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The effect of 5-bromodeoxyuridine on the differentiation of chick embryo pigment cells
Experimental
Cell Research 83 (1974) 1599165
THE EFFECT OF 5BROMODEOXYURIDlNE OF CHICK J. ZIMMERMAN,
EMBRYO
ON THE DIFFERENTIATION
PIGMENT
J. BRUMBAUGH,
CELLS
J. BIEHL and H. HOLTZER
Department of Anatomy, University of Pennsylvania, Philadelphia, Pa 19174, and Department of Zoology, University of Nebraska, Lincoln, Nebr., USA
SUMMARY Cultures of (a) dispersed presumptive melanoblasts from chick somites and (b) organ cultures of retinal melanoblasts were grown in control medium and medium containing Sbromodeoxyuridine (BUdR). The somite cell cultures at time zero had no evidence of melanogenic organelles; the eye cultures, from embryos of the same stage, stage 16-18, contained cells showing the initial stages of melanogenesis. In the presence of BUdR, presumptive trunk melanoblasts failed to pigment, while controls became heavily pigmented, whereas cells in the retina1 pigment epithelium made melanin but in reduced amounts when compared with controls. These results suggest different methods of control over synthetic programs depending upon whether synthesis has, or has not, been initiated when the cells are exposed to BUdR.
5-Bromodeoxyuridine (BUdR) has been shown to block terminal differentiation of many kinds of cells without significantly altering replication rates [l-4]. How BUdR exerts its differential effect on the synthesis of terminal luxury molecules in eukaryotes is as yet unknown. A better understanding of how the analog affects differentiation might be forthcoming if at least under some conditions BUdR has an ‘all-or-none’ qualitative effect upon replicating cells, rather than a quantitative effect in depressing the kinds of molecules a given cell synthesized. For example, in the case of chondroblasts or amnion cells it was reported that BUdR reduced the accumulation of mucopolysaccharide to 10% of that of the control cells [5, 61. In these cases, however, it was impossible to determine whether 10% of the cells synthesized their normal complement of glycosaminoglycans, 90% being totally inactive, or whether all II--731809
cells synthesized the glycosaminoglycans but at one-tenth their normal rate-assuming constant ratios of turn-over in treated and control cells. On the other hand, by combining fluorescein-labelled antibodies and electron microscopy, it has been shown that above a threshold concentration, BUdR will suppress the synthesis of myosin, actin, or tropomyosin for myofilaments in 100 y0 of the replicating myogenic cells [4, 7, 81. In brief, to determine whether a species of molecule is or is not being synthesized by all cells in a population, techniques must be employed that permit the inspection of single cells, and the molecules in question must allow unequivocal cytological identification within single cells. Melanogenesis in replicating normal pigment cells, and in melanoma cells, is suppressed by BUdR [9, lo]. Melanin granules are readily recognized both under the phase Exptl Cell Res 83 (1974)
160 J. Zimmrman
ct al.
/?
and electron microscopes. In addition, under the EM. premelanosomes, the complex organelles consisting of a protein matrix and tyrosinase are also readily identified. These two characteristic markers of pigment cells, the formation of the premelanosome, and the subsequent deposition of melanin, can be followed in individual cells, and their response to BUdR determined. The following experiments performed on trunk presumptive melanoblasts of neural crest origin suggest that, in fact, the suppression of melanogenesis can be total, and that the suppressive action of BUdR extends to the assembly of the definitive premelanosome. Observations of the effect of BUdR on ongoing melanogenesis have also been made with retinal pigment cells.
MATERlALS
AND METHODS
Culture of‘ trunk pigment cells Somites from Hamburger-Hamilton stage 16.. 18 White Leghorn chick embryos are rich in migrating presumptive melanoblasts. Accordingly, the somites were dissected free of surrounding tissue by the use of 2.5”,, trypsin for I min, followed by 3 washings in balanced salt solution. Following this they were incubated for 20 min in 0.25”,, trypsin solution in Ca-Mg free medium and then dissociated and plated (IO6 cells/h0 mm dish), and reared as monlayers as described in Abbott, Mayne & Holtzer [I]. Under these culture conditions the presumptive melanoblasts adhered, replicated and underwent normal melanogenesis. As a consequence, after 7 or 8 days in culture anywhere from 50 to 70”,, of the cells were typical, small, replicating, dendritic pigment cells usually in colonies. Half the cultures received BUdR continuously from the time of plating, at a dosage of 10 jcg/ml. Many cultures were fixed after 8 days. To test for reversibilitv of the BUdR effect. after 8 davs in the analog, representative cultures w&e trypsinized and the cells subcultured in normal medium for periods up to 2 weeks.
Fig. 2. Trunk melanocyte from 8 day control culture possessing both immature completed melanosomes (m). / 24 000. Fig. 3. Trunk melanocyte from 8 day BUdR culture containing membranous structures (II), filaments (f), and microtubules (t). x 24 000. Exptl Cell Res 83 (1974)
premelanosomes whorl
(pm) and
(w), lysosomal-like
162 J. Zimmzrmw
et al.
Organ culture of retinal pigment cells Eye anlagen, freed as completely as possible from surrounding mesenchyme, were removed from stage 16-18 White Leghorn embryos. Some were fixed immediately; others were organ cultured on Millipore paper rafts [l]. Eagle’s minimum essential medium with 10% embryo extract, 10:; horse serum, and I :,, L-glutamine was added to the level at which the Millipore was moistened, so that the organ culture would not float off the paper. The cultures were fixed after 4 days. All reagents, except embryo extract, were obtained from GIBCo, Grand Island, New York. Embryo extract was made from 1l-day chick embryos.
Electron microscopy The trunk pigment cell cultures were fixed in situ with 3 (I” glutaraldehyde, osmicated, stained in uranyl acetate and dehydrated in a graded series of alcohols. They were then infiltrated with an Eoon 812 mixture. using a graded series of hydroxypropyl methacrylate (HPMA)-water and HPMA-Eaon mixtures. After &ring the Epon sheet was sepaiated from the plastic culture dish and suitable area cut out and mounted on Epon pegs. Sections were parallel to the plane of the culture dish. Eye cultures were sliced in half before fixation and processed for electron microscopy as described by Brumbaugh [I 11.All sections were s&ined with lead citrate and examined with an RCA EMU3B (modified) electron microscope.
RESULTS Trunk pigment cells During the first 2 or 3 days of culture large numbers of somite cells degenerated. During this period presumptive melanoblasts could not be distinguished from surviving somite cells. Dividing groups of melanoblasts were not identified under the phase microscope until day 4 or 5. The presence of significant numbers of somite cells (i.e. presumptive myoblasts, chondroblasts and fibroblasts) and the fact that they could not be distinguished from presumptive melanoblasts, precluded using the electron microscope for monitoring the transition of presumptive melanoblasts into frank melanoblasts. The number of dendritic melanoblasts increased with time, so that after 8 days in culture often several hundred
relatively isolated colonies containing both melanoblasts and melanocytes were present in a single 60 mm Petri dish (fig. I a). At this time the numbers of somite cells proper has diminished to approx. 3Ol& of the total number of cells in the dish. The number of cells per colony varied from less than 10 to over 80. The compact arrangement of many of these colonies suggested they were clones derived from a single cell. Sections of cells in such colonies demonstrated the type of melanogenesis characteristic of this White Leghorn genotype (fig. 2). All stages of melanogenic organelles were present, from immature premelanosomes to totally electron-dense melanosomes. In contrast, cultures grown continuously for 8 days in BUdR with virtually all cells incorporating the analog [13], failed to exhibit detectable melanocytes when inspected with the phase microscope (fig. 1b). These otherwise healthy replicating cells tended to flatten relative to control cells. Five colonies each, from two separate BUdR cultures, were then sectioned and observed with the electron microscopy (fig. 3). No evidence of melanin deposition or premelanosomes were found in any of the cells (a 95 Sb confidence level). Instead, membranous whorls were observed in a large number of the BUdR-treated cells. Vacuolar structures, possibly of a lysosomal nature, with matricies of varying densities, were also seen. Lastly, there was an apparent increase in the number of filaments and microtubules, and a decrease in the overall granularity of the treated cells. When cells grown in BUdR for 8 days were trypsinized and subcultured in normal medium for several generations typical pigmented melanocytes emerged. Clearly, as shown with other systems, the effect of BUdR
Fig. 4. Three-day-old, uncultured retinal melanocyte containing an early premelanosome (pm) near the Golgi system indicating that melanogenesis has begun. x 32 000. Fig. 5. Retinal melanocytes from 3 day chick eyes after 4 days in control medium possessing numerous melanogenic organelles all showing deposited melanin. x 24 000. Exptl
Cell Res 83 (1974)
BUdR and melanin synthesis 163
Exptl Cell Res 83 (1974)
164 J. Zimmerman et al.
Fix. 6. Retinal melanocyte from 3 day chick eve after 4 days in BUdR medium possessing only a few, small
melanogenic organelles. -. 24 000.
is reversible providing the suppressed cells are allowed to replicate and yield progeny with normal DNA. Retinal pigment cells Melanogenesis in trunk pigment cells normally occurs around day 8 in vivo. In contrast, melanogenesis in the pigmented cells of the retina was conspicuous under the dissecting microscope in normal 4-day embryos. The pigmented retina of untreated 3-day chick embryos was characterized by a modest number of typical premelanosomes in the Golgi region of the cell (fig. 4). Mature melanosomes were not observed at this stage, indicating this was the approximate time that melanogenesis was initiated in retinal melanoblasts. Whole 3 day eyes, after 2 days in conExptl Cell Res 83 (1974)
trol medium, were pigmented when inspected under the dissecting microscope. Electron micrographs of organ-cultured retinal melanocytes, after a total of 4 days in normal medium, demonstrated large numbers of melanogenic organelles, all showing melanin deposition (fig. 5). The cells of the BUdR-treated eyes, with almost IOO~b of the cells incorporating the analog, after 4 days in culture, were lightly pigmented. Electron micrographs of the BUdR-treated retinal pigment cells revealed some melanogenic organelles, probably more than were present in 3-day eye in vivo (fig. 6). When compared with their cultured controls, however, the number and size of comparable melanogenic organelles was markedly reduced.
BUdR and melanin slwthrsis DlSCUSSlON BUdR totally prevents the initiation of melanin synthesis in trunk presumptive melanoblasts. Not only is melanin deposition blocked, but so is the assembly of the definitive premelanosome. Whether the membranous whorls observed in many of the BUdR-suppressed cells were an abortive attempt at formation of premelanosomes, or whether such structures have nothing to do with melanogenesis, could not be determined from these experiments. The effects of BUdR on melanogenesis in trunk pigment cells is similar to its effect on myogenesis. In both instances, the analog prevents the suppressed cells from entering the terminal stage of their respective lineages, co-ordinately suppressing the expression of several luxury molecules. It is also to be stressed that BUdR-suppressed myogenic cells and BUdR-suppressed pigment cells will, if allowed to replicate and form progeny with normal DNA, yield typical muscle and pigment cells respectively. Together, these findings suggest the possibility that BUdR acts on a higher order genomic control initiating the terminal synthetic program for muscle or pigment cells, rather than on the many separate structural genes involved [2], although other possibilities have not been ruled out [14, 151. The action of BUdR on retinal pigment cells that have already initiated the formation of a few premelanosomes, prior to being exposed to the analog, is more equivocal. The reduced number of melanogenic organelles per cell could be due to the parcelling out of pre-existing organelles, synthesized by already activated genetic programs, to daughter cells (i.e. dilution). Alternatively, our results would be consistent with a continuous though reduced rate of synthesis of melanogenic
165
organelies. Experiments measuring melanin synthesis with labelled thiouracil favor the notion that melanin synthesis is not totally blocked by BUdR in those cells that are in the later compartments of the retinal pigment cell lineage [12]. However, until the proportion of retinal pigment cells that actually incorporate the analog, and the threshold value of BUdR substitution necessary to suppress all or some melanogenesis is determined this issue cannot be resolved. This work was supported by grants from the USPHS. (HD-00030), USPHS (HD-00189), and the American Cancer Society (VC-45) to H. H. and by grants from the National Institute of General Medical Sciences (42355) and NIGMS (18969) to J. B. Wrathall et al. (Wrathall, J R, Oliver, C, Silagi S & Essner, E, J cell biol 57 (1973) 406) have recently reported ultrastructural data on the suppression of melanogenesis by BUdR in melanoma cells which agrees with the data reported here for somite cultures. Note added in proof:
REFERENCES 1. Abbott, J, Mayne, R & Holtzer, H, Dev biol 28
(1972) 430. 2. Holtzer, H, Weintraub, H, May, R & Mochan, B, Current topics in developmental biology (ed A Moscona & A Monroy). Academic Press, New York (1972). 3. Wilt, F & Anderson, M, Dev biol 28 (1972) 443. 4. Okazaki, R & Holtzer, H, J histochem cytothem 13 (1965) 726. 5. Schulte-Holthausen, H, Chacko, S, Davidson, E A & Holtzer, H. Proc natl acad sci US 63 (1969) 864. 6. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 7. Bischoff, R & Holtzer, H, J cell biol44 (1970) 134. 8. Holtzer, H, Sanger, J W, Ishikawa, H & Strahs, K, Symp quant biol 37 (1973) 549. 9. Coleman, A W, Coleman, J R, Kunkel, D & Werner, I, Exptl cell res 59 (1970) 319. 10. Silagi, S & Bruce, S, Proc natl acad sci 1JS 66 (1970) 72. 11. Brumbaugh, J, Dev biol 18 (1968) 375. 12. Zimmerman, J & Holtzer, H. Unpublished results. 13. Holtzer, H & Matheson, D W, Chemistry and molecular biology of the intercellular matrix (ed E A Balazs). Academic Press, New York (1970). 14. Lin, S-Y & Riggs, R D, Proc natl acad sci US 69 (1972) 2574. Received May 22, 1973 Revised version received September 5, 1973
Exptl Cell Res 83 (1974)
Cell Research 83 (1974) 1599165
THE EFFECT OF 5BROMODEOXYURIDlNE OF CHICK J. ZIMMERMAN,
EMBRYO
ON THE DIFFERENTIATION
PIGMENT
J. BRUMBAUGH,
CELLS
J. BIEHL and H. HOLTZER
Department of Anatomy, University of Pennsylvania, Philadelphia, Pa 19174, and Department of Zoology, University of Nebraska, Lincoln, Nebr., USA
SUMMARY Cultures of (a) dispersed presumptive melanoblasts from chick somites and (b) organ cultures of retinal melanoblasts were grown in control medium and medium containing Sbromodeoxyuridine (BUdR). The somite cell cultures at time zero had no evidence of melanogenic organelles; the eye cultures, from embryos of the same stage, stage 16-18, contained cells showing the initial stages of melanogenesis. In the presence of BUdR, presumptive trunk melanoblasts failed to pigment, while controls became heavily pigmented, whereas cells in the retina1 pigment epithelium made melanin but in reduced amounts when compared with controls. These results suggest different methods of control over synthetic programs depending upon whether synthesis has, or has not, been initiated when the cells are exposed to BUdR.
5-Bromodeoxyuridine (BUdR) has been shown to block terminal differentiation of many kinds of cells without significantly altering replication rates [l-4]. How BUdR exerts its differential effect on the synthesis of terminal luxury molecules in eukaryotes is as yet unknown. A better understanding of how the analog affects differentiation might be forthcoming if at least under some conditions BUdR has an ‘all-or-none’ qualitative effect upon replicating cells, rather than a quantitative effect in depressing the kinds of molecules a given cell synthesized. For example, in the case of chondroblasts or amnion cells it was reported that BUdR reduced the accumulation of mucopolysaccharide to 10% of that of the control cells [5, 61. In these cases, however, it was impossible to determine whether 10% of the cells synthesized their normal complement of glycosaminoglycans, 90% being totally inactive, or whether all II--731809
cells synthesized the glycosaminoglycans but at one-tenth their normal rate-assuming constant ratios of turn-over in treated and control cells. On the other hand, by combining fluorescein-labelled antibodies and electron microscopy, it has been shown that above a threshold concentration, BUdR will suppress the synthesis of myosin, actin, or tropomyosin for myofilaments in 100 y0 of the replicating myogenic cells [4, 7, 81. In brief, to determine whether a species of molecule is or is not being synthesized by all cells in a population, techniques must be employed that permit the inspection of single cells, and the molecules in question must allow unequivocal cytological identification within single cells. Melanogenesis in replicating normal pigment cells, and in melanoma cells, is suppressed by BUdR [9, lo]. Melanin granules are readily recognized both under the phase Exptl Cell Res 83 (1974)
160 J. Zimmrman
ct al.
/?
and electron microscopes. In addition, under the EM. premelanosomes, the complex organelles consisting of a protein matrix and tyrosinase are also readily identified. These two characteristic markers of pigment cells, the formation of the premelanosome, and the subsequent deposition of melanin, can be followed in individual cells, and their response to BUdR determined. The following experiments performed on trunk presumptive melanoblasts of neural crest origin suggest that, in fact, the suppression of melanogenesis can be total, and that the suppressive action of BUdR extends to the assembly of the definitive premelanosome. Observations of the effect of BUdR on ongoing melanogenesis have also been made with retinal pigment cells.
MATERlALS
AND METHODS
Culture of‘ trunk pigment cells Somites from Hamburger-Hamilton stage 16.. 18 White Leghorn chick embryos are rich in migrating presumptive melanoblasts. Accordingly, the somites were dissected free of surrounding tissue by the use of 2.5”,, trypsin for I min, followed by 3 washings in balanced salt solution. Following this they were incubated for 20 min in 0.25”,, trypsin solution in Ca-Mg free medium and then dissociated and plated (IO6 cells/h0 mm dish), and reared as monlayers as described in Abbott, Mayne & Holtzer [I]. Under these culture conditions the presumptive melanoblasts adhered, replicated and underwent normal melanogenesis. As a consequence, after 7 or 8 days in culture anywhere from 50 to 70”,, of the cells were typical, small, replicating, dendritic pigment cells usually in colonies. Half the cultures received BUdR continuously from the time of plating, at a dosage of 10 jcg/ml. Many cultures were fixed after 8 days. To test for reversibilitv of the BUdR effect. after 8 davs in the analog, representative cultures w&e trypsinized and the cells subcultured in normal medium for periods up to 2 weeks.
Fig. 2. Trunk melanocyte from 8 day control culture possessing both immature completed melanosomes (m). / 24 000. Fig. 3. Trunk melanocyte from 8 day BUdR culture containing membranous structures (II), filaments (f), and microtubules (t). x 24 000. Exptl Cell Res 83 (1974)
premelanosomes whorl
(pm) and
(w), lysosomal-like
162 J. Zimmzrmw
et al.
Organ culture of retinal pigment cells Eye anlagen, freed as completely as possible from surrounding mesenchyme, were removed from stage 16-18 White Leghorn embryos. Some were fixed immediately; others were organ cultured on Millipore paper rafts [l]. Eagle’s minimum essential medium with 10% embryo extract, 10:; horse serum, and I :,, L-glutamine was added to the level at which the Millipore was moistened, so that the organ culture would not float off the paper. The cultures were fixed after 4 days. All reagents, except embryo extract, were obtained from GIBCo, Grand Island, New York. Embryo extract was made from 1l-day chick embryos.
Electron microscopy The trunk pigment cell cultures were fixed in situ with 3 (I” glutaraldehyde, osmicated, stained in uranyl acetate and dehydrated in a graded series of alcohols. They were then infiltrated with an Eoon 812 mixture. using a graded series of hydroxypropyl methacrylate (HPMA)-water and HPMA-Eaon mixtures. After &ring the Epon sheet was sepaiated from the plastic culture dish and suitable area cut out and mounted on Epon pegs. Sections were parallel to the plane of the culture dish. Eye cultures were sliced in half before fixation and processed for electron microscopy as described by Brumbaugh [I 11.All sections were s&ined with lead citrate and examined with an RCA EMU3B (modified) electron microscope.
RESULTS Trunk pigment cells During the first 2 or 3 days of culture large numbers of somite cells degenerated. During this period presumptive melanoblasts could not be distinguished from surviving somite cells. Dividing groups of melanoblasts were not identified under the phase microscope until day 4 or 5. The presence of significant numbers of somite cells (i.e. presumptive myoblasts, chondroblasts and fibroblasts) and the fact that they could not be distinguished from presumptive melanoblasts, precluded using the electron microscope for monitoring the transition of presumptive melanoblasts into frank melanoblasts. The number of dendritic melanoblasts increased with time, so that after 8 days in culture often several hundred
relatively isolated colonies containing both melanoblasts and melanocytes were present in a single 60 mm Petri dish (fig. I a). At this time the numbers of somite cells proper has diminished to approx. 3Ol& of the total number of cells in the dish. The number of cells per colony varied from less than 10 to over 80. The compact arrangement of many of these colonies suggested they were clones derived from a single cell. Sections of cells in such colonies demonstrated the type of melanogenesis characteristic of this White Leghorn genotype (fig. 2). All stages of melanogenic organelles were present, from immature premelanosomes to totally electron-dense melanosomes. In contrast, cultures grown continuously for 8 days in BUdR with virtually all cells incorporating the analog [13], failed to exhibit detectable melanocytes when inspected with the phase microscope (fig. 1b). These otherwise healthy replicating cells tended to flatten relative to control cells. Five colonies each, from two separate BUdR cultures, were then sectioned and observed with the electron microscopy (fig. 3). No evidence of melanin deposition or premelanosomes were found in any of the cells (a 95 Sb confidence level). Instead, membranous whorls were observed in a large number of the BUdR-treated cells. Vacuolar structures, possibly of a lysosomal nature, with matricies of varying densities, were also seen. Lastly, there was an apparent increase in the number of filaments and microtubules, and a decrease in the overall granularity of the treated cells. When cells grown in BUdR for 8 days were trypsinized and subcultured in normal medium for several generations typical pigmented melanocytes emerged. Clearly, as shown with other systems, the effect of BUdR
Fig. 4. Three-day-old, uncultured retinal melanocyte containing an early premelanosome (pm) near the Golgi system indicating that melanogenesis has begun. x 32 000. Fig. 5. Retinal melanocytes from 3 day chick eyes after 4 days in control medium possessing numerous melanogenic organelles all showing deposited melanin. x 24 000. Exptl
Cell Res 83 (1974)
BUdR and melanin synthesis 163
Exptl Cell Res 83 (1974)
164 J. Zimmerman et al.
Fix. 6. Retinal melanocyte from 3 day chick eve after 4 days in BUdR medium possessing only a few, small
melanogenic organelles. -. 24 000.
is reversible providing the suppressed cells are allowed to replicate and yield progeny with normal DNA. Retinal pigment cells Melanogenesis in trunk pigment cells normally occurs around day 8 in vivo. In contrast, melanogenesis in the pigmented cells of the retina was conspicuous under the dissecting microscope in normal 4-day embryos. The pigmented retina of untreated 3-day chick embryos was characterized by a modest number of typical premelanosomes in the Golgi region of the cell (fig. 4). Mature melanosomes were not observed at this stage, indicating this was the approximate time that melanogenesis was initiated in retinal melanoblasts. Whole 3 day eyes, after 2 days in conExptl Cell Res 83 (1974)
trol medium, were pigmented when inspected under the dissecting microscope. Electron micrographs of organ-cultured retinal melanocytes, after a total of 4 days in normal medium, demonstrated large numbers of melanogenic organelles, all showing melanin deposition (fig. 5). The cells of the BUdR-treated eyes, with almost IOO~b of the cells incorporating the analog, after 4 days in culture, were lightly pigmented. Electron micrographs of the BUdR-treated retinal pigment cells revealed some melanogenic organelles, probably more than were present in 3-day eye in vivo (fig. 6). When compared with their cultured controls, however, the number and size of comparable melanogenic organelles was markedly reduced.
BUdR and melanin slwthrsis DlSCUSSlON BUdR totally prevents the initiation of melanin synthesis in trunk presumptive melanoblasts. Not only is melanin deposition blocked, but so is the assembly of the definitive premelanosome. Whether the membranous whorls observed in many of the BUdR-suppressed cells were an abortive attempt at formation of premelanosomes, or whether such structures have nothing to do with melanogenesis, could not be determined from these experiments. The effects of BUdR on melanogenesis in trunk pigment cells is similar to its effect on myogenesis. In both instances, the analog prevents the suppressed cells from entering the terminal stage of their respective lineages, co-ordinately suppressing the expression of several luxury molecules. It is also to be stressed that BUdR-suppressed myogenic cells and BUdR-suppressed pigment cells will, if allowed to replicate and form progeny with normal DNA, yield typical muscle and pigment cells respectively. Together, these findings suggest the possibility that BUdR acts on a higher order genomic control initiating the terminal synthetic program for muscle or pigment cells, rather than on the many separate structural genes involved [2], although other possibilities have not been ruled out [14, 151. The action of BUdR on retinal pigment cells that have already initiated the formation of a few premelanosomes, prior to being exposed to the analog, is more equivocal. The reduced number of melanogenic organelles per cell could be due to the parcelling out of pre-existing organelles, synthesized by already activated genetic programs, to daughter cells (i.e. dilution). Alternatively, our results would be consistent with a continuous though reduced rate of synthesis of melanogenic
165
organelies. Experiments measuring melanin synthesis with labelled thiouracil favor the notion that melanin synthesis is not totally blocked by BUdR in those cells that are in the later compartments of the retinal pigment cell lineage [12]. However, until the proportion of retinal pigment cells that actually incorporate the analog, and the threshold value of BUdR substitution necessary to suppress all or some melanogenesis is determined this issue cannot be resolved. This work was supported by grants from the USPHS. (HD-00030), USPHS (HD-00189), and the American Cancer Society (VC-45) to H. H. and by grants from the National Institute of General Medical Sciences (42355) and NIGMS (18969) to J. B. Wrathall et al. (Wrathall, J R, Oliver, C, Silagi S & Essner, E, J cell biol 57 (1973) 406) have recently reported ultrastructural data on the suppression of melanogenesis by BUdR in melanoma cells which agrees with the data reported here for somite cultures. Note added in proof:
REFERENCES 1. Abbott, J, Mayne, R & Holtzer, H, Dev biol 28
(1972) 430. 2. Holtzer, H, Weintraub, H, May, R & Mochan, B, Current topics in developmental biology (ed A Moscona & A Monroy). Academic Press, New York (1972). 3. Wilt, F & Anderson, M, Dev biol 28 (1972) 443. 4. Okazaki, R & Holtzer, H, J histochem cytothem 13 (1965) 726. 5. Schulte-Holthausen, H, Chacko, S, Davidson, E A & Holtzer, H. Proc natl acad sci US 63 (1969) 864. 6. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 7. Bischoff, R & Holtzer, H, J cell biol44 (1970) 134. 8. Holtzer, H, Sanger, J W, Ishikawa, H & Strahs, K, Symp quant biol 37 (1973) 549. 9. Coleman, A W, Coleman, J R, Kunkel, D & Werner, I, Exptl cell res 59 (1970) 319. 10. Silagi, S & Bruce, S, Proc natl acad sci 1JS 66 (1970) 72. 11. Brumbaugh, J, Dev biol 18 (1968) 375. 12. Zimmerman, J & Holtzer, H. Unpublished results. 13. Holtzer, H & Matheson, D W, Chemistry and molecular biology of the intercellular matrix (ed E A Balazs). Academic Press, New York (1970). 14. Lin, S-Y & Riggs, R D, Proc natl acad sci US 69 (1972) 2574. Received May 22, 1973 Revised version received September 5, 1973
Exptl Cell Res 83 (1974)