Histological and fine structural changes during chondrogenesis in micromelia induced by 6-aminonicotinamide

DEVELOPMENTAL

BIOLOGY

Histological

28, 555-572(1972)

and Fine Structural

in Micromelia

Induced

Changes

during

Chondrogenesis

by 6-Aminonicotinamide’

ROBERT E. SEEGMILLER,~DENNIS 0. OVERMAN,~ AND MEREDITH N. RUNNER Department

of Molecular,

Cellular and Developmental Biology, Boulder, Colorado 80302

University

of Colorado,

Investigations of drug-induced congenital deformity can lead to better understanding of developmental mechanisms. An analog of nicotinamide, 6-aminonicotinamide (6-AN), given in a dose of 10 pg to chick embryos on day 4 of incubation, resulted in micromelia as diagnosed by reduced cartilage and bone in 100% of embryos. Histologically, chondrogenic cells at day 5 were compactly arranged and the extracellular matrix showed reduced metachromatic staining with toluidine blue. Electron microscopy of these cells showed a deficiency of rough endoplasmic reticulum on days 5 and 6 with dilated cistemae by day 8. In the Golgi apparatus at day 5, vacuoles, which normally collect and transport mucopolysaccharides to the extracellular matrix, were small or absent. Degenerative changes occurred in the core of the chondrogenic rudiment on days 5 to 8, whereas cells and matrix subjacent to the developing perichondrium by day 7 appeared normal in staining and ultrastructural properties. The Golgi apparatus of chondrogenic cells was atypical by virtue of reduced numbers of collecting vacuoles. Necrosis of the central region of the chondrogenic rudiment was transitory; the damage to the rudiment was incompletely repaired by synthesis of matrix by cells near the perichondrial layer. These observations suggest that the limb malformation induced by 6-AN is brought about by inhibition of special synthesis of matrix by chondrogenic cells in addition to necrosis of core cells of the chondrogenic rudiment. INTRODUCTION

Teratogens can assist in elucidating mechanisms of development. The analog of nicotinamide, 6-aminonicotinamide (6AN), was used for study of chondrogenesis in development of the limb. Previously 6AN has been used for studying development of the nervous system, heart, palate, and skeletal system of birds and mammals (Landauer, 1957; Pinsky and Fraser, 1960; Landauer and Clark, 1962; Chamberlain, 1970; Overman and Beaudoin, 1971; Turbow et al., 1971; Caplan, 1971). A dose of 10 pg, when given to day-4 (stage 24, Hamburger and Hamilton, 1951) chick embryos in ovo causes a reduction in body size, shortening of the beak, and dispro‘Supported by NIH Grants HD-02282 and DE198 and a Fellowship from the Pharmaceutical Manufacturers Association Foundation. z Present address: Department of Zoology, Brigham Young University, Provo, Utah 84112. 3 Present address: Department of Anatomy, West Virginia University, Morgantown, West Virginia

26506.

portionate shortening of the limbs. Landauer (1957), after experimentally treating embryos with 6-AN, showed that skeletal tissue of the limb is a primary target of the teratogen in causing micromelia. Researchers have reported seeing necrosis of the condensing mesoderm of presumptive cartilage (Shepard et al., 1968; Seegmiller et al., 1971b,c) and have shown effects on differentiation of cartilage in vitro (A. Caplan, personal communication). The effect of 6-AN as an inhibitor of 35S-sulfate incorporation into mucopolysaccharides of embryonic heart of the rat was reported by Overman and Beaudoin (1971) and incorporation of 35S in the early limb bud of the chick embryo was reported by Searls (1965a). Recently Overman et al., 1971, 1972) reported the effect of 6-AN on incorporation of a chondroitin sulfate precursor, Na,Y30,, into limbs and cartilage rudiments of the chick embryo. It is generally assumed that 6-AN forms 6-aminonicotinamide adenine dinucleotide which replaces the normal

555 Copyright All rights

0 1972 by Academic Press, Inc. of reproduction in any form resenwd.

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DEVELOPMENTAL BIOLOGY

coenzyme nicotinamide adenine dinucleotide (NAD) (Landauer, 1957; Dietrich et al., 1958; KShler et al., 1970), and presumably causes a general reduction in oxidative phosphorylation. A better understanding of the causal relationship between the presumed production of an inhibiting analog of NAD and the occurrence of micromelia, was sought by experiments designed to study the effects of the drug on limb development in the chick. Observations, with light and electron microscopy, on the effects of 6AN during the expression of micromelia are reported in this communication. These observations showing early pathogenesis correlate with 6-AN induced changes in biochemical processes of chondrogenesis (Overman et al ., 1972). MATERIALS

AND

METHODS

Hatchery eggs obtained from Hy-Line Poultry Farms, Des Moines, Iowa were incubated at 38°C in a Jamesway single stage incubator. At day 4 (96 hours of incubation, i.e., Hamburger-Hamilton stage 24) 1Opg of 6-AN in 50 ~1 of distilled water were injected into the extraembryonic coelom between the inner shell membrane and the vascular splanchnopleure. Distilled water alone was injected into control eggs. Embryos were recovered 6 hours to 6 days later for light and electron microscope studies of hind limbs and 4 to 16 days later for observing gross and skeletal malformation. Whole embryos for skeletal observations were stained with toluidine blue and alizarin red and were cleared with glycerin according to Burdi and Flecker (1968). Approximately 100 treated and control embryos of various stages were examined. Hind limbs were prepared for light microscopy by fixing in Carnoy’s solution, dehydrating in alcohol, and embedding in paraffin. Sections were cut at 7 p and stained with toluidine blue. Whole or portions of hind limbs were

VOLUME 28, 1972

prepared for electron microscopy according to the method of Sheldon et al. (1970) by fixing with 3% glutaraldehyde buffered at pH 7.3 with sodium phosphate, postfixing with 2% osmium tetroxide, and dehydrating in alcohol. Tissues thus prepared were embedded in Epon, cut with a diamond knife, stained with uranyl acetate and Reynolds lead citrate and photographed with a Philips 300 microscope. In some instances tissues were stained en bloc with 2% uranyl acetate. Thick sections cut at 2 p were stained with 0.5% toluidine blue in 0.5% sodium borate for light microscope comparison with serially cut thin sections. Estimates of the effect of treatment on the Golgi apparatus and rough endoplasmic reticulum were made by comparing approximately 215 electron micrographs of treated and control chondrogenic tissues. OBSERVATIONS

Gross and Skeletal Morphology

Malformations were seen 100% of the time following treatment with 10 pg of 6-AN at day 4. Embryos thus treated and observed at day 6 were distinguishable from controls because the distal part of the hind limb projected anteriorly. Fore and hind limbs of day-10 embryos showed severe micromelia (Fig. 1). The severity and frequency of micromelia depended on the dosage and timing of the drug. For example, doses less than 10 pg administered at day 4 resulted in a variety of types from outwardly malformation normal to severely malformed limbs (Fig. 2). Administration of 6-AN to embryos older than 5 days caused the distal portion of the hind limb to bend posteriorly without markedly affecting the orientation of the proximal portion. Partial to complete protection resulted when nicotinamide was administered, the severity and frequency of malformation depending upon dosage and timing of nicotinamide admin-

SEEGMILLER, OVERMAN, AND RUNNER

Chondrogenesis

and Micromelia

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i&ration (Overman et al., 1972). Visual inspection of whole amounts stained with alizarin and toluidine blue showed that on days 8-19 cartilage and mineralized bones were markedly reduced in limbs from treated embryos (Fig. 2). Histology of Cartilage Differentiation in Normal Embryos

FIG. 1. Embryos fixed in Bouin’s at day 10. All embryos referred to in this and subsequent figures were treated at day 4. Treatment with 6-AN results in a reduction in body size, a shortened beak, and micromelia. Hind limbs of day 6 embryos were distinguishable from controls because they pointed anteriorly. Fore and hind limbs of day 10 treated embryos had severe micromelia.

Hind limb buds at day 4 (stage 24) from nontreated embryos consisted of undifferentiated mesenchymal cells and an epithelial covering thickened at the distal or apical ridge. By day 5 (stage 27) chondrogenic changes in the central, proximal region of the limb bud had begun: the cells had enlarged, the cytoplasmic to nuclear ratio had increased, and the cells were aligned with their long axes perpendicular to the long or proximodistal axis of the limb. There was appreciable intercellular space filled with matrix, and at day 5 the matrix stained weakly with toluidine blue.

FIG. 2. Day-14 embryos stained with alizarin red and toluidine blue to depict bone and cartilage. Embryos receiving less than 1Org of 6-AN (center) show retardation in growth of cartilage and ossified bone. The embryo on the right, typical of those treated with the usual 10 pg dose, did not stain well with either stain, suggesting a marked reduction of cartilage and bone.

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In the area of the prospective knee-joint aggregation of macrophages and some necrosis were noted on days 4 and 5. Muscle differentiation, occurring peripheral to the chondrogenic area, was indicated by day 5 by the presence of multinucleate cells with intracellular, striated fibrils. By day 6, tissue of the central region had differentiated into cartilage as indicated by extensive amount of intercellular matrix (Fig. 3) and by intense, positive staining reaction (metachromasia) with toluidine blue. The period of observation in untreated limb buds from days 4 to 6 included changes from precartilaginous mesenchyme to characteristic appearance of embryonal cartilage. Histology of Cartilage Differentiation in 6-AN Treated Embryos Presumptive cartilage cells by day 5 and later were compactly arranged, i.e., the amount of intercellular space was less than controls (Figs. 4 and 5). The matrix as determined by staining with toluidine blue was weakly positive for acid mucopolysaccharides. The diagrams in Fig. 6 from treated and control embryos depict differences in amounts of cellular material in chondrogenic rudiments relative ,to the quantities of matrix. The lesser amount of intercellular space one to 6 days after treatment correlates with reduced metachromatic staining by toluidine blue. Cell counts from randomly selected areas of treated and control rudiments showed that those treated with 6-AN had 1.5-2

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times the numbers of cells per unit area thereby accounting for the compact appearance of treated chondrogenic tissue. Cells subjacent to the perichondrial layer appeared normally dispersed on days 7 and 8 (arrows Fig. 5). The matrix surrounding these cells stained positively with toluidine blue. The prospective region of the knee joint from day 5 hind limbs showed extensive necrosis. On days 5, 6, 7, and 8 (Fig. 5; Fig. 15, inset) necrosis extended the entire length of the rudiments and appeared to extend across the knee-joint region. Cells in the developing perichondrium appeared to be unaffected by treatment. Treatment with 6-AN at day 4 prevented cellular dispersal expected at day 5. Compactly arranged cells were associated with weak metachromasia and central necrosis. Concomitant with these alterations the peripheral area appeared to be resistant to 6-AN and showed a tendency for typical cellular dispersal and metachromasia. Electron Microscopy of Cartilage Differentiation in Normal Embryos Cells in the prechondrogenic mesenthyme of day 4 hind limb buds contained Golgi bodies and rough endoplasmic reticulum which were small or reduced in amount. Numerous free ribosomes were present. The nucleus showed uniformly dispersed chromatin and a large, darkly stained nucleolus. At day 5 cells of the central region of the limb showed cyto-

FIG. 3. Cartilage from the proximal portion of hind limb of a day-6 control embryo. Chondrocytes are shown with their long axes perpendicular to the proximodistal axis of the limb. The cells are dispersed by matrix which exhibits metachromasia after staining with toluidine blue. x 425. FIG. 4. Chondrogenic rudiment from the proximal portion of hind limb of a day-6 embryo treated with 6-AN. The tissue shows only weak metachromatic staining and compactness of cells due to reduced intercellular space. The cells are not typically arranged as shown in Fig. 3. Normal appearing muscle is present in lower right. x 425. FIG. 5. Cartilage from the distal portion of hind limb of a day-8 embryo treated with 6-AN. Slightly oblique longitudinal section shows variation in cell morphology and arrangement. Cells are generally compactly arranged except for those near the perichondrium (arrows) which are dispersed by a normal staining matrix. True longitudinal sections show that the central core of necrosis extends the entire length of the rudiment (cf. Fig. 15, inset). Nearby degenerating cells are darkly stained and show vacuolation of the cytoplasm. Development of the periosteum is detectable by presence of osteoid material (oat). x 450.

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FIG. 6. Drawing of chondrogenic cells (stippled) embedded in Epon depicts the relative areas of intercellular space (unstippled). Treated tissue shows more cellular material and less intercellular space per area.

plasmic differentiation indicative of chondrogenesis (Fig. 7). Namely, the Golgi complex showed an increase in number of large vacuoles distended with stainable material (cf. arrows, Figs. 9 and 11). Also, the rough endoplasmic reticulum had increased in amount over previous stages, as measured by increased numbers and length of profiles of reticulum. The rough endoplasmic reticulum showed some dilation of the cistemae. By day 6 cytoplasmic organelles showed further development with vacuoles of the Golgi complex as large

as 2000 A in diameter (arrows Figs. 9 and 11) and with further elaboration of rough endoplasmic reticulum (Figs. 9 and 13). The intercellular matrix of precartilage became noticeable in day-5 limb buds (Fig. 7). This matrix contained fewer collagen fibrils than were seen at day 6 and later, at which time numerous unbanded fibrils of 200 A diameter were dispersed uniformly in the matrix. In summary, observations with light and electron microscopy of the central region of the day 5 control hind limb revealed cell-

FIG. 7. Electron micrograph of cartilage from the hind limb of a day-5 control embryo. Chondrocytes show an increased amount of cytoplasm and organelles compared with prechondrocytes of earlier stages. Nuclear chromatin is homogeneously dispersed, there is relatively little heterochromatin, and the nucleolus is a prominent structure. The intercellular matrix contains collagen fibrils. x 6000. 31apllarly with control. There is more cell contact and less intercellular matrix than in the control. x 6000.

SEEGMILLER,

OVERMAN,

AND RUNNER

ular enlargement and alignment of elongated cells with their axes perpendicular to the longitudinal axis of the limb. Intracellular organelles showed changes during the period of observation such as enlargement of vacuoles of the Golgi complex and elaboration of the rough endoplasmic reticulum, both being indicative of synthesis of product for export. This indication is confirmed by an increase of intercellular, stainable, collagenous matrix (Figs. 7, 9, and 13). These processes became exaggerated on day 6 and thereafter. Electron entiation

Microscopy of Cartilage Differin 6-AN Treated Embryos

Electron micrographs showed that, whereas intercellular contact of controls was limited to a few cytoplasmic processes, considerably more intercellular contact was observed on days 5 to 8 (Figs. 8, 10, and 14) in compactly arranged cells from treated embryonic limbs. In compactly arranged cells of treated embryos nuclear chromatin frequently was less uniformly dispersed than that of control (Fig. 8). The collecting vacuoles of the Golgi apparatus were either smaller or absent and did not contain the usual amount of stainable material found in the controls (Figs. 10, 12; cf. control Figs. 9 and 11). The appearance of undeveloped Golgi in compactly arranged cells remained through days 6, 7, and 8. There were fewer profiles of rough endoplasmic reticulum in compactly arranged cells on day 5. The cisternae of the rough endoplasmic reticulum in embryos 6, 7, and 8 days old were dilated more than control cisternae, and on day 7 and 8 either contained stippled material or appeared as clear vacuoles (Fig. 14; cf. control Fig. 13).

Chondrogenesis

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The stippled material appeared similar to that of controls; the clear vacuoles were atypical. Cells of the central necrotic area on day 5 (Fig. 15) contained lipofuscin droplets. Mitochondria (arrows) were enlarged and lacked the usual array of cristae. The intercellular matrix of the necrotic area was deficient in collagen fibrils. Concomitant with central degeneration, the fine structure of cartilage matrix and chondrocytes subjacent to the perichondrial layer appeared normal (Fig. 16). Limb buds exposed to 6-AN at day 4 and compared with control limb buds on succeeding days showed smaller bone rudiments, compactly arranged cells, less stainable matrix, fewer collecting vacuoles of the Golgi apparatus, less rough endoplasmic reticulum with enlarged cisternae, and, by day 7, extensive central necrosis. This retardation and regression of cells in the chondrogenic rudiment contrasted with cells subjacent to the developing perichondrium. The perichondrial region showed normal chondrocytes embedded in a matrix which stained positively with toluidine blue. Normal chondrogenesis in the peripheral or cortical region of the rudiment, subsequent to regressive changes in the central region, was indicative of attempted repair by replacement. Effects of 6-AN on central chondrogenic cells are seen on day 5 by reduced Golgi and endoplasmic reticulum and little or no matrix. These atypical cells are destined to die on day 6 or 7. Whether the changes seen on day 5 represent early degenerative effects or whether the day-5 changes are independent of degenerate consequences of 6-AN have yet to be determined.

FIG. 9. Control chondrocyte from a day-6 hind limb shows a large Golgi complex with distended lamellae and vacuoles. Rough endoplasmic reticulum (rer) is often dilated and is shown here associated with the Golgi complex. x 16,000. FIG. 10. Compact chondrogenic cells from a treated day-6 hind limb show reduced amount of rough endoplasmic reticulum and absence of vacuoles of the Golgi complex. x 16,000.

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FIG. 13. Survey electron micrograph of chondrocytes from day-8 control limb rudiment. Several profiles of rough endoplasmic reticulum in the cytoplasm are shown. x 5000. FIG. 14. Survey electron micrograph of chondrogenic cells from day-8 treated limb rudiment. Frequent dilation of the rough endoplasmic reticulum in compactly arranged cells is shown. x 5000.

DISCUSSION

Limb Ontogeny Review of some of the special aspects of normal ontogeny of fore and hind limbs relevant to the timing of teratogenic effects of 6-AN is pertinent. The central, proxi-

ma1 region of embryonic limbs at stages 22-25, days 3 s-41/2, shows increased rate of incorporation of 3”S0, relative to periphera1 cells (Searls, 1965a,b). Searls and Janners (1969) and Janners and Searls, (1970) also showed a smaller proliferative index in the central cells by stage 22. Mes-

FIG. 11. High magnification of a control day-6 chondrocyte. The Golgi apparatus is comprised of lamellae, small vesicles and large vacuoles. The vacuoles frequently contain stainable material believed to be chondroitin and tropocollagen. x 50,006. FIG. 12. High magnification of a day-6 chondrogenic cell shows that large vacuoles of the Golgi complex are absent after treatment with 6-AN. The endoplasmic reticulum (rer), showing only a few attached ribonucleoprotein particles, is shown in close proximity to the Golgi complex. x 50,000.

SEEGMILLER, OVERMAN, AND RUNNER

odermal cells before stage 24 apparently are not fully committed to become chondrogenic cells because the fates of different mesodermal cells are reversible. Reversibility was demonstrated by grafting labeled presumptive chondrogenic mesoderm to chondrogenic and to myogenie regions of unlabeled host limbs of the same stage (Searls, 1967). Irrespective of their origin, cells in grafts differentiated in conformity with the cells of the region to which they were grafted. Zwilling (1966, 1968) suggested that stabilization of specialization in limb cells occurs relatively late, at stage 25, because both prospective myogenic and chondrogenie cells cultured from stage 22, 23, and 24 limb buds, form cartilage equally well. These studies indicate that before stage 24 myogenic and chondrogenic tissues were interchangeably labile. Cells from stage 25 and older limbs appear irrevocably stabilized because in tissue transplants one sees chondrogenesis but not myogenesis (Searls and Janners, 1969). In our experiments 6-AN was administered at stage 24 (day 4) when cells of the central region of the limb, undergoing stabilization, had begun a transition from mesenchymal to chondrogenic cells. This transition is characterized by appearance of chondroitin sulfate and collagen, appearance of matrix and elaboration of the Golgi complex and the rough endoplasmic reticulum. Cytodifferentiation

and Histogenesis

Appearance of enlarged Golgi vacuoles and elaboration of rough endoplasmic reticulum of cells of the central region of the control limb, (Figs. 9 and 11) observed

Chondrogenesis

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at stage 25 (day 4%-5), is associated with developmental stabilization and production and export of macromolecules such as collagen and chondroitin sulfate, i.e., chondrogenesis. Metachromasia in the central region of the limb, observed in tissue sections by stage 25, confirms observations of Fell and Canti (1934). The metachromatic staining property of matrix is due to the presence of acid mucopolysaccharides, primarily chondroitin-4 and -6 sulfates. Tropocollagen, another product of chondrogenic cells, is believed to polymerize extracellularly into collagen fibrils which seldom exceed 200 A in diameter; their size and interspersion depend upon the amount of chondroitin sulfate present (Matthews and Decker, 1968; Toole and Lowther, 1968; Seegmiller et al., 1971a). The visualization of metachromasia as well as appearance of collagen fibrils by stage 25 correlates with stabilization of cartilage. The Golgi apparatus is essential for synthesis and secretion of mucopolysaccharides (Fewer et al., 1964; Godman and Lane, 1964), and mucoproteins (Neutra and Leblond, 1966) and transport of products, such as collagen and MPS, from their site of synthesis at the rough endoplasmic reticulum to the extracellular space (Revel and Hay, 1963). The Golgi apparatus is reported to be the site of sulfation of mucopolysaccharides (Horowitz and Dorfman, 1968; Weinstock and Young, 1972). The presence of compactly arranged cells as seen in Figs. 4, 5, 8, 10, and 14 and demonstration of below normal metachromasia 24 hours after treatment with 6-AN suggest that production of

FIG. 15. Electron micrograph showing compactly arranged cells located near the central necrotic region. In the cytoplasm of compact cells there is a paucity of organelles; however, the mitochondria are intact. Necrotic cells show accumulation of darkly stained lipofuscin pigment granules and degeneration of cytoplasmic organelles, particularly the mitochondria (arrows). x 6000. Inset: Light micrograph of the distal hind limb of a treated embryo shows the region of chondrogenic cells from which electron micrograph was taken. Note the central region of necrosis extending the length of the rudiment. x 200. FIG. 16. This electron micrograph of hind-limb cartilage from a day-8 treated embryo shows that the fine structure of cells and matrix subjacent to the perichondrium is normal. x 10,000.

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protein-polysaccharides is subnormal in the presumptive chondrogenic region. The absence of Golgi vacuoles in prechondrocytes 24 hours after treatment correlates with suppression of matrix production. Failure to see products in the Golgi and failure of these cells to label with 35S-sulfate (Overman et al., 1972) reinforce the interpretation of inhibition of production of matrix. The absence of Golgi vacuoles in chondrogenic cells of the chick limb after treatment with 6-AN differs from the presence of unusually large Golgi vacuoles in tracheal chondrocytes of chondrodystrophic fetal mice (Seegmiller et al., 1972). Chondrodystrophic mice seem to show accumulation in the Golgi complex of precursors to matrix products, but in chondrodystrophy transport of materials to the matrix may be impaired. Thus subnormal chondrogenesis in hereditary chondrodystrophy and in 6-AN induced micromelia appear to have different mechanisms of expression.

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ment with 6-AN, impaired Golgi may be incapable of accumulating and/or sulfating the product prior to export. Alternatively, accumulated product in the rough endoplasmic reticulum might not be transported to the Golgi. (Also it may be that macromolecules, synthesized for intracellular use and not designed for extracellular transport, accumulate under the influence of 6-AN.) Thus, accumulation of product in the rough endoplasmic reticulum and interference with subsequent transport to the extracellular space may be due to a primary defect at the Golgi, such as a defect in sulfation or packaging of acid mucopolysaccharides, or may be due to abnormal synthesis in the rough endoplasmic reticulum. In the latter case the Golgi apparatus would shut down secondarily. The present data do not permit distinction between these alternatives. Necrosis. Macrophages associated with cell death are normally present on day 5 in the area of the future knee joint. SaunMechanisms for 6-AN Induced Microders (1966) has suggested that necrosis melia plays an important role in sculpturing of Matrix production-synthesis for ex- the limb. The occurrence of extensive port. The Golgi apparatus is closely as- necrosis in the central area of 6-AN sociated with the rough endoplasmic re- treated cartilage rudiments on days 5-8 ticulum, possibly to the extent of inter- (Figs. 5 and 15) could be an extension of connection of the membrane system the normal zone of degeneration in the (Goel, 1970). Occurrence of unusually dis- joint, similar to that reported occurring tended cisternae of the rough endoplasmic in chick limbs after treatment with insulin reticulum and absence of Golgi vacuoles (Zwilling, 1959). The central area of nein compactly arranged cells from days 7 crosis induced by 6-AN was larger on days and 8 treated limbs suggest that some ma- 6 and 7 than on day 5 suggesting that terials accumulate in the cisternae of the the compactly arranged cells seen on day rough endoplasmic reticulum and are not 5 degenerated by days 6 or 7. Therefore transported to the Golgi. Since products it appears that, in addition to extending accumulate in the cisternae of the rough the normal zone of necrosis at the joint, endoplasmic reticulum under the in- 6-AN induced central degeneration. Our fluence of 6-AN it appears that protein observations however do not permit this synthesis is not entirely blocked. This ob- distinction. Energy state. How does 6-AN interfere servation is supported by isotope labeling experiments using amino acids (Overman with production of matrix? Dietrich et al. et al., 1972). This suggests that after treat- (1958) recovered 6-AN analogs of NAD

SEEGMILLER, OVERMAN, AND RUNNER

from 6-AN treated mice. It has been shown that 6-amino NAD impairs utilization of glucose by interference with dehydrogenase-linked reactions (Kijhler et al., 1970). Landauer as early as 1948 reported protection from teratogenesis by high energy molecules. Oxidation reduction reactions involved with energy exchange have been implicated in 6-AN teratogenicity by genetic studies in mice (Verrusio et al., 1968) and by protection studies in the chick using energy sources such as glycerophosphate as antiteratogens (Landauer and Sopher, 1970). Dietrich et al. (1958) also reported that 6-AN markedly lowered the level of ADP and ATP in tumors, presumably by interfering with NAD-dependent steps in oxidative phosphorylation. Interference with NAD functions leading to reduced production of ATP also has been reviewed by Landauer and Sopher (1970). Estimates of ATP in hind limbs of chick embryos have showed that 24 hours after 6-AN treatment tissue levels of ATP were reduced (unpublished observations), thereby providing direct measurement confirming the conclusions of Landauer and Sopher. If the predominant pathway for energy source of chondrogenic cells is the NADrequiring glycolysis converting glucose to pyruvate, as suggested by glycerophosphate as an antiteratogen, the pathway would be sensitive to the 6-amino analog of NAD and would result in energy insufficiency for chondrogenesis. Runner (1959) suggested that interference with carbohydrate metabolism and concomitant reduction of high energy phosphate after various teratogenic treatments with insulin, iodoacetic acid, xmethyl-folic acid, and fasting adversely affects differentiation of precartilaginous mesenchyme. Interference with chondrogenesis also has been reported to occur as a result of riboflavin deficiency (Aksu et al., 1968) and folic acid deficiency

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(Chepenik et al., 1970), both of which were correlated with a reduction of embryonic levels of ATP. Synthesis and utilization of high energy phosphate by chondrocytes has been the subject of numerous investigations. For example, it has been shown that chondrocytes primarily depend upon anaerobic metabolism for their energy supply (Whitehead and Weidmann, 1959), i.e., the conversion of glucose to lactate requiring NAD cofactor. The necessity of ATP in activating sulfate (Lash et al., 1964) and the dependency upon energy metabolites in the synthesis of mucopolysaccharides (Marzullo and Lash, 1970) have been documented. If chondrogenic cells after treatment with 6-AN have reduced capability for oxidation-reduction energy exchange they would produce insufficient high energy bonds required for synthesis and secretion of normal quantities of matrix. Presumably, they would also lack the products necessary for cytodifferentiation of organelles and cellular membranes. The hypothesis is proposed that, following treatment with 6-AN, the deficiency of matrix, paucity of rough endoplasmic reticulum and appearance of atypical Golgi of chondrogenic cells from day-5 to day-6 embryonic limbs (Figs. 4, 5, 8, 10, and 12) reflect reduced availability of high-energy phosphate. Selectivity of effects and localization of restoration. The effects of 6-AN on chon-

drogenesis are selective because central chondrogenic cells, in particular, show impaired production of intercellular matrix and eventually nuclear pycnosis and degeneration. Peripheral compactly arranged chondrogenic cells, by contrast, show neither nuclear pycnosis nor abnormal appearing mitochondria. Some cells, particularly those nearest or in the developing perichondrium, appear to recover from the effects of 6-AN and account for attempts to restore limb chon-

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Differential inhibition and recovery may be consistent with in vitro observations indicating that, under conditions of limiting levels of energy metabof tissue specific olites, production (Marzullo and Lash, 1970) or luxury (Holtzer, 1968) molecules, nonessential to survival, are discontinued yet the cells are capable of undergoing cell division. The selective effects of 6-AN on peripheral and central chondrogenic cells are correlated with proximity to peripheral blood vessels and nature of energy exchanges. Centrally located cells in the chondrogenic region, depending to a greater degree on less efficient anaerobic metabolism, may become energy deficient by virtue of subsisting closer to the critical threshold. The consequence is to cease producing tissue specific macromolecules and succumb to the cytotoxic effects of 6-AN. The appearance of less uniformly dispersed nuclear chromatin (Fig. 8) suggests that these cells are undergoing a degenerative process. Further experiments are necessary to show that 6-AN has interfered with NADP-dependent steps in DNA synthesis (KZihler et al., 1970). Cells near or in the perichondrial region, and thereby closer to the circulatory system, presumably have greater capability of oxidative pathways to produce high energy bonds. These cells presumably well above the deficiency threshold, do not undergo extensive cellular degeneration and can recover from reduced levels of ATP. Normal-appearing cartilaginous tissue was seen 2-3 days after treatment with 6-AN in the area subjacent to the developing perichondrium. The cells appeared capable of dividing and giving rise to matrix-secreting chondrocytes. Thus, at days 6 and 7, i.e., 2 to 3 days after treatment, repair by replacement of the central degenerated tissue by cells near or in the perichondrial layer indicated that the teratogenic effect of 6-AN had subsided. dmgenesis.

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Prkcis of 6-AN Interference Synthesis

with

Matrix

The following processes normally occurring in the central region of the limb are associated with the morphologic changes under investigation: (1) reduction in rate of cell division, (2) increased incorporation of 35S-sulfate, (3) elaboration of the Golgi complex and rough endoplasmic reticulum, (4) embedment of chondrogenic cells in matrix, (5) isolation of these cells from the circulatory system, and (6) dependence upon anaerobic glycolysis. Two intracellular sites appear affected by 6-AN, viz., rough endoplasmic reticulum and Golgi. (a) The amount of rough endoplasmic reticulum in differentiating chondrogenic cells of treated day-5 to day6 embryonic limbs was subnormal and the cisternae by days 6-7 were dilated. These observations suggest that initial synthesis of protein-polysaccharide and tropocollagen and subsequent transport to the Golgi are inhibited. (b) The Golgi apparatus, where protein-polysaccharides are sulfated and products are packaged prior to export shows fewer vacuoles. Thus the primary site for potential deficiency of phosphorylation and consequently reduced matrix may be either or both rough endoplasmic reticulum and/or Golgi. Assuming that 6-AN interrupts phosphorylation in the limb by interfering with NAD-dependent reactions (Dietrich, et al., 1958), it appears reasonable that 6-AN interferes with energy requirements for production of tissue-specific macromolecules. We interpret that after treatment with 6-AN changes seen in chondrogenic rudiments, viz., compact orientation of cells, paucity of matrix and smaller rudiments, are sequelae to those morphological changes seen in the Golgi and rough endoplasmic reticulum. Failure of chondrogenic cells treated with 6-AN to make a normal quantity of

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