Stephen Meier

Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production.

Abstract The primary stroma of the cornea of the chick embryo consists of orthogonally arranged collagen fibrils embedded in glycosaminoglycan (GAG) produced by the epithelium under the early inductive influence of the lens. The experiments reported here were designed to test whether or not the collagen of the lens basement lamina is capable of stimulating corneal epithelium to produce primary stroma. Enzymatically isolated 5-day-old corneal epithelia were grown for 24 hr in vitro in the presence of 35SO4 or proline-3H on various substrata. Epithelia cultured on lens capsule synthesized 2.5 times as much GAG (as measured by incorporation of label into CPC precipitable material) and almost 3 times as much collagen (assayed by hot TCA extraction or collagenase sensitivity) as when cultured on Millipore filter or other noncollagenous substrata. A similar stimulatory response was observed when epithelium was combined with chemically pure chondrosarcoma collagen, NaOH-extracted lens capsule, vitreous humor, frozen-killed corneal stroma or cartilage, or tendon collagen gels; in the latter case, the magnitude of the effect can be shown to be related to concentration of the collagen in the gel. All of the collagenous substrata stimulate not only extracellular matrix production, but also polymerization of corneal-type matrix, as judged by ultrastructural criteria and by the association of more radioactivity with the tissue than the medium. Since purified chondrosarcoma collagen is as effective as lens capsule, the stimulatory effect on collagen and GAG synthesis by corneal epithelium is not specific for basal lamina (lens capsule) collagen.

Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern.

Abstract The mesoblast of the primary organizer region of the developing chick embryo at the early head process stage was examined with the scanning electron microscope. It was found that the mesoblast layer is patterned from its inception at the primitive streak. Viewed dorsally, the mesoblast region most recently traversed by Hensens node is metameric. Each segment consists of two 175-μm-diameter circular buttons of paraxial mesoblast (somitomeres) and an enclosed axial region. These tripartite segments are stacked tandemly and mark precisely, in the ectoderm above, the limit of neural plate formation. Viewed ventrally, the metameric pattern of the mesoblast is most closely mimicked by underlying endoblast, which shows corresponding radially arranged wedge-shaped cells in somitomere-sized circular patches. At this stage of development, each paraxial somitomere is a slightly hollowed, squat cylinder, composed of tapering mesenchymal cells whose long axes are directed toward the core center. Closely timed with neurulation, somitomeres undergo morphogenesis, being first converted to triangular wedges and, finally, condensed into cubes. Anteriorly, somitomeres participate in branchiomeric development, while posteriorly, they develop into somites. Examination of segmental plates shows that they consist of about 11 tandem somitomeres not visible by light microscopy. The most mature somitomeres, closest to the emerging somites, are delineated from one another by cellular orientations and the progressive buildup of fibrous extracellular matrix. The least mature somitomeres are not as well defined, but appear initially just posterior to Hensens node and merge medially with the notochordal process. The observations suggest that the emergence of somitomeres from the paraxial mesoblast of the primitive streak is the result of its association with nodal cells. Further, this combined association of the mesoblast heralds primary induction and establishes the metameric pattern of the basic body plan.

A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

In this investigation, Triton X-100 extraction was utilized to examine the cytoskeleton of ascidian eggs and embryos. The cytoskeleton contained little carbohydrate or lipid and only about 20-25% of the total cellular protein and RNA. It was enriched in polypeptides of molecular weight (Mr)54, 48, and 43 x 10(3) Mr polypeptide was identified as actin based on its Mr, isoelectric point, and affinity for DNase I. Electron microscopy of the detergent-extracted eggs showed that they contained cytoskeletal domains corresponding to colored cytoplasmic regions of specific morphogenetic fate in the living egg. A yellow crescent cytoskeletal domain in the myoplasm was examined and shown to consist of a plasma membrane lamina (PML) and a deeper lattice of filaments which appeared to connect the yellow crescent pigment granules to the PML. The PML probably consists of integral membrane proteins stabilized by an underlying network of actin filaments since NBD-phallacidin stained this area of the egg cortex and the PML was extracted from the cytoskeleton by DNase I treatment. The yellow crescent cytoskeletal domain was found throughout the cortex of the unfertilized egg. During ooplasmic segregation it progressively receded into the vegetal hemisphere and was subsequently partitioned to the presumptive muscle and mesenchyme cells of the 32-cell embryo. It is suggested that contraction of the actin network in the yellow crescent cytoskeletal domain is the motive force for ooplasmic segregation. This structure may also serve as a framework for the positioning of morphogenetic determinants involved in muscle cell development.

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

Abstract The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when 3 H-glucosamine is used to label GAG as when 35 SO 4 is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.

The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo

Abstract In recent studies of chick embryos, the cranial paraxial mesoblast was found to be patterned into segmental units termed somitomeres. Anterior to the first segmental cleft, seven contiguous segments are aligned, with somitomeric interfaces forming grooves at right angles to the midline. In this study, the morphological relationship between the migratory pathways of cranial neural crest cells and patterned primary mesenchyme was analyzed with the scanning electron microscope, utilizing stereo imaging. In addition, the development of neuromeres in the adjacent neural tube was monitored. It was found that cranial neural crest first appears along the dorsal midline as a ridge of cells which loosens from the wall of the neural tube and migrates laterally as discrete populations. The mesencephalic crest appears first, immediately following neural tube fusion at that level, and migrates over the dorsal surface of the adjacent third somitomere and into the grooves formed by its juncture with the second and fourth somitomeres. Later, the addition of prosencephalic and rostral rhombencephalic crest extends the mesencephalic population to form a shelf of crest which spreads over the dorsal surface of the first four somitomeres. Component cells of this most cranial crest shelf become oriented and mimic the metameric pattern of the subjacent somitomeres. Crest cells adjacent to the fifth somitomeres appear along the midline, but do not migrate, creating a gap anterior to the otic crest. By stage 9, a narrow finger-like segment of the otic crest migrates from a specific neuromere into the grooved interface between the fifth and sixth somitomeres, delimiting the rostral border of the otic placode in the ectoderm above. By the end of stage 9, crest cells delimiting the caudal border of the placode have migrated along the interface of the seventh and eighth somitomeres. The crest cells adjacent to the sixth and seventh somitomeres, between the rostral and caudal otic populations, appear but do not migrate, remaining condensed along the midline. Thus, otic crest cells form a ring which circumscribes the invaginating otic placode. This study suggests that the precise distribution of cranial neural crest cells may result from their introduction at specific times, as specific populations from specific brain regions (neuromeres), onto a patterned mesodermal layer.

Development of the embryonic chick otic placode. II. Electron microscopic analysis.

At the 7–8 somite stage of embryonic chick development (29‐31 hours of incubation), a slightly elliptical island of thickened ectoderm appears laterally on either side of the most distal constricture of the rhombencephalon at the level of the anterior intestinal portal. The appearance and extent of this auditory placode is precisely correlated with the subjacent accumulation of neural crest cells. By 33 hours of incubation, there is a distinct depression in the developing otic placode, and by 40 to 45 hours, the placode is visibly invaginated, forming an epithelial vesicle or otocyst. Carefully staged embryos were serially sectioned, and the area underlying the developing otic placode was traced with a planimeter. It was found that placode size (area 60,000 μm2) is nearly unchanged from 30 to 42 hours of development. During this time interval, the placode cells first become columnar, show nuclear orientation, and then pseudostratify. The increase in placode cell number during this time interval is not likely to be the result of localized, accelerated cell division: the population doubling time of placode cells is eight and one‐half hours and the mitotic index of 2.5% is similar to that of cells in an equivalent area of adjacent, non‐placode forming head ectoderm. A model of otic placode formation is proposed which suggests that by 30 hours of development, a discrete population of placode forming cells is segregated from head ectoderm. Subsequent epithelial pseudostratification results from accumulation of this dividing population within the limits of the placode.

Development of the chick embryo mesoblast: morphogenesis of the prechordal plate and cranial segments

A scanning electron microscopic (SEM) examination of the mesoblast layer in the region of the primary organizer of the chick embryo shows that the mesoderm lying on either side of the anteriormost end of the primitive streak adjacent to Hensens node is organized into circular patches, 175 μm in diameter. Both of these squat cylinders of mesoblat (somitomeres) are joined together by an intervening axial segment laid down by the regression of Hensens node between them. Since pairs of somitomeres are added tandemly by node regression down the streak, they form the basis for metamerism in the embryonic axis. This study reports that as pairs of somitomeres accumulate, they change architecturally, undergoing morphogenesis in a cranial-to-caudal sequence that is coordinate with neurulation. Ultimately, cranial somitomeres expand under the medullary plate and participate in branchiomeric development, while caudal ones condense and develop into somites. At the light level, the first segmental cleft actually appears between the seventh and eighth somitomeres and is apparently the result of somitomeric contraction. While the seventh somitomeres form “somites” in the sense that their cells are radially oriented, their cranial border merges with the sixth pair. The sixth somitomeres are less condensed than the seventh and lie at the constricture of metenand mylencephalon. The first five somitomeres remain abutted in tandem, but enlarge as component mesenchymal cells become stellate. Finely granular extracellular matrix (ECM) accumulates around these cells and particularly in intersomitomeric interfaces. At the cranialmost end of the embryonic midline lies prechordal plate mesoderm, bordering both notochord and portions of the first somitomeres. At stage 4 the prechordal plate emerges as a 140-μm-diameter disk of condensed, radially oriented mesodermal cells lying at the head of the primitive strek. During subsequent headfold formation and neurulation, the disk grows and undergoes morphogenesis but maintains its radial organization. By stage 8+, the mesoderm in the cranial region adjacent to the neural tube is distinctly patterned.

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix: II. Further studies on the nature and site of transfilter “induction”

Abstract Corneal epithelial differentiation (primary stroma production) is dependent on the underlying extracellular matrix (ECM), for if the developing epithelium is enzymatically removed from the embryo, it fails to produce stroma in vitro unless it is cultured on collagenous ECM. We have previously shown that the stimulatory effect is mediated across Nucleopore filters in direct proportion to the surface area created by epithelial cell processes traversing the filter to contact ECM. Since collagenous ECM is insoluble under physiological conditions, transfilter stimulation of stroma production is probably due to an interaction of the epithelial cell surface with “inducer” ECM (killed lens capsule or purified collagen). We grew 5-day-old corneal epithelia on Nucleopore filters atop [ 3 H]proline-labeled lens capsules and used both autoradiography and scintillation counting to show that radioactive collagen does not enter the epithelial cells in detectable amounts. We also show here that the stimulatory effect of collagen on collagen synthesis is not dependent on trapping of serum or binding of conditioned medium factors by ECM. Finally, we demonstrate that the stimulatory effect is reduced by removal of transfilter ECM after 6–12 hr in vitro . By 18–24 hr, however, cultured epithelium is less dependent on the substratum, probably because it has produced its own ECM. We conclude that: (1) the contact mediated collagen-cell surface interaction under study here requires the continuous presence of collagen in vivo and in vitro for maintenance of “stimulated” epithelial stroma synthesis; (2) the collagenous “inducer” interacts directly with epithelium rather than indirectly via trapped intermediates; (3) collagen acts at the epithelial cell surface without entering the cells.

A conditioned medium (CM) factor produced by chondrocytes that promotes their own differentiation

Abstract This report presents evidence demonstrating that chick embryo chondrocyte cultures release into the culture medium a factor(s) which itself can act on chondrocytes to promote their own differentiation. Conditioned medium (CM) stimulates the synthesis of both sulfated mucopolysaccharides, as shown by increased incorporation of 35 SO 4 or glucose- 14 C into hyaluronidase-sensitive material, and collagen. However, protein synthesis, DNA synthesis, and cell number are not affected. While the identity of the factor is not yet known, it is nondialyzable, trypsin-and heat-sensitive. The factor is evidently a specialized product of chondrocytes, because it is not made by unexpressed chondrocytes or differentiated pigmented retina cultures. CM works rapidly on test cultures and has a significant effect on 35 SO 4 incorporation after 2 hr of treatment. In addition, the effect is relatively stable and is not reversed when CM is replaced with fresh medium. The results are significant in that they demonstrate that chondrocytes produce a factor that promotes their own differentiation, as defined in terms of the synthesis of two distinct specialized products.

The migration of myogenic cells from the somites at the wing level in avian embryos

This study is concerned with establishing a morphological basis for the initiation of migration of putative myogenic cells from the somites into the presumptive wing bud in avian embryos. At the 22 somite stage (stage 14) vasculogenesis is a prevalent activity. By use of a quail specific monoclonal antibody to vascular endothelial cells, vascular cells are recognized in the lateral plate, on the intermediate mesoderm, and on somite surfaces. Cells that are found between the lateral plate mesoderm and somites are shown to be vascular endothelial cells. The lateral body folds progressively bring the lateral plate mesoderm close to the lateral margin of the somites and vascular elements disappear from surface view. It is not until the 24 somite stage (stage 15) that some cells in the ventral lateral margin of somites at the wing level can be seen in scanning electron micrographs to extend basal cell processes toward adjacent vascular tubes. These results provide a morphological basis for the early migratory behavior of myogenic cells and demonstrate their close proximity to the prepatterned vascular network.