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The content of water, protein, DNA, RNA and sugar in fetal rat palatal processes during reorientation and fusion
Arc’i oral Btol Vol 24, pp. 601 to 61 I Peqamon Press Ltd 1979. Prmted m Great Britain
THE CONTENT OF WATER, PROTEIN, DNA, RNA AND SUGAR IN FETAL RAT PALATAL PROCESSES DURING REORIENTATION AND FUSION R. S. BARATZand N. A. ZENATI Department of Anatomy, Boston University School of Medicine; Department of Oral and Maxillofacial Surgery, Boston University Henry M. Goldman School of Graduate Dentistry; Boston, MA 02118, U.S.A. and Dtpartement d’ OrthopCdie Dento-faciale, Institut d’ Odonto-stomatologie, HBpital Mustapha, Alger, Algerie
Summary--Growth of developing palatal processes was studied from the time the processes first appeared to their reorientation and fusion to form the secondary palate. Wet weight, dry weight and macromolecular content of the processes were quantified at half-day intervals. There was a steady decrease in the relative water content of the processes, despite a tenfold increase in total mass. Total protein, DNA and RNA increased steadily and maintained their relative abundance. The accumulation of hexose sugars, however, was erratic, a sharp rise in relative sugar content corresponded to the period of process reorientation before fusion. The results suggest that growth of the palatal processes is continuous and that a period of rapid growth cannot account for process reorientation; they do not support the hypothesis that increased turgor due to hydration of polysaccharides provides the active force for process reorientation.
INTRODUaION The palatal processes of most vertebrates undergo reorientation during development, moving from a position parallel to the mid-sagittal plane and lateral to the tongue to a position perpendicular to the midsagittal plane and dorsal to the tongue. They subsequently fuse with each other and, together with other elements, form the secondary palate. The many theories advanced to explain the reorientation movements of the palatal processes fall into two categories: (1) those which focus primarily upon activity within the processes themselves, e.g. differential growth (Zeiler, Weinstein and Gibson, 1964), build-up of mucopolysaccharides (Larsson, Bostrom and Carlsoo, 1959), increased turgor pressure (Pratt, Larsen and Johnston, 1975), and growth of an extracellular matrix network (Walker and Fraser, 1956; Hughes, Furstman and Berdick, 1967); (2) those which focus on changes in the surrounding craniofacial structures, e.g. withdrawal of the tongue (Wragg, Smith and Boden, 1972), growth of the mandible (Zeiler et al., 1964), reflexes of the fetus, such as swallowing and opening the mouth (Humphrey, 1969), and straightening of the crania1 base (Smiley, Hart and Dixon, 1971; Taylor and Harris, 1973). Although many studies involve aspects of the second category, there have been few on the growth of the palatal processes or the association between this growth and the reorientation of the processes. We wished to characterize the mass, water and macromolecular content of palatal processes at the time of reorientation and fusion. As growth can be defined as increase in cell number (i.e. increased DNA) or as increase in dry weight (represented chiefly by protein), we chose to measure these macromole-
cules at the time of reorientation and fusion. We also examined total RNA, because its increase suggests a future increase in protein synthesis. Because histochemica1 studies demonstrated an increase in carbohydrates in the developing palate at the time of fusion (Pratt et al., 1973) and rapid accumulation of carbohydrates was associated with swelling and movement of embryonic tissue (Greene and Pratt, 1976), we quantified the hexose content, as an index of total sugar accumulation.
MATERIALSAND METHODS Animals
Pregnant Sprague-Dawley albino rats were supplied by Charles River Breeding Laboratories, Wilmington, Mass. To ensure proper age dating of fetuses, all matings occurred within one hour on the same afternoon and embryos were collected at the following ages: 15,15.5, 16,16.5 and 17 days. Embryos were regarded as one-day-old 24 h after conception. At least 3 litters were pooled to constitute each age group. Pregnant rats were anaesthetized with ether, the gravid uteri were aseptically removed and placed immediately in sterile dishes containing Hanks Balanced Salt Solution. Embryos were dissected free from maternal membranes and decapitated. The mandible, tongue and posterior parts of the cranium were trimmed away with a razor blade. Palatal processes were then cut free from the maxilla and placed on plastic-coated paper (Parafilm) on ice. Collected palatal processes were weighed on an analytical balance and put in glass tubes, which were immersed in liquid nitrogen for 30min. Frozen tissue was then lyophi-
R. S. Baratz and N. A. Zenati
608
lised and the dry material reweighed before further processing.
17 -
Materials
16 -
Sodium dodecyl sulphate (SDS), and purified calf thymus deoxyribonucleic acid (DNA) were obtained from Sigma Chemical Co., St. Louis, MO. Bovine serum albumin (BSA) was obtained from Miles Laboratories, Bloomington, IN. Anthrone, thiourea and Folin phenol reagent were obtained from Fisher Scientific, Fairlawn, NJ. Hanks Balanced Salt Solution was supplied by IS1 Biologicals, Cary, IL. All other chemicals were reagent grade.
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Protein assay
Tissue was analysed for total protein content by the method of Lowry et al. (1951). One mg of powdered, dry material was dissolved in l.Oml of 10 per cent SDS and heated in boiling water for 60min. This treatment dissolved the material; no precipitate was found after centrifugation at 3000 x g for 20 min. For each of the selected developmental ages, duplicate samples of various concentrations were made and read in the spectrophotometer. The readings were compared with known concentrations of bovine serum albumin. RNA and DNA assays
Other duplicate tissue samples, 3 mg dry weight, were homogenized 10 times in a Potter-Elvehjem tissue grinder in l.Oml, 0.3 M ice-cold perchloric acid (PCA), removed to a test tube, and centrifuged at 3000 x g for 15 min. The supernatant was discarded, and the precipitate was suspended in a fresh 0.3 M ice-cold PCA and centrifuged again. This procedure was repeated 3 times and the resulting pellet was then analysed as follows: Lipids were extracted with ethanol according to the procedure of Schmidt and Tannhauser (1945). The RNA was then extracted by the method of Edelman et al. (1969) and measured by ultraviolet absorbance using an extinction coefficient of 30.6ml mg-‘cm-’ (Fleck and Begg, 1965; Edelman et a/.., 1969). Remaining material was dissolved by heating in 0.3 M PCA and analysed for DNA by the method of Giles and Myers (1965). Purified calf thymus DNA was used to prepare standard solutions against which the unknowns were compared.
\
‘A. ut
I 15
I 15 5
I 16
I 16.5
I 17
Fetal age
Fig. 1. The ratio of wet weights to dry weights of the palatal processes from day 15 to 17 in utero. Determination of neutral sugars
The quantity of neutral sugars in a sample was measured using a modification of the anthrone reaction of Roe (1955). Instead of measuring sugars directly from tissue, l.Omg of dried tissue was first dissolved in 1.0 ml 72 per cent sulphuric acid at 100°C for 60min. This heating prevents later non-specific colour development. The rest of the Roe procedure was followed and readings from unknowns were compared with a dextrose standard.
RESULTS Wet weight and dry weight determinations
The wet weights and dry weights of the palatal processes are listed in Table 1. The wet weight of each lateral process increased approximately two-fold every 12 h. The dry weight also increased but at a slightly higher rate. The net effect was a decrease in the wet weight:dry weight ratio over the time when
Table 1. Wet and dry weight of palatal processes between day 15 and 17 of gestation Age in utero in days
Number of pooled processes examined
Wet weight in mg per process
Dry weight in mg per process
15 15.5 16 16.5 17
65 48 70 48 44
0.80 1.50 2.75 5.18 10.00
0.05 0.11 0.23 0.49 1.00
The values per process were computed as follows: (1) Pooled samples for each age were weighed twice and in all cases the two measurements differed by less than 1 per cent. (2) The numerical average of these two measurements was divided by the number of processes.
Growth of rat palatal processes
609
35Or
.
L% % E
E
.-. 100
\.
./
I
i I 15 5 Fetal
I 16
I 165
I 17
age
Fig. 3. Values for hexose accumulation in palatal processes from day 15 to 17 in urero. Duplicate 1.0 mg samples of pooled dried material were analysed on separate occasions. For each duplicate sample, one measured value differed from the other by less than 1 per cent. The values represent the numerical average of the two determinations. Carbohydrate Fetal age Fig. 2. Results of protein, DNA, and RNA assays of palatal processes from day 15 to 17 of gestation. Duplicate I .Omg samples of pooled, dried material from each age were analysed on separate occasions for total protein, DNA and RNA as described in the text. For each duplicate sample, one measured value differed from the other by less than 1 per cent. The values represent the numerical average of the two determinations. Values for 15.5 days fell where expected on the curves but were excluded because they came from embryos bred on a date different from the other samples.
the palatal processes change their orientation
and fuse
(Fig. 1). Protein, DNA and RNA content in palatal processes day 15 to 17 in utero
from
Results for each age group (except 15.5 days) are expressed in Fig. 2 and Table 2. DNA, RNA and protein all increased two-fold from day 15 to 17. The rate of increase, however, was similar for each macromolecular species. The contribution of these components to dry weight and the ratios of the components to one-another also remained constant (Table 2).
assay
As an index of sugar, we measured the hexose content of developing palatal processes. Unlike the other components analysed, the hexose content fluctuated over time (Fig. 3). Although the amount of hexoses increased with time, the rate of accumulation was not constant. Accumulated sugar doubled between days 15 and 15.5 and days 16.5 and 17. Between these times, little accumulation occurred. DISCUSSION
Many mechanisms have been proposed to account for the reorientation before fusion of the palatal processes. Two of them, rapid growth of the processes and increased turgor of the processes, have not been previously explored experimentally. It is common to find reports on palatal fusion in the literature where the term “shelf’ is used for the palatal process and “elevation” to denote process reorientation. Both of these terms are misleading because a process does not become shelf-like until reorientation is complete and the events of reorientation involve complex movements. Shelf elevation implies a simple transposition of a planar object to a “higher” position. On the contrary, the developing palatal processes are not planar, and the head of the fetus rests on the pericardial area
Table 2. Ratios (pg/mg dry weight to pg/mg dry weight) of protein: DNA, protein: RNA and DNA: RNA between day 15 and day 17 of gestation Age in days in utero
15 16 16.5 17
Ratio of protein: DNA
Ratio of protein: RNA
Ratio of DNA :RNA
50.6 49.0 51.2 47.9
168.4 164.9 171.6 160.3
0.299 0.297 0.298 0.298
Computed from the values in Fig. 2.
610
R. S. Baratz and N. A. Zenati
so that the processes move anteriorly, not superiorly as the term “elevate” would imply. For these and other reasons that we shall discuss below, caution should be exercised in interpreting the reorientation events in terms of theories based on simple mechanical concepts of movement. Movements of embryonic processes require coordination of cellular events in both the mesenchyme and the covering epithelium. Mitotic activity, production of intra- and extracellular macromolecules, cellular movement, and the localisation and coordination of all of these activities with regard to each other, result in a gross movement of an embryonic process. In the developing palatal processes, Pratt et al. (1973) found that increased amounts of extracellular sugars, chiefly hyaluronic acid, appeared coincident with reorientation of the processes. Because increased hydration is often associated with hyaluronate (Laurent, 1970), Fergusson (1977) proposed that tissue swelling might account for the movement of the processes. If this were true one would expect a rise in the water content of the processes before fusion. We examined the relative degree of hydration over time, and found in contrast that it decreased steadily from the time when the processes first appeared to the time of fusion (Fig. 1). In fact, the water content fell by about 25 per cent each day from day 15 to 17 in utero. These findings suggest that the hydration hypothesis needs re-evaluation. As the processes became less hydrated, both their total dry mass and their relative DNA content increased (Table 2). A rise in both is indicative of growth. One of the many proposed mechanisms for palatal process reorientation is that a spurt of growth provides the necessary force for movement (Walker and Fraser, 1956). Our data, however, give no support to this hypothesis. Although we found that the processes grew from the time they first appeared until fusion, there was no detectable spurt of growth, either preceding or coinciding with reorientation (Fig. 2). Our data do not support the view that there is a growth to the final size and shape and a later reorientation, as proposed by Lazzaro (1940) and Coleman (1965), the so-called barn-door hypothesis. Fergusson (1977) reported that mitotic figures were evenly distributed throughout the mesenchyme of developing processes. However, he did not quantify either the rate or sites of mitotic activity. The role, if any, of focal differences in growth (not measured in our study of total growth) in reorientation, remains an open question. The increase in dry mass of each process seemed to be equally divided among the different macromolecular species examined, except the hexose sugars. Accumulated protein, DNA and RNA all increased but maintained their relative abundance. Developing processes, do not show signs of differentiation of the mesenchyme (i.e. appearance of cartilage, bone and large collagen bundles) before fusion (Fergusson, 1977). Similarly, the epithelium does not differentiate until after fusion has occurred. Thus, a large increase in total protein or RNA (reflecting accumulated extracellular proteins of the connective tissue, intracellular proteins of the covering epithelia, or their imminent synthesis) would not be expected in the developing palate. Our data are consistent with this view as the protein:DNA ratio (amount of protein per cell) and
the DNA:RNA ratio (reflecting the rate of protein synthesis) both remained constant over time (Table 2), suggesting that the hypothesis that a large amount of collagen synthesis and/or collagen polymerization are responsible for reorientation (Hassel and Orkin, 1976) also needs re-evaluation. The only increase we found in a constituent which corresponded with reorientation was the accumulation of hexoses (Fig. 3). Cartilage and bone appear in the palate just after fusion and it is likely that their matrix polysaccharides account for the increase we observed between day 16.5 and 17. Greene and Kocchar (1974) found histochemical evidence of carbohydrate-rich material in the epithelium on the medial edge of the processes only just before fusion. A number of studies (reviewed by Fergusson, 1977) suggest that carbohydrates play a significant role in palatal reorientation. Larsson (1962), for example, postulated that a decrease in mucopolysaccharide synthesis is the mechanism by which cortisoneinduced cleft palate occurs. Other cleft-causing agents such as high doses of salicylates, avitaminosis-A and hypervitaminosis-A, and Roentgen radiation disrupt polysaccharide synthesis. Pratt et al. (1973) found increased amounts of extracellular sugars, chiefly hyaluronate, at the time of reorientation of the processes, and our data are consistent with their findings. The continual decrease we found in the wet weight:dry weight ratio contradicts their idea that hydration of accumulated large chain polysaccharides may be the mechanism by which reorientation occurs. It may, however, be possible that hydration of polysaccharides is the active agent in process reorientation as our study was of the water content of the entire process and did not examine the content of selected regions. The hydration theory focuses on swelling due to carbohydrate hydration; perhaps the role of polysaccharides can be viewed as follows: when the large chain sugars are formed, polymerization shrinkage, rather than swelling, occurs. Knowledge of the orientation, location and concentration of these macromolecules, and the temporal sequence of their appearance, should provide insight into their role in process reorientation. Acknowledgements-This work was supported in part by the Boston University Henry M. Goldman School of Graduate Dentistry and by a graduate fellowship (to Dr. Zenatij from the Ministrv of Hieher Education of The Republic of Algeria. _ ” We thank J. Hinds for helpful suggestions with the manuscript, and Janet Henry for typing.
REFERENCES
Coleman R. D. 1965. Development of the rat palate. Anat. Rec. 151, 107-118. Edelman H., Hirsch C. A., Hiatt H. H. and Fox M. 1969. Apparent changes in mouse liver DNA content due to interference by non-DNA diphenylamine-reacting cytoplasmic material. Biochem. b&phys. Acta 179, 172~1%. Fereusson M. W. J. 1977. The mechanism of nalatal shelf elevation and the pathogenesis of cleft palate. Virchows Arch. A Path. Anat. and Histol. 375, 97-113. Fleck A. and Begg D. 1965. The estimation of ribonucleic acid using ultraviolet absorption measurements. Biothem. biophys. Acta 108, 333-339.
Growth of rat palatal processes Giles K. W. and Myers A. 1965. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206, 93. Greene R. M. and Kocchar D. M. 1974. Surface coat on the epithelium of developing shelves in the mouse as revealed by electron microscopy. J. Embryol. exp. Morph. 31, 683-692. Greene R. M. and Pratt R. M. 1976. Developmental aspects of secondary palate formation. J. Embryo/. exp. Morphol. 36, 225-245. Hassel J. R. and Orkin R. W. 1976. Synthesis and distribution of collagen in the rat palate during shelf elevation. Deul Biol. 49, 80-88. Hughes L. V., Furstman L. and Berdick S. 1967. Prenatal development of the rat palate. J. dent. Res. 46, 373-379. Humphrey L. V. 1969. The relation between human foetal mouth opening reflexes and closure of the palate. Am. J. Anat. 125, 317-344. Laurent T. C. 1970. Structure of hyaluronic acid. In: Chemistry and Molecular Biology of the Intracellular Matrix. Vol. 2, pp. 703733. Academic Press, New York. Larsson K. S. 1962. Studies on the closure of the secondary palate. IV. Autoradiographic and histochemical studies of mouse embryos from cortisone-treated mothers. Acta Morph. neerl.-scan. 4, 369-386.
Larsson K. S., Bostrom H. and Carlsoo S. 1959. Studies on the closure of the secondary palate. Exp. Cell Res. 16, 379-383. Lazzaro C. 1940. Sul meccanismo di chiusura de1 palato secondario. Monk. zool. ital. 51, 249-265. Lowry 0. H., Rosebrough N. J., Farr A. I. and Randall R. J.
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1951. Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Pratt R. M., Goggins R. R., Wilk A. L. and King C. T. G. 1973. Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation aid lusion. Devl Biol. 32; 2%%237. Pratt R. M.. Larsen M. A. J.. Johnston M. C. 1975. Miaration of cranial neural crest cells in a cell-free hyaluronate-rich matrix. Devl Biol. 44, 298-305. Roe J. H. 1955. Determination of total hexoses by anthrone reaction. J. biol. Chem. 212, 335. Schmidt G. and Tannhauser S. J. 1945. A method for determination of deoxyribonucleic acid, ribonucleic aCid and phosphoproteins in tissue. Meth. Enzym. VIII, 225. Smiley G. R., Hart J. C. and Dixon A. D. 1971. Growth of the craniofacial complex during formation of the secondary palate. J. dent. Res. 50, 1506-1507. Taylor R. G. and Harris J. W. S. 1973. Growth and spatial relationships of the cranial base and lower jaw during closure of the secondary palate in the hamster. J. Anat. 115, 149-150. Walker B. E. and Fraser F. C. 1956. Closure of the secondary palate in three strains of mice. J. Embryo/. exp. Morph. 4, 176-189.
Wragg L. E., Smith J. A. and Boden C. S. 1972. Myoneural maturation and function of the fetal rat tongue. Archs oral Biol. 17, 673-682.
Zeiler K. B., Weinstein S. and Gibson R. D. 1964. A study of the morphology and the time of closure of the palate in the albino rat. Archs oral Biol. 9, 545554.
THE CONTENT OF WATER, PROTEIN, DNA, RNA AND SUGAR IN FETAL RAT PALATAL PROCESSES DURING REORIENTATION AND FUSION R. S. BARATZand N. A. ZENATI Department of Anatomy, Boston University School of Medicine; Department of Oral and Maxillofacial Surgery, Boston University Henry M. Goldman School of Graduate Dentistry; Boston, MA 02118, U.S.A. and Dtpartement d’ OrthopCdie Dento-faciale, Institut d’ Odonto-stomatologie, HBpital Mustapha, Alger, Algerie
Summary--Growth of developing palatal processes was studied from the time the processes first appeared to their reorientation and fusion to form the secondary palate. Wet weight, dry weight and macromolecular content of the processes were quantified at half-day intervals. There was a steady decrease in the relative water content of the processes, despite a tenfold increase in total mass. Total protein, DNA and RNA increased steadily and maintained their relative abundance. The accumulation of hexose sugars, however, was erratic, a sharp rise in relative sugar content corresponded to the period of process reorientation before fusion. The results suggest that growth of the palatal processes is continuous and that a period of rapid growth cannot account for process reorientation; they do not support the hypothesis that increased turgor due to hydration of polysaccharides provides the active force for process reorientation.
INTRODUaION The palatal processes of most vertebrates undergo reorientation during development, moving from a position parallel to the mid-sagittal plane and lateral to the tongue to a position perpendicular to the midsagittal plane and dorsal to the tongue. They subsequently fuse with each other and, together with other elements, form the secondary palate. The many theories advanced to explain the reorientation movements of the palatal processes fall into two categories: (1) those which focus primarily upon activity within the processes themselves, e.g. differential growth (Zeiler, Weinstein and Gibson, 1964), build-up of mucopolysaccharides (Larsson, Bostrom and Carlsoo, 1959), increased turgor pressure (Pratt, Larsen and Johnston, 1975), and growth of an extracellular matrix network (Walker and Fraser, 1956; Hughes, Furstman and Berdick, 1967); (2) those which focus on changes in the surrounding craniofacial structures, e.g. withdrawal of the tongue (Wragg, Smith and Boden, 1972), growth of the mandible (Zeiler et al., 1964), reflexes of the fetus, such as swallowing and opening the mouth (Humphrey, 1969), and straightening of the crania1 base (Smiley, Hart and Dixon, 1971; Taylor and Harris, 1973). Although many studies involve aspects of the second category, there have been few on the growth of the palatal processes or the association between this growth and the reorientation of the processes. We wished to characterize the mass, water and macromolecular content of palatal processes at the time of reorientation and fusion. As growth can be defined as increase in cell number (i.e. increased DNA) or as increase in dry weight (represented chiefly by protein), we chose to measure these macromole-
cules at the time of reorientation and fusion. We also examined total RNA, because its increase suggests a future increase in protein synthesis. Because histochemica1 studies demonstrated an increase in carbohydrates in the developing palate at the time of fusion (Pratt et al., 1973) and rapid accumulation of carbohydrates was associated with swelling and movement of embryonic tissue (Greene and Pratt, 1976), we quantified the hexose content, as an index of total sugar accumulation.
MATERIALSAND METHODS Animals
Pregnant Sprague-Dawley albino rats were supplied by Charles River Breeding Laboratories, Wilmington, Mass. To ensure proper age dating of fetuses, all matings occurred within one hour on the same afternoon and embryos were collected at the following ages: 15,15.5, 16,16.5 and 17 days. Embryos were regarded as one-day-old 24 h after conception. At least 3 litters were pooled to constitute each age group. Pregnant rats were anaesthetized with ether, the gravid uteri were aseptically removed and placed immediately in sterile dishes containing Hanks Balanced Salt Solution. Embryos were dissected free from maternal membranes and decapitated. The mandible, tongue and posterior parts of the cranium were trimmed away with a razor blade. Palatal processes were then cut free from the maxilla and placed on plastic-coated paper (Parafilm) on ice. Collected palatal processes were weighed on an analytical balance and put in glass tubes, which were immersed in liquid nitrogen for 30min. Frozen tissue was then lyophi-
R. S. Baratz and N. A. Zenati
608
lised and the dry material reweighed before further processing.
17 -
Materials
16 -
Sodium dodecyl sulphate (SDS), and purified calf thymus deoxyribonucleic acid (DNA) were obtained from Sigma Chemical Co., St. Louis, MO. Bovine serum albumin (BSA) was obtained from Miles Laboratories, Bloomington, IN. Anthrone, thiourea and Folin phenol reagent were obtained from Fisher Scientific, Fairlawn, NJ. Hanks Balanced Salt Solution was supplied by IS1 Biologicals, Cary, IL. All other chemicals were reagent grade.
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Protein assay
Tissue was analysed for total protein content by the method of Lowry et al. (1951). One mg of powdered, dry material was dissolved in l.Oml of 10 per cent SDS and heated in boiling water for 60min. This treatment dissolved the material; no precipitate was found after centrifugation at 3000 x g for 20 min. For each of the selected developmental ages, duplicate samples of various concentrations were made and read in the spectrophotometer. The readings were compared with known concentrations of bovine serum albumin. RNA and DNA assays
Other duplicate tissue samples, 3 mg dry weight, were homogenized 10 times in a Potter-Elvehjem tissue grinder in l.Oml, 0.3 M ice-cold perchloric acid (PCA), removed to a test tube, and centrifuged at 3000 x g for 15 min. The supernatant was discarded, and the precipitate was suspended in a fresh 0.3 M ice-cold PCA and centrifuged again. This procedure was repeated 3 times and the resulting pellet was then analysed as follows: Lipids were extracted with ethanol according to the procedure of Schmidt and Tannhauser (1945). The RNA was then extracted by the method of Edelman et al. (1969) and measured by ultraviolet absorbance using an extinction coefficient of 30.6ml mg-‘cm-’ (Fleck and Begg, 1965; Edelman et a/.., 1969). Remaining material was dissolved by heating in 0.3 M PCA and analysed for DNA by the method of Giles and Myers (1965). Purified calf thymus DNA was used to prepare standard solutions against which the unknowns were compared.
\
‘A. ut
I 15
I 15 5
I 16
I 16.5
I 17
Fetal age
Fig. 1. The ratio of wet weights to dry weights of the palatal processes from day 15 to 17 in utero. Determination of neutral sugars
The quantity of neutral sugars in a sample was measured using a modification of the anthrone reaction of Roe (1955). Instead of measuring sugars directly from tissue, l.Omg of dried tissue was first dissolved in 1.0 ml 72 per cent sulphuric acid at 100°C for 60min. This heating prevents later non-specific colour development. The rest of the Roe procedure was followed and readings from unknowns were compared with a dextrose standard.
RESULTS Wet weight and dry weight determinations
The wet weights and dry weights of the palatal processes are listed in Table 1. The wet weight of each lateral process increased approximately two-fold every 12 h. The dry weight also increased but at a slightly higher rate. The net effect was a decrease in the wet weight:dry weight ratio over the time when
Table 1. Wet and dry weight of palatal processes between day 15 and 17 of gestation Age in utero in days
Number of pooled processes examined
Wet weight in mg per process
Dry weight in mg per process
15 15.5 16 16.5 17
65 48 70 48 44
0.80 1.50 2.75 5.18 10.00
0.05 0.11 0.23 0.49 1.00
The values per process were computed as follows: (1) Pooled samples for each age were weighed twice and in all cases the two measurements differed by less than 1 per cent. (2) The numerical average of these two measurements was divided by the number of processes.
Growth of rat palatal processes
609
35Or
.
L% % E
E
.-. 100
\.
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I
i I 15 5 Fetal
I 16
I 165
I 17
age
Fig. 3. Values for hexose accumulation in palatal processes from day 15 to 17 in urero. Duplicate 1.0 mg samples of pooled dried material were analysed on separate occasions. For each duplicate sample, one measured value differed from the other by less than 1 per cent. The values represent the numerical average of the two determinations. Carbohydrate Fetal age Fig. 2. Results of protein, DNA, and RNA assays of palatal processes from day 15 to 17 of gestation. Duplicate I .Omg samples of pooled, dried material from each age were analysed on separate occasions for total protein, DNA and RNA as described in the text. For each duplicate sample, one measured value differed from the other by less than 1 per cent. The values represent the numerical average of the two determinations. Values for 15.5 days fell where expected on the curves but were excluded because they came from embryos bred on a date different from the other samples.
the palatal processes change their orientation
and fuse
(Fig. 1). Protein, DNA and RNA content in palatal processes day 15 to 17 in utero
from
Results for each age group (except 15.5 days) are expressed in Fig. 2 and Table 2. DNA, RNA and protein all increased two-fold from day 15 to 17. The rate of increase, however, was similar for each macromolecular species. The contribution of these components to dry weight and the ratios of the components to one-another also remained constant (Table 2).
assay
As an index of sugar, we measured the hexose content of developing palatal processes. Unlike the other components analysed, the hexose content fluctuated over time (Fig. 3). Although the amount of hexoses increased with time, the rate of accumulation was not constant. Accumulated sugar doubled between days 15 and 15.5 and days 16.5 and 17. Between these times, little accumulation occurred. DISCUSSION
Many mechanisms have been proposed to account for the reorientation before fusion of the palatal processes. Two of them, rapid growth of the processes and increased turgor of the processes, have not been previously explored experimentally. It is common to find reports on palatal fusion in the literature where the term “shelf’ is used for the palatal process and “elevation” to denote process reorientation. Both of these terms are misleading because a process does not become shelf-like until reorientation is complete and the events of reorientation involve complex movements. Shelf elevation implies a simple transposition of a planar object to a “higher” position. On the contrary, the developing palatal processes are not planar, and the head of the fetus rests on the pericardial area
Table 2. Ratios (pg/mg dry weight to pg/mg dry weight) of protein: DNA, protein: RNA and DNA: RNA between day 15 and day 17 of gestation Age in days in utero
15 16 16.5 17
Ratio of protein: DNA
Ratio of protein: RNA
Ratio of DNA :RNA
50.6 49.0 51.2 47.9
168.4 164.9 171.6 160.3
0.299 0.297 0.298 0.298
Computed from the values in Fig. 2.
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R. S. Baratz and N. A. Zenati
so that the processes move anteriorly, not superiorly as the term “elevate” would imply. For these and other reasons that we shall discuss below, caution should be exercised in interpreting the reorientation events in terms of theories based on simple mechanical concepts of movement. Movements of embryonic processes require coordination of cellular events in both the mesenchyme and the covering epithelium. Mitotic activity, production of intra- and extracellular macromolecules, cellular movement, and the localisation and coordination of all of these activities with regard to each other, result in a gross movement of an embryonic process. In the developing palatal processes, Pratt et al. (1973) found that increased amounts of extracellular sugars, chiefly hyaluronic acid, appeared coincident with reorientation of the processes. Because increased hydration is often associated with hyaluronate (Laurent, 1970), Fergusson (1977) proposed that tissue swelling might account for the movement of the processes. If this were true one would expect a rise in the water content of the processes before fusion. We examined the relative degree of hydration over time, and found in contrast that it decreased steadily from the time when the processes first appeared to the time of fusion (Fig. 1). In fact, the water content fell by about 25 per cent each day from day 15 to 17 in utero. These findings suggest that the hydration hypothesis needs re-evaluation. As the processes became less hydrated, both their total dry mass and their relative DNA content increased (Table 2). A rise in both is indicative of growth. One of the many proposed mechanisms for palatal process reorientation is that a spurt of growth provides the necessary force for movement (Walker and Fraser, 1956). Our data, however, give no support to this hypothesis. Although we found that the processes grew from the time they first appeared until fusion, there was no detectable spurt of growth, either preceding or coinciding with reorientation (Fig. 2). Our data do not support the view that there is a growth to the final size and shape and a later reorientation, as proposed by Lazzaro (1940) and Coleman (1965), the so-called barn-door hypothesis. Fergusson (1977) reported that mitotic figures were evenly distributed throughout the mesenchyme of developing processes. However, he did not quantify either the rate or sites of mitotic activity. The role, if any, of focal differences in growth (not measured in our study of total growth) in reorientation, remains an open question. The increase in dry mass of each process seemed to be equally divided among the different macromolecular species examined, except the hexose sugars. Accumulated protein, DNA and RNA all increased but maintained their relative abundance. Developing processes, do not show signs of differentiation of the mesenchyme (i.e. appearance of cartilage, bone and large collagen bundles) before fusion (Fergusson, 1977). Similarly, the epithelium does not differentiate until after fusion has occurred. Thus, a large increase in total protein or RNA (reflecting accumulated extracellular proteins of the connective tissue, intracellular proteins of the covering epithelia, or their imminent synthesis) would not be expected in the developing palate. Our data are consistent with this view as the protein:DNA ratio (amount of protein per cell) and
the DNA:RNA ratio (reflecting the rate of protein synthesis) both remained constant over time (Table 2), suggesting that the hypothesis that a large amount of collagen synthesis and/or collagen polymerization are responsible for reorientation (Hassel and Orkin, 1976) also needs re-evaluation. The only increase we found in a constituent which corresponded with reorientation was the accumulation of hexoses (Fig. 3). Cartilage and bone appear in the palate just after fusion and it is likely that their matrix polysaccharides account for the increase we observed between day 16.5 and 17. Greene and Kocchar (1974) found histochemical evidence of carbohydrate-rich material in the epithelium on the medial edge of the processes only just before fusion. A number of studies (reviewed by Fergusson, 1977) suggest that carbohydrates play a significant role in palatal reorientation. Larsson (1962), for example, postulated that a decrease in mucopolysaccharide synthesis is the mechanism by which cortisoneinduced cleft palate occurs. Other cleft-causing agents such as high doses of salicylates, avitaminosis-A and hypervitaminosis-A, and Roentgen radiation disrupt polysaccharide synthesis. Pratt et al. (1973) found increased amounts of extracellular sugars, chiefly hyaluronate, at the time of reorientation of the processes, and our data are consistent with their findings. The continual decrease we found in the wet weight:dry weight ratio contradicts their idea that hydration of accumulated large chain polysaccharides may be the mechanism by which reorientation occurs. It may, however, be possible that hydration of polysaccharides is the active agent in process reorientation as our study was of the water content of the entire process and did not examine the content of selected regions. The hydration theory focuses on swelling due to carbohydrate hydration; perhaps the role of polysaccharides can be viewed as follows: when the large chain sugars are formed, polymerization shrinkage, rather than swelling, occurs. Knowledge of the orientation, location and concentration of these macromolecules, and the temporal sequence of their appearance, should provide insight into their role in process reorientation. Acknowledgements-This work was supported in part by the Boston University Henry M. Goldman School of Graduate Dentistry and by a graduate fellowship (to Dr. Zenatij from the Ministrv of Hieher Education of The Republic of Algeria. _ ” We thank J. Hinds for helpful suggestions with the manuscript, and Janet Henry for typing.
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