Advances in Cellular Neurobiology: Volume 2

Advances in Cellular Neurobiology

Sergey Fedoroff

Leif Hertz

College of Medicine, University of Saskatchewan, Saskatoon, Canada

ISSN  0270-0794

Volume 2 • Number Suppl. (PB) • 1981

Table of Contents

Cover image

Title page

Copyright page

List of Contributors

Preface

Contents of Volume 1

Section 1. Cell Differentiation and Interaction

Section 2. Aging and Pathology

Section 3. Methodologies

Section 1. Cell Differentiation and Interaction

Apud Cells and Paraneurons: Embryonic Origin

Publisher Summary

I APUD and Paraneuron Concepts

II Embryonic Origin of APUD Cells and Paraneurons

The Origin and Nature of Microglia

Publisher Summary

I Microglia as a Separate Cellular Entity

II Ameboid Microglia (Ameboid Cells)

III Hypotheses on the Origins of Microglia

IV Recent Experimental Data

V Reappraisal of the Hypotheses on the Origins of Microglia

VI Microglia and Neural Macrophages

VII Conclusions

Acknowledgments

Physiology and Pharmacology of Mammalian Central Neurons in Cell Culture

Publisher Summary

I Introduction

II Methods of Preparing and Studying Cell Cultures

III Morphology and Physiology of Cultured Neurons

IV Amino Acid Pharmacology of Cultured Spinal Neurons

V Pharmacology of Clinically Important Drugs on Cultured Spinal Neurons

VI Summary and Conclusions

Trophic and Specifying Factors Directed to Neuronal Cells

Publisher Summary

I Introduction

II Nerve Growth Factor

III Other Factors Directed to Neurons

IV Conclusions and Projections

Acknowledgments

Section 2. Aging and Pathology

Cellular Aspects of Human Brain Tumors (Gliomas)

Publisher Summary

I Cellular Components of Gliomas

II Cell, Tissue, and Organ Cultures

III Clonogenicity

IV Cellular Kinetics

V Mitosis and Chromosome Analysis

VI Flow Cytometry and DNA Distribution

Acknowledgments

Lipofuscin and its Relation to Aging

Publisher Summary

I Introduction

II Morphology

III Staining Reactions

IV Autofluorescence

V Lipofuscin and Neuromelanin

VI Ceroid

VII Biochemistry

VIII Distribution

IX Lipofuscin in Specific Organs

X Lipofuscin in Disease

XI Genesis of Lipofuscin

XII The Fate of Lipofuscin

XIII Functional Significance of Lipofuscin

XIV Summary and Conclusions

The Reactive Astrocyte

Publisher Summary

I Introduction

II Ultrastructure of Astrocytes

III Response of Astrocytes To Dorsal Root Injuries

IV Cellular Response to CNS Injury

V Phagocytic Role of Neuroglia in Removal of Myelin

VI. Astrocytic Proliferation in CNS Injury

VII Glycogen Accumulation in Reactive Astrocytes

VIII Enzyme Histochemistry of Reactive Astrocytes

IX Astrocytic Reactions in Pathological Conditions

X Astrocytic Response and Aging

XI Conclusions

Acknowledgments

The Astrocyte in Liver Disease

Publisher Summary

I Introduction

II Glial Functions

III Etiology and Pathogenesis

IV Astrocyte Alterations in Hepatic Encephalopathy

V Summary and Conclusions

Acknowledgments

Section 3. Methodologies

Radioenzymatic Methods for Analysis of Neurotransmitters

Publisher Summary

I Introduction

II Norepinephrine, Epinephrine, and Dopamine

III Serotonin

IV. Acetylcholine

V Octopamine

VI Application of Radioenzymatic Assays to Neurotransmitter Analysis in Cells

VII Advantages and Limitations

VIII Conclusion

Application of Immunofluorescence in Studies of Cytoskeletal Antigens

Publisher Summary

I Introduction

II Methods

III Application of the Immunofluorescent Technique to Cytoskeletal Fiber Systems in Cells from Nervous Tissues

IV Prospects for the Future

Acknowledgments

Separation of Cell Types from the Cerebellum and Their Properties

Publisher Summary

I Introduction

II Dissociation and Fractionation of Cells

III Properties of the Isolated Cells and Cell Fractions

IV Culture of Cerebellar Cells

V Discussion and Conclusions

Acknowledgments

Pineal Cells in Monolayer Culture

Publisher Summary

I Introduction

II Methods

III Results

IV Discussion

Acknowledgments

Subject Index

Copyright page

COPYRIGHT © 1981, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED.

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List of Contributors

Ruben Adler(115),     Department of Biology and School of Medicine, University of California—San Diego, La Jolla, California 92093

Ann Andrew(3),     Department of Anatomy, Medical School, University of Witwatersrand, Johannesburg 2001, South Africa

R. Balázs(461),     MRC Developmental Neurobiology Unit, Institute of Neurology, London WC1N 2NS, England

Jeffery L. Barker(83),     Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205

Joe A. Connolly*(393),     Histology Division, Department of Anatomy, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada

C.L. Dolman(205),     Department of Pathology, University of British Columbia, Vancouver, British Columbia V52 1M9, Canada

J. Garthwaite†(461),     MRC Developmental Neurobiology Unit, Institute of Neurology, London WC1N 2NS, England

Takao Hoshino(167),     Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, California 94143

Vitauts I. Kalnins(393),     Histology Division, Department of Anatomy, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Eng-Ang Ling(33),     Department of Anatomy, Faculty of Medicine, National University of Singapore, Sepoy Lines, Singapore 0316, Republic of Singapore

P.M. MacLeod(205),     Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V52 1M9, Canada

Doris R. Nathaniel(249),     Department of Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

Edward J.H. Nathaniel(249),     Department of Anatomy, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada

Michael D. Norenberg(303),     Laboratory Service, Veterans Administration Medical Center and Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80220

James Parr(491),     Department of Neurology and Ralph L. Smith Mental Retardation Research Center, College of Health Sciences and Hospital, University of Kansas Medical Center, Kansas City, Kansas 66103

S.R. Philips(355),     Psychiatric Research Division, University Hospital, Saskatoon, Saskatchewan S7N 0X0, Canada

Bruce R. Ransom(83),     Department of Neurology, Stanford University Medical Center, Stanford, California 94305

Vernon Rowe(491),     Department of Neurology and Ralph L. Smith Mental Retardation Research Center, College of Health Sciences and Hospital, University of Kansas Medical Center, Kansas City, Kansas 66103

Valerie Steinberg*(491),     Department of Neurology and Ralph L. Smith Mental Retardation Research Center, College of Health Sciences and Hospital, University of Kansas Medical Center, Kansas City, Kansas 66103

Silvio Varon(115),     Department of Biology and School of Medicine, University of California—San Diego, La Jolla, California 92093

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

*Present address: Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.

†Present address: Department of Veterinary Physiology and Pharmacology, University of Liverpool, Brownlow Hill, P.O. Box 147, Liverpool L69 3BX, England.

*Present address: Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002.

Preface

Sergey Fedoroff and Leif Hertz

Despite tremendous advances in biomedical research in the last 20 years, understanding of the nervous system has lagged behind. The topographical anatomy and functional relation of the brain and spinal cord were already established at the turn of the century, but the structure and function at the cellular level and such higher brain functions as learning, memory, and intelligence are still poorly understood.

The central nervous system is a heterogeneous system composed of many small populations of cells, each differing from the other in numbers, size, types, and density of cells, and each occupying a distinct topographical area of the brain. At the same time, these distinct cell populations (nuclei) are developmentally and functionally integrated into one nervous system. Each of these areas of the central nervous system is composed of several types of neuronal and glial cells with different biochemical, biophysical, and pharmacological characteristics. Apart from electrophysiological recording, techniques to study the characteristics of the individual cell types have become available only very recently, establishing a new area of neurobiology that could appropriately be named cellular neurobiology and that draws on some aspects of morphology, biochemistry, pharmacology, physiology, endocrinology, embryology, and genetics.

Discussion with many neurobiologists convinced us that it would be very useful, at a time when neurobiology is expanding exponentially, to have a publication that would regularly review new information in the area of cellular neurobiology. As a result, Advances in Cellular Neurobiology was undertaken. It differs from existing publications in the area of neurobiology in that attention is focused on the cellular basis of neurobiological events rather than on molecular organismic organizations and functions of the nervous system.

Each volume will have three subdivisions: cell differentiation and interaction, aging and pathology, and methodology. We intend that the series will provide a compendium of the rapidly increasing body of information about neural cells and, as an interface between normal and abnormal (pathological) reactions of the cells, that it will stimulate new ideas and research. At the same time, the transfer of basic information to clinical situations and application may be facilitated. Advancement of research in any field is dependent on the methodologies available, and such has certainly been the case in the development of cellular neurobiology. For this reason we have included a section on methods; the intention is to review both methods currently in use and those from other fields that might possibly be applied in neurobiology.

Contents of Volume 1

Section 1. Cell Differentiation and Interaction

SPECIALIZATIONS OF NONNEURONAL CELL MEMBRANES IN THE VERTEBRATE NERVOUS SYSTEM

M. W. Brightman, J. J. Anders, and J. M. Rosenstein

EFFECTS OF NEUROHORMONES ON GLIAL CELLS

Dietrich van Calker and Bernd Hamprecht

RETROGRADE AXONAL TRANSPORT

M. A. Bisby

BIOCHEMICAL CHARACTERISTICS OF INDIVIDUAL NEURONS

Takahiko Kato

Section 2. Aging and Pathology

CEREBELLAR GRANULE CELLS IN NORMAL AND NEUROLOGICAL MUTANTS OF MICE

Anne Messer

CELL GENERATION AND AGING OF NONTRANSFORMED GLIAL CELLS FROM ADULT HUMANS

Jan Pontén and Bengt Westermark

AGE-RELATED CHANGES IN NEURONAL AND GLIAL ENZYME ACTIVITIES

Antonia Vernadakis and Ellen Bragg Arnold

GLIAL FIBRILLARY ACIDIC (GFA) PROTEIN IN NORMAL NEURAL CELLS AND IN PATHOLOGICAL CONDITIONS

Amico Bignami, Doris Dahl, and David C. Rueger

Section 3. Methodologies

IN VITRO BEHAVIOR OF ISOLATED OLIGODENDROCYTES

Sara Szuchet and Kari Stefansson

BIOCHEMICAL MAPPING OF SPECIFIC NEURONAL PATHWAYS

E. G. McGeer and P. L. McGeer

SEPARATION OF NEURONAL AND GLIAL CELLS AND SUBCELLULAR CONSTITUENTS

Fritz A. Henn

SEPARATION OF NEURONS AND GLIAL CELLS BY AFFINITY METHODS

Silvio Varon and Marston Manthorpe

Subject Index

Section 1. Cell Differentiation and Interaction

Apud Cells and Paraneurons: Embryonic Origin

Ann Andrew,     Department of Anatomy, Medical School, University of the Witwatersrand, Johannesburg, South Africa

Publisher Summary

This chapter reviews the main aspects of the concept of amine precursor uptake and decarboxylation (APUD) that was aimed at grouping diverse cell types whose main common feature was the ability to synthesize the so-called biogenic or monoamines. In the period of the development of the APUD concept, it was noticed that the ability to take up amine precursors such as dopa and 5-HTP and decarboxylate them with the formation of monoamines such as catecholamines and serotonin was common to a number of cell types. Criteria for the membership of the APUD series have changed in the present. This concept was extended to the paraneuron concept to emphasize the close relationship between conventional neurons and neuron-like cells, paraneurons, some of which are APUD cells located in the amphibian cutaneous glands, the placenta, the thymus, and the APUD neurons of the hypothalamus and sympathetic ganglia. Cells such as the monoamine-storing cells in the avian aortic wall are regarded as paraneurons. The study of such similarities between APUD and paraneurons cells has helped in the recognition of the fact that neurons and polypeptide-secreting endocrine cells are not completely disparate entities.

I APUD and Paraneuron Concepts

A Development of the APUD Concept

B The Paraneuron Concept

II Embryonic Origin of APUD Cells and Paraneurons

A Hypothesis of a Common Derivation

B Evidence for the Origin of APUD Cells and Paraneurons

C Conclusion

References

In 1966, a unifying concept was proposed that brought together diverse cell types whose main common feature was the ability to synthesize so-called biogenic or monoamines. This is the APUD (Amine Precursor Uptake and Decarboxylation) system devised by Pearse (1966a, 1968). It has attracted widespread attention and much discussion. Perhaps its major contribution has been the attention it has drawn to the relationship between polypeptide-secreting endocrine glands and the nervous system. This kinship has been the focal point of the paraneuron concept of Fujita (1976), which has evolved out of the APUD idea. The implications of the APUD theory in particular have had a considerable impact in pathology, an impact that has hinged on the proposed embryonic derivation of the members of the APUD series.

The APUD and paraneuron concepts have been reviewed on a number of occasions: here, the main tenets will be surveyed only briefly as a prelude to a critical discussion of evidence bearing on the origin of the cells concerned.

I APUD and Paraneuron Concepts

A Development of the APUD Concept

Pearse (1966b, 1968) noticed that the ability to take up amine precursors (such as dopa and 5-HTP) and decarboxylate them with the formation of monoamines (such as catecholamines and serotonin) was common to a number of cell types: thyroid C (calcitonin-secreting) cells; chromaffin cells of the suprarenal medulla; corticotropes and melanotropes of the hypophysis; and pancreatic B, enterochromaffin, and mast cells. Other cytochemical features (the possession of certain enzymes and a high side-chain carboxyl content) were shared by members of the group, as were production of polypeptide hormones and the presence of the related ultrastructural features (Pearse, 1966a, 1968, 1969). Since then the membership of the series has been expanded to include melanocytes (Rost et al., 1969); other pancreatic and gut endocrine cells (Pearse, 1968, 1969, 1971, 1975); carotid body type I cells (Pearse, 1971); Merkel cells (mechanoreceptors in the epidermis) (Winkelmann, 1977); the small, intensely fluorescent cells of sympathetic ganglia (SIF cells) (Pearse, 1977); sympathetic ganglion cells and accessory chromaffin tissue, endocrine cells in the thymus and the urogenital tract (Pearse and Takor Takor, 1979); and also, aortic body type I cells (Hansen, 1978) and such cells as the bipolar cells in the amphibian pulmonary artery (Haller and Rogers, 1978). At first, Pearse expressly excluded hypothalamic neurosecretory cells, but later he brought them into the fold (Pearse, 1975). Parathyroid chief cells and hypophyseal thyrotropes and gonadotropes were recalcitrant; they could not be provoked to take up and decarboxylate an amine precursor. Eventually, however, all adenohypophyseal cell types were included, Pearse (1975) considering them to have lost their APUD mechanism. Kruse (1971) shared the notion that the APUD ability might be atavistic, having shown that it was not affected by the process of hormone secretion in thyroid C cells. Later additions to the series were the polypeptide-producing cutaneous glands of Amphibia and placental endocrine cells (Pearse, 1977). So at last, virtually all peptide hormone-producing cells were encompassed, as well as cells secreting hormone-like substances (neurosecretions and neurotransmitters) and in addition, some cells not known to elaborate such substances.

Thus, criteria for membership of the APUD series have changed. Some of the cytochemical features were admitted early on to be inconstant (Pearse, 1971; see Andrew, 1976a); the ability to synthesize amines is no longer a sine qua non. Polypeptide hormone production has become the discriminating factor, but not all such cells have been included (see Bennett, 1977). Although many neurons are peptide- and/or amine-producing, Pearse has not accepted them into his system lest it become too cumbersome (Pearse and Takor Takor, 1979). However, the realization that some of the same peptides occur in cells from different systems—the nervous system, the gastrointestinal tract, and the skin of Amphibia (Pearse, 1976a, 1977)—brought to the fore the possibility of a relationship between these cells. This state of affairs provoked Pearse to regard APUD cells as nerve cells that had acquired a specialized (endocrine) function (Pearse and Takor Takor, 1976). The recent demonstration of neuron-specific enolase in neurons and various APUD cells (hypothalamic cells, pinealocytes, adenohypophyseal and suprarenal medullary cells, cells in the position of thyroid C cells, and pancreatic islet cells) by Schmechel et al. (1978) has emphasized this relationship. It may be noted that gut endocrine cells were not tested.

B The Paraneuron Concept

Building on the APUD concept, Fujita (1976, 1977) and Fujita and Kobayashi (1978, 1979) have extended it to emphasize the close relationship between conventional neurons and neuron-like cells, which they have called paraneurons. Some of these are APUD cells; many, however, do not show amine-handling ability and, according to these authors, had been artificially excluded from the assembly of related cells.

APUD cells that have not been considered paraneurons are those located in the amphibian cutaneous glands, the placenta, the thymus, and, obviously, the APUD neurons of the hypothalamus and sympathetic ganglia. Such cells as the monoamine-storing cells in the avian aortic wall are regarded as paraneurons (Ookawara, 1977). On the borderline between paraneurons and neurons are gustatory cells, hair cells of the inner ear and lateral line organs, retinal photoreceptors, olfactory cells, and liquor-contacting neurons (Fujita, 1977).

The common characteristics of paraneurons center around structure, function, and metabolism. As recorded by Fujita (1976, 1977) and Fujita and Kobayashi (1979), they comprise the production of neurotransmitters or neurotransmitter-like substances and of peptide hormones or candidate hormones; the possession of synaptic vesicle-like or neurosecretory-like granules and the usual organelles associated with protein synthesis; and the release of secretory products in response to stimulation of receptor sites.

Thus, the formation of monoamines is not an obligatory criterion for paraneurons and there is no difficulty about the inclusion of cells such as thyrotropes, gonadotropes, and parathyroid chief cells. Cells synthesizing melanin from an amine precursor (dopa)—melanocytes—or melatonin from serotonin—pinealocytes—are regarded as paraneurons, as are mast cells and gustatory cells that show amine-handling properties but do not secrete peptide hormones (Fujita, 1977). Merkel cells, which lack monoamines but contain granules suggested to be a neurotransmitter (Kurosumi, 1976), may be considered paraneurons.

Some substances apparently serving as neurotransmitters are produced by such paraneurons as gut endocrine cells. These include substance P, neurotensin, and somatostatin (Fujita and Kobayashi, 1978), all of which may serve also as local hormones (Fujita, 1977). Serotonin too may act in both ways. Hence, the distinction between neurotransmitters, neurosecretions, and hormones is now blurred, and there is gradation among paraneurons from neuron-like to endocrine cells, and from mainly sensory and receptor to mainly secretory cells. At one extreme are the most neuron-like—retinal rods and cones, olfactory cells, and hair cells of the inner ear—and at the other, suprarenal chromaffin cells, carotid body chief cells, and thyroid C cells. In between fall gut, pancreatic, and lung endocrine cells.

Similarly, there is no sharp boundary between neurons and paraneurons: both show stimulus-secretion coupling, and there is evidence that they share similar receptors. The excitability of the plasma membranes of some paraneurons, e.g., suprarenal medullary cells (Tischler et al., 1977) and pancreatic B cells (Dean and Matthews, 1970), may be related to their secretory activity (Tischler et al., 1976; Kanno, 1977) and emphasizes their affinity with neurons. The probable similarity of secretory granule and synaptic vesicle contents (amines and peptides, ATP, and other nucleotides) in some neurons and paraneurons and of the mechanism of their release is also commented upon (Fujita and Kobayashi, 1978).

Some paraneurons are interposed in a chain of neurons (interneurons such as SIF and carotid body cells), others are sensory cells at the beginning of a chain (such as olfactory and visual cells), and yet others lie at the effector end of the chain (suprarenal medullary cells). In some species, adenohypophyseal and pancreatic endocrine cells are not innervated; gut endocrine, parathyroid chief, and thyroid C cells also do not appear to be directly associated with nerve fibers (Fujita, 1976). It is suggested, however (Fujita, 1977), that the transmitter-like products of, for instance, gut endocrine cells diffuse into the lamina propria and there stimulate afferent fibers participating in reflex arcs or linked to the central nervous system. Melanocytes may be innervated in lower vertebrates, and mast cells are said to be intimately related to nerve fibers (see Fujita, 1977; Fujita and Kobayashi, 1979).

Even morphological features formerly considered specific for neurons—chromidial substance (Nissl bodies) and neurofibrils—are not regarded by Fujita and Kobayashi (1978, 1979) as distinctive since they occur in many cells in the form of rough endoplasmic reticulum, microtubules, and microfilaments.

Thus, we see that paraneurons embrace practically all the APUD cells and include a number of more neuron-like cells as well. The traditional dividing line between endocrine cells and nerve cells has been effaced by the evolution of these two concepts. This becomes more acceptable when it is realized that the phylogenetic ancestor of these two kinds of cells was a common cell type that performed the functions of both (Scharrer, 1975).

II Embryonic Origin of APUD Cells and Paraneurons

A Hypothesis of a Common Derivation

An integral part of the APUD concept has been concerned with the embryonic origin of APUD cells. The idea of a common ancestor was put forward at the very beginning, even though it was realized that cells of accepted ectodermal, mesodermal, and endodermal origin had been included (Pearse, 1966a,b, 1968). Amine storage and the presence of cholinesterase in APUD cells suggested a neural source, perhaps the neural crest. The inclusion of melanocytes and suprarenal chromaffin cells was in line with the idea of a neural crest derivation; with the claim that thyroid C cells and carotid body type I cells were of such an origin, confidence in this idea was gained. It did not take long to become entrenched in the literature.

Certainly some APUD cells arise from the neural crest, but the claim that gut and pancreatic endocrine cells were so derived proved untenable. With the inclusion of hypothalamic neurosecretory cells and pinealocytes in the APUD series, it was evident that the source had to be expanded to embrace neural ectoderm. Neuroectoderm thus became the favored origin (Pearse, 1975; Pearse and Takor Takor, 1976). Parathyroid and cutaneous glands of Amphibia were attributed to the inner neural or placodal layer of ectoderm, the adenohypophysis and hypothalamus to the ventral neural ridge, and the endocrine placenta to neuroectodermal epiblast or ectoblast of the yolk sac, stated to be continuous with the epiblast or ectoblast of the neural plate (Pearse, 1976, 1977).

Gut and pancreatic endocrine cells were still misfits. Pearse (1977, 1979) accommodated them by proposing that all APUD cells are derived from neuroendocrine-programmed epiblast or ectoblast; further, he suggested that gut endocrine cells might drop out of the epiblast after the definitive gut endoderm had separated from it or they might arise from the endoderm itself by a process of isolated induction of neuroendocrine abilities.

The idea that all APUD cells share a common origin has proved very appealing, especially to pathologists (see, for instance, Tischler et al., 1977; Guillemin, 1978; Gould et al., 1979; Klapdor, 1979). The reason for its appeal is that apparent interconvertibility of cell types evidenced by some pathological situations is neatly explained in such a way. The idea applies, for instance, as Pearse (1973) and Pearse and Welbourn (1973) have pointed out, to multiple endocrine adenomatosis and to ectopic hormone production. In the latter case, a tumor of one APUD cell type synthesizes a hormone normally characteristic of another. Carcinoids (enterochromaffin cell tumors) may produce gastrin, calcitonin, VIP, or secretin; ACTH is secreted by a variety of tumors such as carcinoids, oat cell carcinomas of the lung, islet cell tumors and pheochromocytomas (suprarenal chromaffin tumors); and a pheochromocytoma may produce insulin or VIP (Omenn, 1970; Pearse and Welbourn, 1973; Whitman, 1977; Skrabanek and Powell, 1978). However, this phenomenon is not limited to APUD cell tumors: for instance, parathormone is secreted by tumors of the lung and kidney (Omenn, 1970) and calcitonin by non-APUD as well as APUD tumors (Abe et al., 1977). In multiple endocrine adenomatosis, a number of different polypeptide hormone-secreting cells are neoplastic in the same individual. In MEA I, adenohypophyseal cells, pancreatic and gut endocrine cells, parathyroid chief cells, and sometimes other APUD cells may be involved (Pearse, 1973; Bolande, 1974) and in MEA II, thyroid C cells, suprarenal chromaffin cells, carotid body chief cells, and parathyroid chief cells (Pearse, 1973; Montgomery and Welbourn, 1975). Less differentiated or dedifferentiated cells with like potentialities available to them could explain these phenomena.

A neuroectodermal origin of all members was originally one of the principles of the paraneuron concept (Fujita, 1976), which could therefore explain the pathological findings cited above equally well as does the APUD theory. However, at the time, the possibility was mooted that this criterion might be abandoned; this indeed proved to be so (Fujita and Kobayashi, 1978, 1979).

B Evidence for the Origin of APUD Cells and Paraneurons

Pertinent evidence on the embryonic origin of cell types included in the APUD and/or the paraneuron series is reviewed in this section. It is more fully and more critically discussed when the issue is contentious, as is especially the situation with gut and pancreatic endocrine cells.

1 Sympathetic Ganglia

Although sympathetic ganglia are now generally accepted to be neural crest derivatives, the evidence has not distinguished unequivocally between the neural crest and the neural tube as their source. Some findings favor the neural crest alone (e.g., Hammond and Yntema, 1947; Hammond, 1948, 1949; Nawar, 1956), whereas others support contributions to a greater or lesser degree from the ventral half of the neural tube (e.g., Staudacher, 1940; Jones, 1941; Strudel, 1952, 1953). However, in deletion experiments, conflicting results are often attributable to incomplete extirpation or to the possible infiltration of the operated region by crest cells from adjoining areas.

Evidence from experiments in which labeled neural crest has been transplanted to unlabeled hosts is more reliable. Raven (1937) has shown in this way that some cells in urodele amphibian ganglia are of neural tube origin; Chibon (1967) has demonstrated the neural crest derivation of sympathetic ganglion cells in a urodele by transplanting crest labeled with tritiated thymidine. In chicks, Weston (1963) found sympathetic ganglia to arise from radioactively labeled grafted neural tube; when the dorsal part of the tube, which contains the neural crest, was removed, the ganglia were absent. He thought it unlikely that they originate in the neural tube itself, and considered them neural crest derivatives. Weston wondered whether the cells of neural tube origin that Raven found in the sympathetic ganglia could not be supporting elements.

In any event, sympathetic ganglion cells are neuroectodermal in origin. It can only be surmised that SIF cells have the same origin: they have not yet been investigated from this point of view.

2 Suprarenal Chromaffin Cells

The chromaffin cells of the suprarenal medulla have long been recognized to be neural crest derivatives. Recent confirmatory evidence has come from Weston’s (1963) transplantation of neural tube and crest from tritiated thymidine-labeled chick embryos to unlabeled hosts. Labeled cells were present in the suprarenal medulla. Also in chick embryos, Polak et al. (1971) described cells showing dopa-provoked formaldehyde-induced fluorescence (FIF) characteristic of APUD cells, close to the ventrolateral surface of the neural tube and alongside the notochord. Subsequently, such cells were present in the position of sympathetic ganglia and ultimately in the suprarenal glands. The neural crests themselves did not show FIF, but this study does provide confirmation that precursors of the chromaffin cells reach the suprarenal gland by way of the sympathetic ganglia. However, the level at which fluorescent cells were first seen—opposite somites 6 to 12—is not the region of neuroectoderm that Chevalier (1972) has shown to have the potency to produce suprarenal medullary cells. He combined cortical anlagen, deprived of medullary cells by irradiation of the neural tube, with various levels of normal neural tube and showed that the neuroectoderm at the levels of somites 12 to 29 only was competent to give rise to suprarenal chromaffin cells. Teillet and Le Douarin (1974) replaced segments of the neural tube (containing neural crest) of chick embryos with the equivalent region of quail neural tube. They found that levels opposite somites 18 to 24 gave rise to catecholamine-containing cells exhibiting the large Feulgen-positive nucleoli Le Douarin had shown to be characteristic of quail cells. They could thus corroborate the contention that suprarenal chromaffin cells are of neuroectodermal origin.

3 Aortic Bodies

On the basis of morphological studies of cat embryos, Hammond (1941) maintained that cells of neural origin migrate along the aortic depressor nerve, a branch of the vagus, and accumulate to form the aortic arch bodies; he noted that a minor contribution may enter the bodies from sympathetic fibers joining the nerve later. Boyd (1961) undertook a similar study on human embryos. He attributed the aorticopulmonary bodies (included among aortic bodies) to neuroblast-like cells of a similar vagal source.

Thus, the chemoreceptor cells of aortic bodies are considered neuroectodermal derivatives. There is no confirmatory experimental evidence available.

4 Carotid Body Chief Cells

In a morphological investigation of embryonic sheep material, Batten (1960) concluded that the parenchymatous cells of the carotid body were derived largely from an epibranchial placode and to a lesser extent from the petrosal ganglion; the latter he held to arise from neural crest cells that associated with the placode. Cells were believed to migrate from these sources along the carotid branch of the glossopharyngeal nerve and then enter a mesenchymal condensation to form the carotid body. These findings tie in to some extent with the experimental evidence.

In chick embryos with quail rhombencephalic grafts, Le Douarin et al. (1972) and Pearse et al. (1973a) found that the carotid bodies consisted mainly of quail cells with large Feulgen-positive nucleoli. The FIF exhibited by these cells was yellow-green, whereas that of the carotid body cells in chick embryos is yellow. The presence of chick cells in a few embryos indicated that migration of chick neuroectodermal cells had begun before transplantation. It was concluded that the majority of carotid body cells—types I and II—arise as late emigrants from the anlage of the rhombencephalon, probably from the neural crest. In any case, the cells had been shown to be neuroectodermal.

A neuroectodermal origin for carotid body cells was implicit in the contention of Korkala and Hervonen (1973) that cells showing FIF enter the carotid body by way of a process connecting it with the sympathetic trunk. However, this association with the sympathetic trunk would indicate derivation from trunk levels of the neuroectoderm, which in turn would be at variance with the more cogent evidence for rhombencephalic neuroectoderm.

5 C Cells of the Thyroid Gland and Ultimobranchial Body

In 1967, Pearse and Carvalheira found dopa-provoked FIF in rodent embryos, successively in the fourth pharyngeal pouch, its ventral portion, the ultimobranchial body (derived from pouch IV), and eventually within the thyroid gland once the ultimobranchial bodies had fused with it. At this time, they suggested that C cells might originate outside the pharynx. Later, in younger mouse embryos, Pearse and Polak (1971a) described fluorescent cells, first in a position between the epidermis and the neural tube, then in a more ventral situation, and finally within the ventral portions of the fourth pouches. Taking the results of both studies together, they concluded that C cells were of neural crest origin.

Meanwhile, Le Douarin and Le Liévre (1970) had transplanted the caudal third of the brain up to the level of somite 5, from quail to chick embryos. They subsequently encountered quail cells in the ultimobranchial bodies and identified the quail cells by electron microscopy as C cells. That some chick cells were present was taken as an indication that migration of crest cells could already have been underway at the time of operation. Later, after processing tissues from similarly operated embryos for dopa-provoked FIF to demonstrate the APUD nature of C cells, Le Douarin et al. (1974) showed that the fluorescent cells contained quail nuclei and again in some cases, chick nuclei. Finally the two groups of investigators collaborated (Polak et al., 1974) to demonstrate that C cells showing calcitonin-immunoreactivity in chick embryos with quail rhombencephalic transplants contained mostly quail nuclei. This is conclusive evidence that C cells in the avian ultimobranchial body are of neuroectodermal origin.

6 Melanocytes

The origin of pigment cells from the neural crest is well established in vertebrates. Experiments have involved deletion of the neural crest, its transplantation to a different site or species, or explantation (Dorris, 1938, 1939, 1941; du Shane, 1935; Ris, 1941; Rawles, 1947; Newth, 1950; Niu, 1947, 1954; Stevens, 1954). Teillet and Le Douarin (1970), using quail to chick transplants of neural tube and neural crest, produced White Leghorn chicks with bands of pigmented feathers of quail pattern, thus confirming the neuroectodermal derivation of melanocytes.

7 Mast Cells

The literature on the origin of mast cells deals almost exclusively with the question of whether they arise directly from mesenchyme (see, for instance, Kiernan, 1974) or from another cell type such as the lymphocyte (Csaba et al., 1961; Ginsberg et al., 1963, 1967; Combs et al., 1965). Macrophages (Muller-Hermelink et al., 1971) and plasma cells (see Hottendorf et al., 1966) are also contenders. Evidence for the derivation of some mast cells from bone marrow is put forward by Kitamura et al. (1977). The cell types concerned are all accepted mesenchymal derivatives and are likely to be considered mesodermal in origin. It is known, however, that the neural crest is the source of much mesenchyme in the head (see, for instance, Le Liévre, 1974) and it is believed to contribute to the dermis generally.

The possibility that mast cells are derived from the neural crest was mooted by Okun (1967) on the grounds that pigment appeared in cultures of mast cells, especially when corticotropin was present in the medium, and that melanocytes of albinos reacted in the same way. However, Hirano et al. (1974) found no evidence for a neural crest origin of mast cells in amphibian transplantation and deletion experiments.

8 Merkel Cells

The suggestion has been made that the epidermal Merkel cell may be derived from the neural crest. This possibility is based on the contention that Merkel cells differ in cytology from keratinocytes and on the observation, in embryos, of Merkel cells in the mesenchyme underlying the epidermis or apparently crossing into the epithelium together with nerve fibers (Breathnach and Robins, 1970; Hashimoto, 1972; Winkelman and Breathnach, 1973). Other investigators, however, have found that Merkel cells appear in the epidermis before nerve fibers do (Lyne and Hollis, 1971; Tweedle, 1978).

The only experimental work in this area seems to have been that of Tweedle, who eliminated the entire presumptive neural tube and crest from urodele embryos without any effect on the presence of Merkel cells. Furthermore, he found no Merkel cells in the underlying mesenchyme in unoperated embryos.

The alternative hypothesis is that the cells differentiate from epidermal cells. The possession of desmosomes linking Merkel cells with keratinocytes is regarded by some as evidence for this view (Lyne and Hollis, 1971; Kurosumi, 1976) but English (1974) has pointed out that even melanocytes, which are undoubtedly of extrinsic origin, may become linked to keratinocytes by desmosomes. Cells with structural features intermediate between those of Merkel cells and keratinocytes have been described and regarded—with caution—as suggestive of an epidermal origin of the former (Nafstad, 1971; English, 1974, 1977).

9 Pinealocytes

The pineal gland is a recognized derivative of the roof of the forebrain: its neuroectodermal origin has not been contested.

10 Liquor-Contacting Neurons

There is no reason to doubt that these arise from the neural ectoderm.

11 Sensory Cells

Apparently, some sensory cells of mammalian taste buds are ectodermal and some, endodermal (Hamilton and Mossman, 1972). The origin of the olfactory cells is assumed to be in keeping with the ectodermal derivation of the nasal mucosa from the olfactory placode; likewise, the ectodermal otic placode gives rise to the hair cells of the inner ear. The hair cells in lateral line organs of lower vertebrates also stem from ectodermal placodes. Such placodes have not been considered neuroectodermal up to now, but a case might be made for classifying them in this way. The rods and cones of the retina are indubitably neural in origin.

These types of sensory cells are classed among paraneurons, but they have not been considered to be APUD cells, except for gustatory cells, which were listed in this way by Andrew (1976a).

12 Amphibian Cutaneous Glands

Amphibian cutaneous glands are regarded by Pearse (1975) as arising from neural or placodal ectoderm: he claims that they develop from the inner neural (sensorial) layer of the ectoderm, from which placodes originate. The sensorial layer gives rise to the stratum germinativum, from which the adult epidermis develops (Nieuwkoop and Faber, 1967), and is therefore doubtless involved in the formation of the cutaneous glands. There seems to be, however, no good reason to associate this process with neural development such as occurs in the induction of the sensorial layer to form the neural plate, neural crest, and sensory placodes.

13 Parathyroid Chief Cells

Classically, the parathyroid glands are regarded as endodermal, being derived from the third and fourth pharyngeal pouches. In seeking to establish whether they might be neuroectodermal in origin, Pearse and Takor Takor (1976), using serial sections, found it impossible to decide whether, in rat and chick embryos, these glands arise from the endoderm of the pharyngeal pouches or from the ectoderm of the visceral clefts. In the frog, however, they adjudged the glands to be derived from the ectodermal lining of the opercular cavity, since when first recognizable, they were situated close to the ectoderm and at some distance from the ectoderm-endoderm junction. This was at a stage when the pouches had long since opened into the opercular cavity. The possibility is not excluded, however, that the primordia of the glands separated from the endoderm at an earlier stage and moved to their definitive positions as they do in mammals. Pearse and Takor Takor maintain that in the frog, the parathyroid glands arise from the inner neural ectodermal layer mentioned above. They therefore consider the parathyroid gland to be a derivative of neural or placodal ectoderm (Pearse, 1975), but the evidence presented is not convincing.

14 Adenohypophyseal Cells and Hypothalamic Neurosecretory Cells

The adenohypophysis has long been regarded as arising from the hypophyseal (Rathke’s) pouch and the latter, as an outgrowth of the ectoderm of the stomodeum.

In 1974, Ferrand et al. isolated mesenchyme-free hypophyseal pouches from 4- to 5-day chick embryos and cultured them as chorioallantoic grafts. ACTH was not demonstrable by immunocytochemistry in the explants on retrieval. Pouches from 5.5- to 6.5-day embryos could not be separated from mesenchyme; when they had been cultured, ACTH cells were identifiable in half the grafts. The authors offered several explanations. The preferred one was that neural crest cells—known from quail to chick grafts to contribute to cephalic mesenchyme—enter the developing adenohypophysis after the fifth day and differentiate into ACTH cells. A less favored explanation was that the mesenchyme or the hypothalamus induces the development of ACTH cells in the pouch after the fifth day. A reason for considering this second possibility less likely was that the various adenohypophyseal cell types are able to differentiate in hypophyseal pouches grown with heterologous mesenchyme. If the first hypothesis were correct, one wonders where the neural crest cells would have come from in the latter situation. More convincing would be evidence of quail nuclei in the adenohypophysis following quail to chick transplantation of neural tube and crest at mid- and hind-brain levels [since these provide mesenchyme in the head (Le Douarin, 1975)].

Takor Takor and Pearse (1975) became less ardent in their support of the idea of a neural crest origin for ACTH cells when they described in chick embryos a ventral neural ridge that, they contended, gave rise to the hypophyseal pouch and to the floor of the diencephalon, possibly including neurosecretory cells of the hypothalamus. They maintained that the neural ridges that flank the neural plate extended beyond the tip of the anterior neuropore onto the ventral surface of the head and reached the stomodeum as a ventral neural ridge. This was held to form a groove, the floor of which perforated through to the underlying prosocoele. When the groove closed, tissue from the ventral neural ridge was reported to become incorporated into the floor of the diencephalon. Thus, the hypothalamus was regarded as probably neuroectodermal. The hypophyseal pouch was considered to arise from the ventral neural ridge between the optic chiasma and the stomodeum.

In a morphological reinvestigation, Levy et al. (1980) could find no evidence for a ventral neural ridge or for incorporation of ventral ectoderm into the diencephalic floor. Also, Silver and Gould (1977) have found the hypophyseal pouch to be separated by a wide interval from the anterior neuropore. Both sets of authors consider that there is no good reason to reject the classical view of the formation of the adenohypophysis from the ectoderm of the stomodeum. The hypothalamus is still best regarded as differentiating from neural ectoderm.

15 Placental Endocrine Cells

The polypeptide hormone-secreting cells of the placenta are part of the trophoblast (not the yolk sac). The ectoderm from which the trophoblastic layer of the chorion arises in mammals is continuous with the embryonic ectoderm, but this in no way entitles the trophoblast or the endocrine cells to be regarded as neuroectodermal in origin.

16 Thymic Endocrine Cells

No investigations have yet dealt with the embryonic origin of the thymic cells having features of APUD cells (Håkanson et al., 1974a) and ultrastructurally visible secretory granules (Töró et al., 1969), though the elegant experiments of Le Douarin and Joterau (1975) have demonstrated that the connective tissue component is derived from neuroectoderm in avian embryos.

17 Endocrine Cells of the Urogenital Tract

Cells resembling endocrine cells of the gut in their general features have been described in the urethra (Dixon et al., 1973; Håkanson et al., 1974b; Ramsdale, 1974), prostate (Håkanson et al., 1974b), and uterine cervix (Fox et al., 1964), but no studies on their embryonic origin have been reported.

18 Endocrine Cells of the Respiratory Tract

Only Pearse and Polak (1971b) have made an attempt to elucidate the origin of the endocrine cells found in the respiratory tract. Their study dealt mainly with the endocrine cells of the pancreas and the gut, and is discussed in Section II,19,b.

19 Pancreatic and Gut Endocrine Cells

a Early Views.

Pancreatic islets have long been accepted to be of endodermal origin (see, for instance, Liegner, 1932; Pictet and Rutter, 1972). Most of the evidence consists of the observed differentiation of islet cells from epithelial elements derived from the endodermal pancreatic buds (for references, see Andrew, 1976b). The likelihood that pancreatic and gut endocrine cells have a common origin was suggested by Feyrter (1943) and Adelson (1971), the latter assuming this to be endodermal.

For many years, the only type of endocrine cell known in the gut was the enterochromaffin cell. Many workers considered it endodermal in origin (for references, see Andrew, 1963) but some favored derivation from the mesoderm. The studies concerned were mostly morphological investigations of enterochromaffin cell differentiation. Those workers supporting an endodermal source had found the first distinguishable enterochromaffin cells in the epithelium; those regarding the source as mesodermal described comparable cells in the lamina propria (Kull 1912, 1925; Dias-Amado, 1925a,b). Tehver (1930) opposed a mesodermal origin since he could see no granular cells in the connective tissue even when they first appeared in the epithelium. Although Monesi (1960) was convinced of the presence of enterochromaffin cells in the lamina propria, he rejected such an origin since at no stage were all the cells extra-epithelial.

Derivation of enterochromaffin cells from autonomic neuroblasts or cells was upheld by Danisch (1924) and Chung (1934) because they found cells showing affinity for silver and acidophilia apparently en route from the submucosal plexus to the epithelium. This finding implied a neuroectodermal origin for enterochromaffin cells. Simard and van Campenhout (1932) attempted experimental verification of the postulated neural origin. They cultured as chorioallantoic grafts, gut of chick embryos excised before nervous elements reputedly entered the gut. Enterochromaffin cells differentiated in the grafts and it was concluded that they were not nervous in origin. However, this conclusion was invalidated when it was shown that neuroectodermal cells in the form of enteric ganglia enter the gut much earlier than had been supposed (Andrew, 1964). The possibility of a neural crest origin for enterochromaffin cells was thus reopened.

b Recent Work.

Pearse and Polak (1971a,b) thought they had provided evidence for the neural crest origin of APUD cells of the respiratory tract, gut, and pancreas. They observed dopa-provoked FIF in mouse embryos in cells believed to be neural crest cells; at subsequent stages, fluorescent cells were seen between the neural tube and pharynx, and later, in the pharynx, stomach, duodenum, dorsal pancreatic bud, trachea, and bronchi. They concluded that at least some respiratory, gut, and pancreatic endocrine cells originate in the neural crest. However, the position of the fluorescent cells identified as neural crest cells in the earlier embryos seems too far lateral, and experimental verification that all the fluorescent cells belonged to the same population was necessary.

i Pancreatic endocrine cells.

Working on the assumption that they had in this way demonstrated primitive endocrine cells of the gut wall to be of crest origin, Pearse et al. (1973b) claimed that these cells gave rise to at least two types of pancreatic endocrine cell (A and D cells). They revealed dopa-provoked FIF in cells with ultrastructurally characteristic pleomorphic granules in the future pancreatic region of the gut, and subsequently, similar granules, also with APUD features, in the cells containing granules diagnostic of the islet cell types.

When seeking APUD neural crest cells in chick embryos, however, Polak et al. (1971) could find none at early stages and no fluorescent cells en route to the gut. These observations have been confirmed for chicks (Andrew, 1975), but unexpected dopa-provoked FIF appeared at very early stages in cells in the roof of the groove that later closes to form the gut tube. At subsequent stages, such cells were concentrated at the site of evagination of the dorsal bud and later on, within the bud. These observations are in line with those of Pearse et al. (1973b), suggesting that progenitors of pancreatic islet cells can be identified in the gut, but they throw no light on the question of whether pancreatic endocrine cells are of neural crest origin.

In 1976, Pictet et al. cultured endoderm and mesoderm of rat embryos from which the ectoderm had been removed before the 4-somite stage. Neural ectoderm was thus excluded, as were neural crest cells that begin to migrate only after this stage. The presence, in the cultures, of pancreatic B cells demonstrated by insulin-immunoreactivity is evidence that they are not neuroectodermal in origin.

At about the same time, a series of experiments was undertaken in chick embryos in which lengths of neural tube containing neural crest were replaced isotopically and isochronically by neural tube and crest originally from chick embryos labeled with tritiated thymidine and subsequently from quail embryos.

In the first experiment (Andrew, 1976b), the host embryos varied in age between the 6- and 24-somite stages; the grafts were made at trunk levels, i.e., caudal to somite 5. For embryos at each stage, it was known at which levels the neural crest had not yet formed and at which levels migration from the crests proper had not yet begun (Andrew, 1963). These were the levels selected for transplantation. Earlier work had shown that such provisions would preclude the possibility that host neural crest cells had emigrated from the transplantation site before the operation was performed. In embryos killed at 3.75 days of incubation, isotope was detected by autoradiography, and quail cells, by Feulgen staining. Donor cells in sympathetic and spinal ganglia showed that neural crest migration from the grafts had occurred normally. Cells showing dopa-provoked FIF in the pancreas bore no isotopic label and contained no quail nuclei. It was concluded that neither the neural crest nor the neural ectoderm of trunk levels had contributed to the pancreatic APUD cells present.

It was, however, not established which pancreatic endocrine cell types could be embraced by this conclusion, and so the experiment was repeated and operated embryos were allowed to develop longer, until A, B, D, and avian pancreatic polypeptide (APP) cells could be distinguished. Electron microscopy and immunocytochemical methods showed that none of these cell types contained quail nuclei (Kramer and Andrew, 1981); therefore, none was derived from trunk levels of neuroectoderm.

Meanwhile, Fontaine et al. (1977) replaced chick neural crest and tube of (presumably) 6- to 9-somite chick embryos with quail grafts at vagal levels (opposite somites 1 to 7). Feather pigmentation and quail enteric ganglia demonstrated successful migration of crest cells from the grafts. In embryos of 13 days’ incubation, quail cells that had invaded the pancreas did not show FIF and proved to be neurons and neurilemmal cells. Other cells exhibited dopa-provoked FIF: these must have been islet cells since chicken A, B, D, and APP cells all have this characteristic. They contained no quail nuclei, nor did islet cells stained with lead hematoxylin. The islet cells present were clearly not derived from vagal levels of the neuroectoderm.

Similar operations were performed by Andrew and Kramer (1979), the grafts extending for varying distances between the mesencephalic–metencephalic junction to the level of somite 5 in 6- to 9-somite embryos. The presence of quail cells in cranial sensory ganglia, enteric ganglia, and cells accompanying parasympathetic pancreatic nerves confirmed normal migration of neuroectodermal cells from the grafts. APUD pancreatic cells demonstrated in operated embryos at 3.75 days of incubation did not show the quail nuclear marker, nor did A, B, and D endocrine cells distinguished by specific stains for light microscopy and by electron microscopy in older embryos. D cells were sparse, but it could be concluded that at least A and B endocrine cells are not derived from rhombencephalic levels of neuroectoderm and probably not from mesencephalic levels.

The validity of the conclusion reached by Fontaine et al. and by Andrew and Kramer depends on elimination of the possibility of neural crest migration having preceded transplantation. This possibility was precluded by the choice of host stages. The neural crest forms first at mesencephalic levels (Holmdahl, 1928; Romanoff, 1960); this formation occurs at the 6-somite stage, at which no migration has taken place (Holmdahl). Migration starts here and at the 8-somite stage (Romanoff). Therefore, operations at mesencephalic levels were performed at and before this stage. On a similar basis, operations at rhombencephalic levels were carried out at or before the 9-somite stage.

The above-cited evidence that at least the major pancreatic endocrine cell types are not neuroectodermal in origin seems incontrovertible. In an experiment designed to distinguish between mesoderm and endoderm as the source of endocrine cells (see the following section and Andrew, 1977), A, B, and D islet cells identified by electron microscopy arose from endoderm in the few cases in which pancreas differentiated, which is in line with the formerly accepted endodermal origin of pancreatic endocrine cells.

ii Gut endocrine cells.

A few years before the emergence of the APUD concept, an experiment was conducted to find out whether enterochromaffin cells were neural crest derivatives (Andrew, 1963). Pieces of chick blastoderm were excised so as to exclude neural ectoderm and neural crest from experimental expiants and include them in controls. The expiants were cultured as chorioallantoic grafts. Elimination of neural crest at the 9-to 22-somite stages was based on a morphological study of neural crest formation and the onset of migration in the breed used; in the case of donors between the 8-somite and the definitive primitive streak stage, explants were excised from caudal blastoderm to exclude that area around the primitive node defined by various workers as potential neural plate and/or neural crest. Exclusion of the crest was confirmed by the absence from experimental grafts of the neural crest derivatives, spinal and enteric ganglia, and melanocytes. However, enterochromaffin cells were in every case present in well-differentiated gut of experimental as well as control grafts, even in the many grafts prepared before the 8- and even the 6-somite stage.

Le Douarin and Teillet (1973) agreed with these findings. In quail-chick chimeras designed to determine the level of origin of enteric ganglia from the neural crest, the enterochromaffin cells found were not derived from either vagal or trunk levels of the crest. They could identify no quail cells in the epithelium of the gut.

Cheng and Leblond (1974a,b), from their studies using tritiated thymidine in an analysis of the renewal of epithelial cell types in the mouse intestine, gained the impression that the labeled endocrine cells were derived from the same columnar cells at the bases of the crypts as were the other cell types. An endocrine cell containing the type of large phagosome that they considered a marker of crypt base cells was regarded as evidence for this view. The occurrence of a few cells containing both endocrine granules and mucous droplets led them to exclude the possibility of a separate precursor cell for endocrine cells. These observations provide rather scanty support for the endodermal origin of gut endocrine cells, but they are in line with this possibility. The same may be said of the findings of Matsuyama and Suzuki (1970). In regenerating murine gastric epithelium consisting ostensibly of immature mucous cells only, they found that cells called argyrophil (identified by membrane-bounded secretory granules) differentiated, as did parietal and chief cells. That undifferentiated separate progenitors of endocrine cells were undetected is, however, theoretically possible.

The promulgation by Pearse of the idea that all endocrine cells of the gastrointestinal tract were of neural crest origin prompted further experimentation (Andrew, 1974). Endoderm and adherent mesoderm constituting presumptive gut, excised from chick embryos between the short head process and the 25-somite stages, were cultured as chorioallantoic grafts. In the gut that differentiated, enterochromaffin cells were present whatever the stage of the donor. It should be noted that more than half the grafts were derived from embryos younger than the 8-somite stage and a goodly proportion of those again, from embryos younger than the 6-somite stage; some donors were even presomite embryos. Since the earliest crest migration starts at the 8-somite stage and that in the head (which was excluded from the expiants) (see Section II,B, 19,b,i), there should not have been any neural crest cells in these grafts. In grafts from embryos of the 10-somite stage and older, neural crest cells were present by intent, enteric ganglia being found as expected from previous work (Andrew, 1964). No enteric ganglia were encountered in the grafts from the younger embryos. This finding in itself supports the argument that