Some Aspects of Research in Developmental BiologyAuthor(s): Philip GrantSource: BioScience, Vol. 17, No. 3 (Mar., 1967), pp. 170-173Published by: Oxford University Press on behalf of the American Institute of Biological SciencesStable URL: http://www.jstor.org/stable/1293835 .

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Some Aspects

of Research

in

Developmental Biology

Philip Grant

University of Oregon

Dr. Grant served as Director of the Developmental Biology Program of the National Science Foundation from 1962 to 1966.

As one surveys the broad trends of the last few years in developmental biology, it can be stated that the most

noteworthy phenomenon is the realiza- tion and acceptance of the notion

among developmental biologists that the complexities of developmental systems are now open to investigation at all levels of organization, from the molecular to the organismic. In 1958 at the Johns Hopkins Symposium on the chemical basis of development, Dr. S. Spiegelman vigorously complained of the pessimism which dominated the

thinking of embryologists and called for a serious investigation of embryo- genesis at the molecular level within the

working hypotheses of microbial ge- netics. Apparently, his persuasive ap- peal has borne fruit. In 1961, Jacob and Monod enunciated the messenger RNA

hypothesis and elaborated on repressor- inducer feedback models to explain differentiation in embryonic systems. At Endicott House, MIT, in 1962, a

group of embryologists met with molec- ular biologists to assess the new models of microbial genetics and molecular

biology as they relate to developing systems. That year may have repre- sented a turning point for develop- mental biology as embryologists, bio- chemists, and molecular geneticists plunged in to analyze development at the molecular level. Also in that year, in a significant appraisal of the impact of molecular biology on development, C. Grobstein illuminated this central issue by stressing the importance of a balanced analysis of developmental problems at all levels. It becomes es- sential to understand how the events at one level are translated into ontogenetic behavior at the next higher level dur-

ing the emergence of the organism. In the 3 or 4 years since the Endi-

cott House meeting, molecular develop- ment has become established as a vig- orous area of investigation, attracting an increasing number of bright gradu- ate students. Moreover, important ob- servations have been made in several

developmental systems which suggest that the microbial models, with modi- fication, may, in fact, apply to em- bryonic systems. Investigations of such systems as those of the sea urchin and

amphibian embryos have led to con- cepts such as "masked messenger RNA," a more stable messenger RNA in contrast to the labile species in

170 BioScience March 1967

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bacteria. In several instances, some evidence of control at the polyriboso- mal level of translation of genetic mes- sages into functioning polypeptides has also been obtained. These, however, represent only quantitative modifica- tions of fundamental molecular mech- anisms postulated for information trans- fer in microbial systems. The concept of the unity of biological systems sug- gests that basic biochemical mechan- isms, particularly as they relate to the behavior of informational macromol- ecules, are common to all organisms, from phage to man. Evolution has simply embellished and compounded this basic theme in eukaryotic forms to accommodate the demands of larger size and greater range of environmental adaptation.

This view, of course, has led to in- vestigations of microbial systems with the avowed purpose of understanding developmental problems. Sporulation and germination in bacteria and fungi are rightfully treated as developmental phenomena, and insight into the mo- lecular interactions occurring here may be applicable to cytodifferentiation in higher forms. Even more exciting are the analyses of R. Edgar of Cal Tech and his colleagues on morphogenesis in phage. By the brilliant application of genetic tools, this group has begun to unravel some fundamental questions of morphogenesis at the macromolecular level. Out of this work will arise some undersanding of the factors responsible for the assembly of highly organized macromolecular complexes (at the level of cell organelles) from genetically controlled polypeptide chains. Here

again, a microbial system serves to

generate models for organellogenesis within the cells of eukaryotic organisms.

It is interesting, in this connection, that a staff member at a well-known institution recently remarked that he suddenly realized that more than 60% of the faculty in his department are involved in developmental biology, whereas 5 years ago he would have identified the majority as being molecu- lar biologists or geneticists. It is cer- tainly evident that the questions now being asked about gene expression and its regulation are relevant develop- mental questions. One can say with enthusiasm that genetics and embryol- ogy have been resynthesized into a more fruitful conceptual framework

catalyzed by molecular biology and bio- chemistry.

Unfortunately, the vigor with which molecular biology has been infused into development has not been accom- panied by rigor in the application of sophisticated technical procedures. Too frequently the literature is being mud- died up by uncritical data and unbridled interpretations as eager individuals, be- cause of naivete, indiscriminantly em- ploy the fashionable technique to de- velopmental systems. The quality of the output on these problems does not gen- erally meet the high standards demand- ed of the biochemist or molecular biolo- gist. In the early phases of exploration of molecular development it is essential that critical, sound information be ob- tained. Otherwise, the observations will only confuse and misdirect those who follow.

Some encouragement, h o w e v e r, should be provided the courageous, the ambitious, those who do not hesitate to jump in with enthusiasm. I believe these individuals are essential to stimulate a new field, dig out the problems, and focus on the issues. Once the situation begins to ferment, with many new investigators involved, then standards may be elevated to insure high quality information. This may be achieved by offering special training courses or workshops for investigators who are now employing or who wish to employ elaborate biochemical procedures to their problems. Certainly the scientific societies, such as the Society for De- velopmental Biology, can sponsor in- strumentation workshops seeking sup- port from appropriate granting agen- cies. Similarly, funds should be pro- vided to individuals who wish to devote a period of study in someone else's laboratory. Such funds 'may be handled in a special small-grants program spe- cifically designed to permit "retreading" of an investigator's outlook and skills. Unless something is done to improve the quality of some of the current out- put in molecular development, the dis- cipline will fail to attract capable inves- tigators so badly needed at this point.

Accompanying these exciting trends of the last 4 years has been some concern that the training of young Ph.D.'s in development may, because of the fashion, overemphasize the value of molecular approaches to develop- ment. Have we leaped too eagerly on

the molecular biology bandwagon, for- getting that embryogenesis involves cel- lular and supracellular phenomena, equally demanding of vigorous quanti- tative analysis? Although this concern may be justifiably directed to certain institutions, it is fortunate that centers of training exist which emphasize cel- lular and tissue level phenomena. More significantly, these areas of investiga- tion have also made significant strides over the last few years, generating as much excitement as those in molecular development. In fact, a realistic ap- praisal of the situation would dispel any real cause for concern. The cellular and tissue organizational levels are progressing rapidly, spurred on by new developments in organ and cell culture. A more balanced discipline, coupling molecular and cellular approaches to development is a major trend in some laboratories. In this last year, for ex- ample, particular success has been achieved in the establishment of in vitro cell populations which either retain their differentiated function as clones or may exhibit in vitro dif- ferentiation from a relatively undif- ferentiated cell type. Retinal pigment cultures, differentiating myoblasts, and the clonal isolation of chondrocytes are but a few examples of the successful in vitro systems being investigated. These same systems, after control of differen- tiation has been achieved, are later amenable to biochemical analyses. Gor- don Sato at Brandeis University has established adrenal tumor cultures which have retained the capacity for steroid biosynthesis. Studies have been

completed on the mechanism of ACTH action on these cells. By means of actinomycin D, pulse labeling with uridine and phenylalanine, and the separation of RNA and proteins, it has been found that ACTH control over adrenal tumor steroid synthesis is mediated by synthesis of a special pro- tein via the activation of a messenger RNA already present in the cell. It is, therefore, possible to investigate bio- chemical regulatory mechanisms relat- ing to the maintenance of the differ- entiated state in culture. One may eval- uate in vitro such factors as nutritional environment (amino acids or hormones, for example), influence of substrate on cell migration, adhesiveness, aggre- gation, cell division, and ultimately cytodifferentiation. These phenomena

BioScience March 1967 171

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at the cellular level still require inten- sive investigation before meaningful questions may be posed at the molecu- lar level.

The underlying faith of some biolo- gists is that such phenomena as cell aggregation, sorting out, and tissue moro- phogenesis may be explained in terms of the synthesis of specific surface com- ponents elicited by controlled gene ex- pression. It is anticipated that the "in- teraction" of cellular and molecular approaches to development will be most productive in carefully controlled in vitro situations.

A number of interesting accomplish- ments appeared last year in this area of in vitro cytodifferentiation and its control. Efforts have continued to pro- duce new clonal systems patterned after Konigsberg's chick myoblast clones which successfully differentiated into striated muscle. Drs. Coon and Zwill- ing at Brandeis University reported successful cloning of chick cartilage as a stable trait persisting in culture over many generations. Embryonic cartilage cells were cloned in a conditioned medium and shown to retain the ability to synthesize cartilage even after a tenfold increase in population, partic- ularly when regrown in dilute culture. Similarly, Dr. Robert Cahn at the Uni- versity of Washington has brown ret- inal pigment cells as clones over 50 cell divisions without loss of pigment. They lose their pigment when grown as mass monolayers, but repigment when grown as clones, indicating the stability of differentiated functions in culture. It is now becoming evident that past failures to demonstrate stability of dif- ferentiated function in long term in vitro culture reflected our ignorance in providing a proper nutritional environ- ment. Other cell lines have been es- tablished and one can expect a signifi- cant expansion of exciting work in this area in the next few years.

An indication of the nature of some of the environmental factors which may condition cytodifferentiation in vitro has come from the work of Dr. C. Grobstein at the University of Cali- fornia at San Diego who has been studying the interactions between epi- thelium and mesenchyme during pan- creas and salivary gland cytodifferentia- tion. Some of his recent results indicate that differentiation in vitro of the sali- vary gland is accompanied by the depo- sition of a collagen matrix between the

interacting tissues prior to the major events of cytodifferentiation. Dr. Grob- stein suggests that collagen may func- tion as an important morphogenetic mediator at inductive interfaces. These observations are particularly significant in the light of a recent report by Konigsberg and Hauschka (1966) that conditioned medium may be replaced by reconstituted collagen films in muscle differentiation of myoblast clones. A coherent story is gradually emerging that the interaction between cell surfaces and their immediate matrix environment or substratum have pro- found effects on cellular behavior dur- ing the course of cytodifferentiation.

The role of the cell surface and un- derlying cortex in development has al- ways been an area of active investiga- tion, particularly as it relates to sorting out, cell recognition, and cell aggrega- tion behavior. Such activities are of considerable importance in morpho- genesis and have been under investiga- tion in a number of laboratories, here and abroad. Recent progress in this field has been excellent and some new leads have been uncovered. In addition to playing a role in cell aggregation, cell surfaces are recognized as playing vital roles in regulating cell and nuclear functions. New interest is being gene- rated in the role of ions as regulators of enzyme function and cellular be- havior and, of course, on changes in membrane permeability as a modulator of ion flow. The importance of ions in growth and differentiation has been elegantly demonstrated by A. Braun at Rockefeller Institute in the crown gall phenomenon of plants. In a bril- liant series of studies initiated more than 10 years ago, the physiological basis for autonomous growth of the crown gall tumor has been fairly well determined. Tumor cells, in contrast to normal cells, have acquired the capacity for autonomous growth be- cause they can synthesize all of the essential growth substances that their normal counterparts require but are incapable of making. The critical hor- mones and growth substances synthe- sized by the tumor result from a se- quential unblocking of biosynthetic pathways which may reflect progressive alterations in the properties of mem- brane systems during the course of the neoplastic transformation. R e c e n t studies have shown that several essen- tial biosynthetic systems unblocked in

the plant tumor are activated by spe- cific ions. These studies demonstrate that progressive functional changes in membrane permeability during cell transformation allow the penetration of essential ions to key metabolic compart- ments in autonomous tumor cells while these are prevented in normal cells. The fully autonomous tumor cells take up ions more efficiently than normal cells, perhaps activating a significant metabolic sequence concerned with cell growth and division. Studies of this type will lead ultimately to fractiona- tion of cellular membranes (as is now possible for some bacterial cells) and their characterization. Models for growth control by regulation of ion flow through modifications of mem- brane permeability may be applicable to other developmental systems and should lead to some exciting opportu- nities for new programs.

The work of Sonneborn and his col- leagues on the inheritance of cortical patterns in Paramecium has renewed interest in the cortex in eggs and em- bryos as bearers of "developmental in- formation." How is information organ- ized and stored within a cortical frame- work? What is the mechanism of its expression during development? How does it interact with other information systems in the nucleus and cytoplasm? These questions will come to dominate our thinking as we strive to evaluate different levels of control from the genome to overt cell behavior during growth and differentiation.

Future trends in development must not exclude the potentialities of studies on organellogenesis - the ontogeny of cellular organelles, such as plastids, mitochondria, flagellae, and cilia. A recent regional Developmental Biology Conference at Ames, Iowa, was ad- dressed specifically to the question and assembled an interesting mixture of presentations on chloroplasts, photo- responsive membrane systems, cilia, and centrioles. Studies of these struc- tures, at the supramacromolecular level of organization are akin to the investi- gations currently directed toward the morphogenesis of phage and tobacco mosaic virus. Moreover, since several such elements may behave as autono- mous genetic systems, another level of regulatory phenomena defining cellular behavior during embryogenesis must be proposed. What is the nature of inheri- tance in such extra-nuclear systems?

172 BioScience March 1967

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How is their organization created and how is it maintained? What is the mechanism of interaction between semi- autonomous cytoplasmic systems and the nuclear genome?

Exciting new possibilities for cellu- lar regulation during development have been suggested in the work of protein chemists studying the function of pro- teins as related to their structure. Dr. D. Koshland of the University of California, Berkeley, has demonstrated the influence of conformational changes on the tertiary structure of protein molecules as expressed in alterations of enzymatic behavior and has proposed models of cellular activity (permea- bility-active transport) based on care-

fully modulated conformational changes in enzymatic and structural proteins. At the level of the ontogeny of protein structure and its modification by small molecules, the problems of intracellular

regulatory mechanisms become unusu-

ally complicated because we lack the essential rationale and approach to in-

telligently attack them within morpho- genetic systems. We shall soon be able to handle these problems in simple systems as protein biochemists success- fully provide the tools and concepts. We have yet to learn to ask the ques- tions that are uniquely answerable by developmental systems.

Two other trends merit mention. Fine structural studies of plant and animal

developing systems are rapidly assum-

ing major importance in many labora- tories. Electron microscopy has pro- gressed rapidly beyond the "picture- taking" phases of the earlier years and has now reached maturity. Critical bio- logical phenomena, impossible to ana-

lyze with the light microscope, are now amenable to attack with electron mi-

croscopy. Problems of cellular motility, contractility, and form changes are

open to meaningful investigation at the fine structural level. The role of micro- fibrils and filaments, the ontogeny of organelles, and particularly the relation between structure and function are ex-

amples of a few dominant issues. Of

equal value are studies of the nature of intercellular connections or associa- tions - the role of "tight junctions" as a morphological basis for physiological continuity between cells. The classical

embroyological concepts of "fields" and

"gradients" may owe their explanation to the existence of distinctive morpho- logical conections among cells allowing for a "community of action" of a par- ticular cell population. More impor- tantly, such cellular associations facili- tate the establishment of "regulatory foci" (the center of a "field," or the

peak of the gradient) which may in- fluence cells at the periphery. Here

again, classic problems are now open to

analysis with modern, sophisticated techniques.

Finally, mention must be made of expanding interest in problems of the ontogeny of neural function and be- havior. As a result of progress made in neural physiology, particularly with the achievements of the microelectrode technique, it is inevitable that these superb techniques would be applied to embryological problems. Problems of specification of nerve pathways from sense organs to the central nervous system during embryogenesis and nerve regeneration have received particular attention over the years and are effec- tively exploited with the new technol- ogy. Although neurophysiologists have traditionally been active in the area of developmental neurophysiology, too few embryologically trained investiga- tors have dared to venture into the

apparent esoteric environment of neu-

rophysiology. Just as other peripheral

fields are now focusing on neural biol- ogy (genetics, biochemistry), so too, the developmental biologist is becom-

ing committed to this area, albeit slowly and certainly not in the numbers re-

quired. No doubt, as the field expands many will be attracted by the opportu- nities for clever experimentation at this level.

It is possible to predict that a fusion of disciplines will occur as the embry- ologist's growing interest in membrane and surface phenomena collides with the neurophysiologist's interest in ex-

tending his microelectrode probes into a wider spectrum of cells. What better way to measure changes in membrane func- tion (at least to certain ions) than by a microelectrode procedure?

As is obvious from the above illus- trations, developmental biology has broadened its outlook in biology. It looks at all levels of biological organiza- tion and exhibits a healthy infusion of new techniques, procedures, and con- cepts from other disciplines. Old lines

separating disciplines have broken down and more sophisticated interdisciplinary approaches have taken the place of tra- ditional attitudes. It is unfortunate, therefore, to realize that training at graduate and particularly at undergrad- uate levels has failed to keep up with these vital trends in research. Only a few institutions have drastically modi- fied their biology curriculum to accom- modate this exciting new synthesis in biology. Traditional courses in tradi- tional administrative organizations (bot- tany and zoology departments, for ex- ample) persist and thrive in spite of the growing clamor for change. I be- lieve in no other discipline is evidence of this ferment more striking than in developmental biology which now seems to represent one of the primary interests of modern biology.

Central States Entomological Society The Central States Entomological Society will hold its 42nd annual meeting May 6 in the Agriculture Build- ing at the University of Missouri, Columbia. (Robert B. Mills, Secretary-Treasurer, Kansas Entomological So- ciety, Department of Entomology, Kansas State Univer- sity, Manhattan, Kansas 66504).

Symposium on Population Biology

The New York State Science and Technology Foundation is sponsoring a Symposium on Population Biology to be held at Syracuse University, June 7-9. Richard C. Lewon- tin, professor of zoology and associate dean of biological sciences, University of Chicago, is the symposium chair- man. For further information, write: Symposium, Depart- ment of Zoology, Lyman Hall, Syracuse University, Syra- cuse, N.Y. 13210.

BioScience March 1967 173

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