Norman K. Wessells

Early pancreas organogenesis: Morphogenesis, tissue interactions, and mass effects☆

The midgut region and adjacent tissues of mouse embryos acquire the ability to form exocrine pancreas tissue in vitro at about the 8-somite stage. By 10 somites a region of the gut itself can be identified as the precursor of the pancreas. The endoderm of this area apparently becomes committed or determined by about 15 somites, since at that time it can form exocrine acini when combined with mouse salivary mesoderm. These alterations occur at stages before condensed pancreatic mesoderm is present or before outgrowth of the pancreas diverticulum occurs. The beginning of pancreatic morphogenesis is marked by appearance of a broad-based upswelling from the dorsal gut. The base of this evagination gradually constricts to give rise to the pancreatic duct. These, and results on the distribution of mesodermal and endodermal cells during early organogenesis, are discussed in relation to the concept that a primary commitment or determination phase is followed by a secondary support phase during which nonspecific mesodermal input is sufficient to permit tissue growth. The role of such growth, as reflected in epithelial mass, was also investigated. Precocious appearance of zymogen granules or amylase is not evoked by even a 24-fold increase in mass of primitive mouse pancreatic epithelium. On the other hand, fractionation of growing epithelia, with resultant decrease in tissue mass, leads to postponement of overt differentiation. Once cells have reached a specific chronological age, they differentiate if left in an undisturbed tissue mass for about 2–3 days. Under such circumstances, differentiation can occur in extraordinarily small tissue masses. Mass per se is not the critical factor leading to cessation of mitosis and commencement of over cytodifferentiation. Operationally, the cells must be sufficiently mature and must be undisturbed, in the sense of not being stimulated to continue dividing, if differentiation is to occur.

Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia

Abstract Parasympathetic neurons from avian embryonic ciliary ganglia survive in low density culture when neurons are free from contact with other cells. A charged substratum, polyornithine, and a conditioned medium permit cell survival and vigorous neurite formation. The heart-conditioned medium must be present continuously and is active after dialysis. Neurites elongate rapidly, branch extensively, and follow patterns of charged substratum provided in the culture dish.

Cell locomotion, nerve elongation, and microfilaments☆

Abstract A basic difference in locomotion between migratory cells and nerves correlates with a difference in distribution of certain microfilament systems. Lattice filaments are present where extension and movement of cell surface occur in both cell types. Bundles of sheath filaments which bind heavy meromyosin, are present in migratory cells, where displacement of the cell soma over the substratum occurs, but absent from nerves, where the cell body and axon remain fixed upon the substratum and “locomotion” is restricted to the axonal tip. It is proposed that the microfilament lattice is involved in the extension phase of locomotion, and the microfilament sheath in the contractile phase.

Morphology and proliferation during early feather development

Abstract The first back feathers of chicken embryos appear in regions where a dense dermis is present. The earliest recognizable stage of the individual feather involves a cluster of elongated epidermal cells which form slightly before or coincident with an early dermal condensation. Thymidine-H 3 incorporation and mitosis are virtually absent from the condensations and overlying epidermis for a period of about 24 hours after formation. DNA synthesis and mitosis commence again in both tissues as feather outgrowth begins. Quantitative studies of dermal cell density, and results of labeling with thymidine-H 3 , suggest that the condensations arise as a result of differential mitotic activity beneath the epidermal feather placodes. Cells in the condensations subsequently remain near the tip of the dermal core, while cells originally at the edge of the condensation, or in the surrounding dermis, make a major contribution of daughter cells to the basal and lateral parts of the dermal core. Recombination experiments indicate that both dermis and epidermis of skin separated at the nonproliferative phase of feather development have the capacity to form feathers in vitro when combined with more primitive back tissues. These and other observations are discussed in relation to the concept of induction in the feather.

An analysis of salivary gland morphogenesis: Role of cytoplasmic microfilaments and microtubules

The roles of microfilaments and microtubules as causative organelles in the cell shape changes required for in vitro morphogenesis of embryonic mouse salivary epithelium have been explored by use of the drugs cytochalasin B and colchicine. Cytochalasin inhibits morphogenesis, causes flattening of the epithelium and loss of the clefts that were present at the time of drug application. These effects correlate with a specific disruption of cytoplasmic microfilaments when viewed with the electron microscope. Removal of cytochalasin results in reappearance of ordered microfilaments and resumption of morphogenesis. Colchicine disrupts microtubules and halts morphogenesis, but does not cause flattening of the epithelium or loss of clefts. Treatment with cytochalasin followed by recovery in the presence of colchicine demonstrates that recovery clefts can form in the absence of microtubules. It is proposed that normal salivary gland morphogenesis includes microfilament participation via contractile activity, in addition to mitosis and to extracellular stabilization processes.

Effects of collagenase on developing epithelia in vitro: Lung, ureteric bud, and pancreas

Abstract Cultured epithelia from embryonic lungs, ureteric buds, and pancreas were treated with collagenase. Lung morphogenesis stopped and ureteric buds lost their characteristic shape after such enzyme treatment. If mesoderm was supplied to the cultures after treatment, lung development continued and some of the ureteric buds maintained their shape. Pancreatic epithelia were unaffected by enzyme treatment and formed exocrine acini and differentiated cells even in the presence of collagenase. Electron micrographs of treated lung tissues show that the basement lamina and other nearby extracellular materials are removed by collagenase. The results imply that some form of collagen is an essential contribution of mesoderm to epithelium in cases where epithelial branching is occurring, but not where acini are forming. The former process is correlated with high mesoderm specificity requirements. The data suggest that collagen is essential for stabilization of epithelial morphology; no evidence demonstrates any inductive properties for collagen in these systems.

Non-equivalence of conditioned medium and nerve growth factor for sympathetic, parasympathetic, and sensory neurons.

Abstract Conditioned medium derived from embryonic chick heart cell cultures (heart conditioned medium; HCM) supports vigorous neurite outgrowth from embryonic sympathetic, sensory, and ciliary neurons. Antiserum to nerve growth factor (NGF) does not reduce neurite outgrowth in response to HCM, and a displacement assay fails to detect binding by components of HCM to the NGF receptor. The HCM activity is resistant to acid treatment but is destroyed by alkali. In both these respects the HCM activity differs from NGF. The activity can be inactivated by heating to 90 °C, and is retained by Amicon PM-50 filters. It is concluded that HCM stimulation of peripheral nervous system cells may not operate via NGF.

Ultrastructural studies of early morphogenesis and cytodifferentiation in the embryonic mammalian pancreas

Summary Early morphogensis and cytodifferentiation of mouse embryonic pancreas have been investigated. The organ forms as a broad-based diverticulum whose base narrows to form the duct. A crescent of cells, believed to be involved in morphogenesis, is found in each lateral wall of the gut. Changes of the crescents with time and the ultrastructure of cells in them is discussed. A model is presented to explain elevation of the pancreatic diverticulum which depends upon (1) alteration in crescent cell cross-sectional shape and (2) upon some relative movement of cells out of the crescent region. Low levels of endocrine and exocrine cell-specific product can first be detected as this morphogenetic process starts ( Rutter et al. , 1968 ). Rough endoplasmic reticulum, Golgi apparatus, and various vesicles are seen in presumed exocrine cells at these times, but no prozymogen granules have been found. Prospective B-cells can first be identified by increased cytoplasmic electron density; soon thereafter β-granules appear. These correlations of structure are discussed in light of the biochemical information. All told, the data suggest a close temporal linkage between initial specific synthesis and initial organ morphogenesis.

Tissue interactions during skin histodifferentiation

Abstract Skin tissues from 11-day chick embryos have been cultured in protein-free nutrients. Under these conditions, epidermis displays columnar basal cells and mitoses only in regions where an intimate junction is established with either dermis or muscle. Epidermis that fails to form a junction, or that is cultured alone, develops abnormally and becomes necrotic by 4 days. Epidermal differentiation to the tonofibril stage occurs only in epidermis-dermis recombinants in regions of a junction. The dermal-epidermal interaction can occur when Millipore filters are inserted between the tissues, or in the absence of visualized periodic acid-Schiff staining at the junction. Intercellular materials form and are randomly distributed in dermal explants. In recombinants, the materials usually appear first in regions distant from the epidermis. Other properties of interacting skin tissues are described, including some aspects of scale morphogenesis.

Nonproliferation in dermal condensations of mouse vibrissae and pelage hairs.

Abstract The early development of mouse embryo vibrissae and pelage hairs has been investigated by labeling with nucleic acid precursors and by organ culture procedures. As soon as dermal condensations form in either hair type, condensation cells fail to incorporate H3-thymidine or show mitoses, although such cells continue to bind H3-uridine into ribonuclease-sensitive material. Condensation cells remain relatively quiescent mitotically during hair peg downgrowth and few adjacent (labeled) dermal cells migrate into the condensation mass. These observations, in conjunction with earlier work, demonstrate remarkable similarities between hairs and feathers in terms of nonproliferation at times when significant tissue interactions are thought to be occurring.