuria showed that the cross-links were significantly decreased as compared with the age-matched controls, supporting the tentative conclusions reached from the in vitro model studies. In addition, the solubility of dermal collagen in non-denaturing solvents was significantly increased in the two patients examined, reflecting a functional defect in collagen cross-linking. Although the concentration of homocysteine used in this study to demonstrate these effects in vitro is clearly higher than that which is observed in homocystinurics plasma, the data do suggest a possible pathogenetic mechanism of connective tissue defect in homocystinuria.

The collagen of chick embryonic notochord

Abstract Notochords, isolated from 2 1 2 day chick embryos, were cultured in the presence of 3H proline and the labeled proteins co-purified with chick skin carrier collagen. The purified material, most of which eluted from CM-cellulose as a single peak in the region of the carrier collagen α1 chain, contained 41% of the incorporated proline as hydroxyproline and from gel filtration measurements had a molecular weight of approximately 100,000 daltons. When the material was chromatographed on DEAE-cellulose with carrier α1 chains from both skin [α1 (I)] and cartilage [α1 (II)], it eluted predominantly with the cartilage chains.

Collagen Synthesis in vitro by Embryonic Spinal Cord Epithelium

Isolated spinal cords from chick embryos (stages 12 to 15) were incubated in vitro with radioactive proline. The proteins synthesized were fractionated by coprecipitation with added carrier collagen, followed by molecularsieve and ion-exchange chromatography. A portion of the isotopically labeled proteins were found to be collagen molecules consisting only of α1 chains.

Müllerian-Inhibiting Substance: An Update

The decades long study of Mullerian Inhibiting Substance by numerous laboratories around the world has been driven, in large part, by pediatric surgeons and pediatric endocrinologists who have a keen interest in the molecular pathophysiology of genital tract defects that are visited upon their patients. A better understanding of the genes involved in the development of the normal reproductive tract in males and females should lead to a more rational analysis of the diseases caused by their abnormal function. Furthermore, a translation of this knowledge from the bench to the bedside may lead to clinically useful advances in the diagnosis and management of intersex patients. The molecular analyses of MIS and MIS receptor gene mutations and persistent Mullerian duct syndrome and the development of MIS ELISAs to evaluate testicular function as well as to follow the progress of gonadal tumors are several clear examples of successes over the years. It will be interesting to see what lies ahead.