James W. Lash

An experimental analysis of the development of the spinal column: VI. Aspects of cartilage induction☆

Abstract Both the embryonic spinal cord and notochord induce the formation of cartilage in a population of chick somite cells. This inducing capacity is relatively specific—most other living and fixed tissues, including living and fixed cartilage, are ineffective. The cartilage promoting activity of the spinal cord appears to be mediated by a factor transmissible through mesenchymal or muscle tissue and through a millipore filter. The cartilage promoting activity of the notochord acts more locally and appears to be associated with the notochordal sheath. The time for the passage of the spinal cord cartilage promoting factor through the millipore filter is approximately eight hours.

Environmental enhancement of in vitro chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein.

Proteoglycan complex extracted from embryonic cartilage (chondromucoprotein) with 4.0 M guanidinium chloride greatly stimulates in vitro somite chondrogenesis. In the presence of exogenous chondromucoprotein (CMP) which consists predominantly of proteochondroitin sulfate, there is a large increase in the amount of differentiating cartilage which can be detected visually in somite explants. There is a 2–3-fold increase in the amount of sulfated glycosaminoglycans (including chondroitin 4- and 6-sulfate) accumulated by somite explants supplied with exogenous CMP complex. These results are of potential significance, since during the period of interaction between the notochord or spinal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan.

Hydroxylation of Proline and the Intracellular Accumulation of a Polypeptide Precursor of Collagen

Autoradiographs of embryonic cartilage indicated that labeled protein accumnulated intracellularly when the tissue was incubated with tritiated proline, and when the hydroxylation of proline was inhibited by anaerobic conditions or by a chelator for ferrous iron. The labeled protein apparently corresponds to protocollagen. the polypeptide precursor of collagen which serves as a substrate for the enzymatic synthesis of hydroxyproline.

Somites in developing embryos

Somitomeres: The primordial body segments.- The metameric organization of the presomitic mesoderm and somite specification in the mouse embryo.- The epiblast origin of avian somite cells.- Aspects of somite formation in the early chick embryo.- The early development of the intermediate mesoderm in the chick.- The effects on vertebral development of removing a single somite from a 2-day old chick embryo.- On the problem of metamerism in the head mesenchyme of chick embryos.- The pattern of communication through gap junctions during formation of the embryonic axis.- A unique population of non-dividing cells in the somites.- Somitogenesis in the frog.- Heat shock effects in chick embryos.- Cell lineage and the formation and maintenance of half somites.- The tail bud and cessation of segmentation in the chick embryo.- Models of segmentation.- A comparison of the adhesiveness of somitic cells from chick and quail embryos.- The adhesion recognition signal of fibronectin: A possible trigger mechanism for compaction during somitogenesis.- Genetic modifications of developmental acts in chick and mouse somite development.- CSAT antibody interferes with in vivo migration of somitic myoblast precursors into the body wall.- Somite chondrogenesis: Extracellular matrix production and intracellular changes.- Initiation of chondrogenesis in somitic, limb and craniofacial mesenchyme: Search for a common mechanism.- Myogenesis: A problem of Cell Distribution and Cell Interactions.- The distribution of somite-derived, myogenic cells during early development of the wing bud.- Somites and neural development.- The programming of vertebral development.

A role for fibronectin in the migration of avian precardiac cells: I. Dose-dependent effects of fibronectin antibody

An anterior-posterior concentration difference of fibronectin associated with the endoderm in early chick embryos has been implicated in the directional migration of precardiac mesoderm cells. We have examined the effect of increasing concentrations of an antibody to fibronectin (FN) to test the essentiality of FN to precardiac cell migration. For controls embryos were incubated in the presence of antibodies produced against several other extracellular components, such as laminin and anti-collagen types I and IV, as well as against integrin, a cell surface FN receptor. Embryos were also incubated in the presence of a high concentration of exogenous FN, as well as in the presence of an RGD-containing synthetic pentapeptide that is recognized by the FN receptor. After incubation of chick embryos in various concentrations of anti-FN (5 to 80 micrograms/ml), a dose-dependent effect of anti-fibronectin was observed, whereby heart development was arrested at high concentrations of anti-FN. Early developmental stages were more susceptible to lower antibody concentrations than later stages. Incubation in the presence of the RGD-containing synthetic peptide resulted in partial cardiabifida. None of the antibodies serving as controls affected cell migration or early heart development. These results support the hypothesis that FN is a major component in the migratory pathway and plays a role in the directional migration of precardiac cells to the embryonic midline.

Precardiac cell migration: Fibronectin localization at mesoderm-endoderm interface during directional movement☆

The pathway of directional movement of chick precardiac mesoderm cells was studied by indirect immunofluorescence and by scanning electron microscopy. Directional movement of the precardiac cells begins at stage 6 from the lateral sides of the embryo at the level of Hensens node. The cells move anteriorly in an arc to the embryos midline. By stage 8 the cells arrive at the lateral sides of the anterior intestinal portal and movement ceases. The interval of this directional movement is approximately 10 hr. During migration the precardiac cells are in close association with the underlying endoderm. As migration proceeds, the cells encounter increasing amounts of fibrils in the substratum at the mesoderm-endoderm interface. Concomitant with increasing fibril formation there is an increase in fibronectin (FN) in the heart-forming region. During stage 5 FN first appears in the lateral heart-forming regions and increases in amount during the period of cell migration. By stage 7 a concentration difference of FN is apparent in the lateral regions with more FN cephalad and decreasing amounts caudad. At stages 7 and 8 large amounts of extracellular FN-associated fibrils are observed at the lateral sides of the anterior intestinal portal where the cells stop moving. The precardiac cells moving into this region are oriented perpendicular to the anterior intestinal portal and in close association with these fibrils. There is no evidence that the fibrillar meshwork forming the substratum of the precardiac mesoderm cells is physically oriented as a guide for directional movement. The correlations between FN distribution at the mesoderm-endoderm interface and directional cell movement suggest that the precardiac cells may migrate by haptotaxis, i.e., by moving along the substratum toward areas of greater adhesiveness.

Notochordal stimulation of in vitro somite chondrogenesis before and after enzymatic removal of perinotochordal materials.

In the present investigation, evidence is presented directly implicating proteoglycans produced by the embryonic notochord in the control of somite chondrogenesis. It has been demonstrated by several histochemical techniques that during the period of its interaction with somites, the notochord synthesizes perinotochordal proteoglycans, and these proteoglycans have been shown to contain chondroitin 4-sulfate (40%), chondroitin 6-sulfate (40%), and heparan sulfate (20%). Dissection of notochords from embryos with the aid of a brief treatment with trypsin results in the removal of perinotochordal extracellular matrix materials including proteoglycans, while dissection of notochords without the aid of enzyme treatment or with a low concentration of collagenase results in their retention. There is a considerable increase in the rate and amount of cartilage formation and a corresponding 2 to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated by somites cultured in association with notochords dissected under conditions in which perinotochordal materials are retained. Treatment of collagenase-dissected or freely dissected notochords with highly purified enzymes (chondroitinase ABC, AC, and testicular hyaluronidase) which specifically degrade proteoglycans causes a loss of histochemically detectable perinotochordal proteoglycans. These notochords are considerably impaired in their ability to support in vitro somite chondrogenesis. In addition, when trypsin-treated notochords are cultured (“precultured”) for 24 hr on nutrient agar (in the absence of somites), perinotochordal material reaccumulates. Somites cultured in association with such “precultured” notochords exhibit considerable increase in the amount of cartilage formed and a 2- to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated as compared to somites cultured in association with trypsin-treated notochords which have not been “precultured.” This observation indicates that trypsin-treated notochords reacquire their ability to maximally stimulate in vitro somite chondrogenesis by resynthesizing and accumulating perinotochordal material. Finally, “precultured” notochords treated with chondroitinase to remove perinotochordal proteoglycans are considerably impaired in their ability to support in vitro somite chondrogenesis. These observations are consonant with the concept that proteoglycans produced by the embryonic notochord play an important role in somite chondrogenesis.

Induction of cell differentiation: I. The in vitro induction of vertebral cartilage with a low-molecular-weight tissue component

Abstract A nucleotide-containing fraction has been obtained from a cold perchloric acid extract of the chick embryonic spinal cord and notochord that simulates the chondrogenic action of the intact inducing tissues. This fraction induces the formation of cartilage in explanted somites. Upon partial purification of this fraction the ability to induce cartilage formation has been restricted to one nucleotide-containing component.

Environmental enhancement of in vitro chondrogenesis

Abstract In most in vitro tissue interaction studies, it is assumed that the negative control of the culture system (i.e., the tissue which does not differentiate when isolated) is representative of an in vivo situation, and that the isolated tissue is quite unable to differentiate without the interacting tissue. It is becoming increasingly obvious that the failure of isolated tissues to differentiate in vitro may be due to the techniques of the experimenter, not necessarily to metabolic deficiencies of the tissue. The results reported here show that while it is possible to demonstrate an effective interaction in which notochord promotes the differentiation of cartilage from somitic tissue, the negative result (somites alone failing to undergo chondrogenesis) applies only to a prescribed set of culture conditions. By substituting fetal calf serum and other nutrient supplements for horse serum, which was previously used in the nutrient medium, the incidence of in vitro chondrogenesis is markedly enhanced in somites cultured in the absence of notochord. The notochord therefore does not impose chondrogenic information upon the somites, it only permits or enhances a preexisting chondrogenic bias of the somites. In addition, DNA synthesis and proliferation were found to have only a desultory relation to chondrogenesis. The primary role of proliferation is to provide new cells for a continuing process of chondrogenic differentiation.

A role for fibronectin in the migration of avian precardiac cells: II. Rotation of the heart-forming region during different stages and its effects☆

Precardiac cells in early chick embryos between stages 5 and 8 of development migrate anteriorly and medially from two well-defined, lateral heart-forming regions to the lateral walls of the developing anterior intestinal portal. Previously, it was shown that an increasing fibronectin (FN) concentration exists at the endoderm-mesoderm interface along the pathway in which the cells move. Thus, a haptotactic mechanism for precardiac cell migration was suggested. To analyze fibronectins role further the FN concentration difference was interrupted by microsurgically rotating both the precardiac region mesoderm and endoderm (ectoderm was left intact) and by perturbation with the use of an antibody to fibronectin. These experiments, reported here, indicated that precardiac cells do follow cues established by a FN concentration difference at the mesoderm-endoderm interface in the lateral region and that anti-fibronectin can inhibit normal cell migration during heart formation.