Thyroxine a n d the Development of the Tibia in the Embryonic Chick B . K. HALL Department o f Biology, L i f e Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada ABSTRACT Embryonic chicks were treated with exogenous L-thyroxine or with thiourea at eight days of incubation and the subsequent development of the tibia studied. The weight of the tibia was 62% lower than that from normal embryos, and the length of the tibia 24% below normal in the embryos treated with thiourea. This reduced rate of growth was shown to be due to a reduction in the rate of maturation of chondroblasts into chondrocytes, reduced chondrocyte hypertrophy and defective deposition of acid mucopolysaccharide into the cartilage matrix. Osteogenesis pet se was unaffected. It was concluded that thyroxine plays a role in the control of chondrocyte maturation and in cartilage matrix production during normal development. The epiphyses of the tibiae from the embryos treated with thiourea were extensively eroded, invaded by marrow and more fragile than those from untreated embryos, indicating that thyroxine is essential for the maintenance of the integrity of the articular cartilage. An abnormal core of bone developed within the proximal epiphysis of these embryos. Exogenous thyroxine at concentrations as low as 100 pg/embryo also reduced the growth of the tibia below that seen in untreated embryos. Evidently the cells of the skeleton are sensitive to both lowered and increased levels of circulating thyroxine. The role played by the hormone thyroxine in the differentiation and growth of embryonic long bones is poorly understood. The present communication deals with the effect of thyroxine on the development of the embryonic chick tibia from the eighth to the eighteenth day of incubation. Embryos were either made hyperthyroid by the application of exogenous thyroxine or were made hypothyroid by the in vivo injection of thiourea, an anti-thyroid agent. Thiourea was first used as an antithyrogenic agent in the embryonic chick by Grassowicz ('46). In 1949 Adams and Bull published an account of the inhibition of general body growth and of the length of the tibia (tibiotarsus) which followed single or multiple injections of 2 mg thiourea or of 0.5 to lmg thiouracil commencing on the eighth day of incubation. They found that multiple injections (at 8, 14 and 18 days) were no more effective than were single injections; that overall ANAT. REC., 176: 49-64. body growth was inhibited by up to 18%; and that the length of the tibia was shorter than normal. No other aspeots of skeletal growth were studied by them. Subsequently, Adams and Buss ('52) provided further information on this reduction in growth in hypothyroid chick embryos and also studied changes in thyroid weight, histology and mitotic activity. Romanoff and Laufer ('56) treated 11 day embryos with from 2 to 10 mg of thiourea and studied the subsequent development of the liver, thyroid and adrenal glands in some detail. No further detailed information on the response of long bones of chick embryos in the in vivo application of antithyroid agents such as thiourea is available. The respnse of whole long bones of the embryonic chick to exogenous uhymxine in vitro has been studied by Fell and Mellanby ('55) who found an initial enhancement of 'cartilage maturation folReceived Oct. 5, '72. Accepted Jan.22, '73. 49 50 B. K. HALL lowed by retardation of bone growth, and by Lawson ('61) who confirmed growth retardation in both the tibia and the radius. Melcher ('71 ) has shown that hypertrophic chondrocytes of Meckel's cartilage maintained in vitro may be induced to commence synthesis of DNA in response to thyroxine, Evidently thyroxine enhances both proliferation and maturation of chondrocytes. The response of chondrocytes from embryonic chicks isolated in vitro and exposed to thyroxine #has been studied by Pawelek ('69) who found an enhancement of the differentiation of non cartilage-making clones in chondrocytes and a concomitant stimulation of the synthesis of chondroitin sulphate by the differentiating cells (see also Dorfman and Schiller, '58). Thus exogenous thyroxine exerts a direct effect on the differentiation and maturation of cartilage in vitro. Thyroxine also enhances the formation of ectopic cartilage and bone (Somogyi and Kovass, '69). The aim of the present study was to render chick embryos hypothyroid by treatment with thiourea, or hyperthyroid by treatment with thyroxine, and to examine the differentiation, growth, histology and gross histochemistry of the tibia in an endeavour to provide information additional to that currently available on the in vivo effects of thyroxine on skeletogenesis and to correlate these data with that obtained in the above in vitro studies. MATERIALS AND METHODS Eggs were incubated in a forced-draft Leahy incubator at 37 k: 0.5"C and 54 & 2% relative humidity. At eight days of incubation 153 eggs were injected with thiourea (Fisher Scientific) and 152 eggs with L-thyroxine (sodium salt, British Drug Houses, lot no. 0.476590). Doses used/embryo were: thiourea 1 pg to 5 mg (see table l ) , and rhyroxine 100 pg to 10 mg (see table 3 ) . The chemicals were dissolved in sterile saline (0.9% NaCl supplemented with 1% ETOH for thyroxine) and injected in a final volume of 0.5 em3 through a pinhole in the shell and shell membrane and onto the chorio-allantoic membrane. All injectiom were carried out with a 2.5 em3 presterilized Tuberculin syringe. The thio- urea-treated embryos were examined at two-day intervals from 10 to 18 days, the thyroxine-treated embryos at 14 and 18 days of incubation. Three hundred and forty eight contml uninjected embryos were also examined. All embryos were examined for survival. The surviving elmbryos were removed from their shells, cleared of adherent yolk and extraembryonic membranes, blotted on filter paper and weighed to the nearest 0.01 g. The tibiae were dissected out, cleaned of adherent connective tissue and muscle, weighed to the nearest 0.01 mg and their lengths measured to the nearest 0.01 mm. Tibiae from embryos at each age examined were treated by the Alizarin Red S method of Evans ('48) to visualize cenlters of ossification, to assess gross morphology and to determine the proportion of bone to cartilage in treated versus untreated embryos (by measurement of the linear dimensions of the osseous shaft and of the unossified cartilage model and epiphyses). At least three tibiae per dose per age were fixed in 80% ethanol, embedded in 52°C m.p. paraffin, serially sectioned and used for histological and histochemical analysis. The Alcian Blue 8GX-Chlorantine Fast Red 5B (ABCR) method of Lison ('54) was used as the routine histological stain. Alcian blue stains the non-sulphated acid mucopolysaccharides of the cartilage matrix bright blue whilst chlorantine red stains bone matrix bright red. Light green in Mason's trichrome procedure (Pantin, '60) was used to visualize the distribution of collagen and to supplement the ABCR method for general hisitology. Further information on the distribution of acid mucopolysaccharides, mucoproteins, glycoproteins and glycogen was obtained from ethanol-stable metachromasia after 0.01% toluidine blue (Ham and Harris, '50) and from the periodic acidSahifF (PAS) procedure (Barka and Anderson, '65). Sites of calcification were visualized with alizarin red S and sites of alkaline phosphatase by the Gomori method (Barka and Anderson, '65). As the final steps in the Gomori methods also visualize calcium, sections were decalcified with 5% EDTA and the dkaline phosphatase reactivated 51 THYROXINE AND THE CHICK TIBIA with 1% sodium veronal (Schajowicz and Cabrini, '56) before the Gomori method was applied. RESULTS Thiourea-treated embryos Body weight At ten days of incubation, i.e. two days post-injection with thiourea, the embryos treated with 1 or 5 mg were significantly smaller ( 1 6 % , P > 0.02) than untreated embryos. Lower doses of thiourea retarded body weight by an average of 8% but were not statistically different from controls. The initial effect of thiourea on embryonic growth was thus restricted to doses above 1 mg/embryo (table 1, fig. 15). By 12 days of incubation all doses (0.001 to 5 mg) significantly retarded body weight below control values by an average of 21% (table 1). Analysis of variance indicated no significant difference between doses, ind&ating that all doses were equally effective in slowing embryonic growth. By 14 days of incubation all doses of thiourea except the lowest used (0.001 mg) retarded body weight below controls by an average of 20% (P > 0.02 or lower, table 1). At 16 and 18 days of incubation the effect of thiourea on body weight could be divided into two dose groups: 1 to 5 mg and 0.1 to 0.001 mg. All doses produced significantly lower body weights than normal but the higher doses produced significantly greater reductions below normal (35% ) than did the lower doses ( 1 8 % , fig. 15). From the fourteenth day of incubation onwards a dose of thiourea as low as 0.01 mg was sufficient to retard body weight by some 1 8 % . The subnormal body weight of 35% of control values seen after doses of thiourea above 1 mg may have been due to pharmacological effelcts of thiourea rather than to its effect on thyroxine levels (see DISCUSSION). Growth of the tibia At ten days of incubation and over the range of doses used,, the tibiae were significantly lighter (by 44% P > 0.001) and TABLE I Survival rate, number of survivors per age and body weight of survivors for embryos treated with thiourea at eight days of incubation Dose Embryonic age (days) 10 mg 5 2.5 1.0 0.5 0.1 0.05 0.01 0.001 Control 5 2.5 1.0 0.5 0.1 0.05 0.01 0.001 Control 1 69 (5) 78 (5) 79 ( 5 ) 84 (4) 87 (6) 100 (20) 12 Survival ( % 48 (9) 40 (4) 71 (9) 75 (9) 77 (10) 77 (7) 75 (4) 96 (24) P > 0.01. UP > 0.02. 7 P > 0.05. 16 18 ) 28 (7) 46 (4) 40 (4) 65 (5) 60 (3) 59 (3) 67 (5) 96 (24) Body weight ( 9 ) 2 3.23f0.10 3 6.08f0.153 3.23f 0.183 1.72?~0.13~ 3.45C00.063 6.50f0.495 3.22k 0.07 6.81 C 0.43 1.8220.11 3.36f0.053 6.80f0.256 6.732 0.39 1.81k0.10 3.38k0.13 3 6.56f0.35 2.01k0.08 3.2920.20 7.23-CO.12 2.04&0.04 4.19f0.05 8.22f0.12 1.69i0.06 No. of survivors examined in parentheses. Mean + Standard error. P > 0.EOOl. 4P > 0.001. 1 2 3 14 30 (4j 92 (12) 9.4720.57 12.37? 0.15 10.952 0.817 13.20k 0.31 12.38% 0.80 16.01 % 0.34 16.28 2 0.44 16.34f 0.95 17.26* 0.71 20.35i0.24 12.30k0.175 12.18k0.834 14.4020.17 52 B. K. HALL shorter (by 20%, P > 0,001) than were those from control embryos. It may be recalled that at this same age body weight was lighter by 16%, indicating that the growth of the tibia was much more affected by thiourea than was the rest of the body (see fig. 15). The doses of thiourea which did not reduce body weight at ten days (i.e., below. 1 mg thiourea) did significantly reduce the weight of the tibia and as a consequence the relative tibia weight (tibia weight (mg)/g body weight-tibia weight) was very much lower than normal (table 2). A similar situation was seen at 12 days of incubation, where the weight and length of the tibia were below control vdves by 45 and 24% respectively and where the relative tibia weight W;LS substantially below normal. By 14 days of incubation the lowest dose of thiourea used (0.001 mg) produced a smaller tibia weight (39% ) than did the higher doses, and this same trend was intensified at 16 and 18 days (cf. the similar effect on growth of the rest of the body). At 18 days of incubation, the weight of the tibia was 62% below normal after treatment with from 1 to 5 mg thiourea and 45% below normal after treatment with lower doses. Length of the tibia displayed the same pattern (figs. 1-3, 15). Even though its size was reduced the form of growth curve for the tibia from the treated embryos paralleled that from control embryos. Thus despite the greater depression in the growth of the tibia compared with that of the remainder of the body the proportionality of tibia weight to body weight was maintained. Over the 12 to 18 day period the reduction in tibia weight exceeded that of the remainder of the body by a factor of 2.1 : 1. The general trend in the relationship between weight and length of the tibia was that at the lower doses (0.001 to 0.1 mg) weight was depressed to a greater extent than was length and that at the higher doses length was more depressed. The effect of thiourea on the rate of ossification of the tibia was assessed by examining the “alizarin preparations” and calculating the percentage of the cartilaginous model replaced by bone. Aside from an early initiation of ossification at ten days in embryos treated with 0.1 to 5 mg thiourea the relative amounts of bone in the tibiae from the treated embryos was essentially normal. Absolute amounts of bone were, of course, less than normal. Histogenesis and histochemisty of the tibia Changes in the morphology of the tibiae from the treated embryos were assessed using the “alizarin preparations” and the histological sections and are summarized in figure 16. At ten days of incubation the proximal epiphyses of a majority of the tibiae were bent, the bending originating at the junction of the articular and proliferating chondrocyte zones (see fig. 4 for zones), A cenltral area of the same zone of the distal epiphysis was eroded and the resting cavity infiltrated by elements of the marrow. In some specimens the cartilaginous shaft had fractured. Such features were not seen in untreated tibiae. Osteogenesis appeared normal. The reduction in the growth rate of the tibia described above was due to a reduction in the number of chondrocytes maturing and undergoing hypertrophy. At ten days of incubation the zone of hypertrophic chondrocytes made up 56% of the total length of the tibia in control embryos but only 46% in the tibiae from the treated embryos. Evidently treatment with thiourea resulted in a slowing of hypertrophy of the chondrocytes. The proliferating zone was little affected indicating that the rate of production of chondroblasts was normal. By 12 days of incubation bone had begun to form within the cavity in the distal epiphysis, the cartilaginous shaft had undergone more erosion than was normal and the articular surface of the proximal epiphysis had broken down. The acid mucopolysaccharides of the cartilage matrix were visualized after alcian blue or toluidine blue. Up to 12 days of incubation their distribution appeared normal, although after toluidine blue the matrix was more fibrillar than usual. From 12 days of incubation onwards the distribution of matrix acid mucopolysaccharides was distinctly abnormal. Alcian blue staining was very patchy and the matrix did not stain uniformly with toluidine blue. Glycogen appeared as the chondrocytes 53 THYROXINE AND THE CHICK TIBIA TABLE 2 Tibia weight ( m g ) , length ( m m ) and weight/g body weight for control embryos and for embryos treated with thiourea at eight days o f incubation. Values are means -C S.E.M. Age (days) Dose ?. 10 6.602 0.61 2.5 1.0 0.5 0.1 0.05 0.01 0.001 Control 5 2.5 1.0 0.5 0.1 0.05 0.01 0.001 Control 5 2.5 1.o 0.5 0.1 0.05 0.01 0.001 Control 1 2 5.75 2 0.37 6.00 2 0.40 12 14 Tibia weight ( m g ) * 10.9720.84 25.602 1.40 12.32k 1.17 12.55f0.40 27.75 f1.20 12.662 0.41 26.8222.00 13.1020.54 28.4022.14 26.4021.74 2 8 . O O k 1.63 13.14C 0.55 13.90k 1.14 35.0022.85 22.96 2 0.65 46.61 2 0.83 16 44.30 2 3.92 77.30 -C 1.78 51.20k 8.21 80.40 rt 2.79 70.25 * 5.42 70.00f 1.62 69.50'1.77 99.65* 1.51 109.7824.47 110.70k 5.34 112.302 5.44 125.00C5.12 207.9823.87 16.17k 0.75 20.27 k 0.23 17.172 1.20 20.85%0.31 18.80f0.33 22.00 C _ 0.60 22.10 k 0.53 22.00 -+- 0.49 22.36 f0.44 26.05 f0.07 ~ 5.752 0.65 6.10k0.38 10.7120.17 6.74 rt 0.23 6.30 2 0.33 6.54 2 0.15 6.722 0.32 6.9720.18 8.36 k 0.15 Tibia length ( m m ) I 9.4220.18 13.20-+-0.10 9.75 f0.30 10.01 & 0.23 12.80 % 0.62 10.30k0.11 12.40k0.55 9.0020.12 12.80~0.21 12.37-+0.22 9.66f 0.23 12.60 k 0.23 9.72f 0.33 14.10%0.15 12.69 2 0.06 16.69 f0.10 18.70f 0.66 18.90 2 0.35 21.74f0.09 2 Tibia ( m g / g body weight - tibia wt. ( m s > > 7.86 6.83 8.49 9.44 7.70 7.34 8.62 6.72 9.44 7.94 7.93 7.87 8.43 6.63 11.48 7.90 7.84 8.61 6.39 11.51 6.10 8.53 9.78 11.54 11.09 11.44 10.52 14.03 All si nificantly different from control: P No. 08ernbryos: see table 1. 18 12.66 12.33 13.90 13.79 13.94 14.70 20.87 > 0.001. underwent hypertrophy as is the normal some it lay alongside the epiphysis, consituation. The distribution of muco- and nected to it by the perichondrium and surglycoproteins as visualized by the PAS re- rounding mesenohyme. In others the proaction was not uniform and paralleled the liferating zone of the distal epiphysis had toluidine blue staining. Calcification and broken down (fig. 8); chondrocytes had alkaline phosphatase distribution was become freed from the intercellular matrix where extensive vacuolization was apparnormal. The severity of the deviations from ent (fig. 5) and free red blood cells had normal increased with increasing em- invaded the zone of several points (fig. 6). bryonic age. Bone was prominent within Long blood vessels running from the base the proximal epiphysis by 14 days and in of the articular cartilage almost to the end some tibiae from 16 day embryos the of the hypertrophic zone were present proximal epiphysis had become detached (fig, 7). The distribution of the acid mucopolyfrom the diaphysis. Erosion of both proximal and distal epiphyses was very exten- saccharides stained by alcian blue was sive (fig. 4). In some specimens rhe articu- most abnormal, especially in the articular lar zone, especially of the distal epiphysis, and proliferating zones (fig. 8). Some had become completely detached from the clumps of chondrocytes had normal remainder of the cartilage (fig. 7) and in amounts of alcian blue - staining mate- 54 B. K. HALL rial surrounding them, others had none, giving the cartilage a mottled appearance (figs. 9, 10). The articular zone stained intensely with toluidine blue. PAS and Masson’s trichrome (fig. 11) - more intensely than did the remainder of the cartilage. The cells of the proliferating zone were widely separated from one another, the matrix consisting of little other than sparsely distributed fibers (fig. 8). By 18 days of incubation the shaft of bone in the center of the distal epiphysis was extensive in all specimens treated with 0.1 or more mg of thiourea (figs. 12, 13). It ran from the base of the articular zone proximally into the marrow cavity where it was intimately connected with the diaphyseal bone. This core of bone was heavily calcified and contained a well developed marrow cavity, partially filled with fibrous material connecting the bone to the epiphyseal cartilage (fig. 14). These fibres stained orthochromatically after toluidine blue, violet after PAS and green after Masson’s trichrome. The histogenesis of the tibiae in embryos treated with thiourea was then characterized by: increased resoprtion of the cartilage model and of the epiphyses, fragile epiphysed articular zones, extensive erosion of the articular surfaces, abnormal deposition of acid mucoplysaccharides into the cartilage matrix and the deposition of a central core of bone within the proximal epiplhysis (fig. 16). Thyroxine-treated embryos L-thyroxine at or a b v e 10 ag/eight-day embryo was lethal in 100% of cases (table 3). The L. Dsowas 100 ng/embryo. At doses below 10 ng/embryo survival was comparable to that of control embryos, Only at one dose (10 ng/embryo) was body weight significantly lower than that of control embryos at 14 days of incubation (table 3). Both the weight and the length of the tibia were significantly below control values for all doses at both 14 and 18 days of incubation (P > 0.001, table 3). Variance analysis on these data indicated no significant difference between doses. The average reduction of 33% in weight and 14% in length was very similar to that obtained after 1 &g thiourea. Thus thyroxine significantly reduced growth of the tibia without affecting growth of the remainder of the body. The cytodflerentiation of the cells of the tibia h m thyroxine-treated embryos appeared normal. Histochemically those embryos treated with doses of thyroxine below 100 ng appeared normal. In those embryos given a dose of 1 &gand examined at 14 days of incubation the distribution of sulphated acid mucopolysaccharides (as visualized by tduidine blue) and of mucoand glycoproteins (as visualized by PAS) appeared normal, but the non-sulphated acid mucopolysaccharides were very unevenly distributed, especially in the articular zone. The matrix surrounding many of TABLE 3 Body weight; weight, length and relative weight of the tibia from control embryos and from embryos treated with thyroxine at eight days of incubation and examined at 14 or 18 days of incubation. Values are means 2 standard error Dose 1 Pg 100 ng 10 n g 1 ng 100 Pg Control I PLg Control 1 2 Tibia N Body weight Weight 2 Lengths 9 mg mm mg/gl 718 7/8 24/25 14 days of incubation 27.42 1.92 7.50f0.28 7.81 % 0.25 33.0& 3.09 6.92f0.522 29.721.71 32.722.00 7.9040.14 7.34 f 0.33 31.32 2.01 8.2240.12 46.62 0.83 14.0C 0.27 14.4 & 0.56 13.9&0.21 14.7& 0.24 14.5 C 0.40 16.7+- 0.10 7.93 8.53 8.66 8.35 8.60 11.47 2/10 12/13 18 days of incubation 13.7140.29* 120.02 7.07 20.3540.24 207.92 3.87 22.6& 0.28 26.0 2 0.07 17.82 20.86 7/18 4/8 7/a Based on wet weight of two tibiae ( m g ) / g body weight Significantly different from control ( P < 0.001). - tibia wt (mg). THYROXINE AND THE CHICK TIBIA the chondrocptes was unstained after alcian blue, indicating minimal deposition of acid mucopolysaccharide. By 18 days of incubation the sulphated acid mucopolysaccharides were also unevenly distributed, especially in the proliferative zone. Thus 1 pg of exogenous thyroxine resulted in impaired deposition of acid mucopolysaccharide into the cartilage matrix. With lower doses the gross histochemistry of the tibia (collagen distribution, calcification, alkaline phosphatase, glycogen distribution) was normal. DISCUSSION It was evident that when thyroxine synthesis was inhibited in the embryonic chick by the in vivo application of thiourea, the growth of the tibia was retarded to a much greater extent than was the growth of other organs (by a factor of 2 : 1). Many tissues of the embryo evidently depend on thyroxine for their normal development. This is no doubt because of the s t i m u l a t q effect which thyroxine has on general metabolism (MacLean and Urist, '68; Romanoff, '60; Pawelek, '69) ; on oxidative metabolism, glycolysis and mucopolysaccharide synthesis (Hoch, '62; Dziewiatkowski, '64; Dorfman and Schiller, '58); and on collagen synthesis (Kivirikko et al., '67; Blumenkrantz and Prockop, '70). The growth of the skeleton may be more affected than other organs in hypothyroid embryos: ( a ) because skeletal cells are especially dependent on thyroxine or have a lower threshold to thyroxine or (b) because the skeleton contains large amounts of acid mucopolysaccharide and collagen, both of which are especially sensitive to circulating levels of thyroxine. The fact that long bones or their constituent cells respond to thyroxine when isolated in vitro (see Introduction) indicates that the hormone can act directly on the skeleton. The direct action of thyroxine on skeletal cells in vivo has been clearly demonstrated by Dratman and Kuhlenbeck ('69). They injected C'"-labelled thyroxine into newborn rats and found intense labelling of fibroblasts and of osteoblasts six minutes postinjection. Subsequently cartilage matrix was labelled, especially near blood vessels and areas of cartilage erosion. Dziewiatkowski ('64) has shown that SS5uptake is 55 diminished after thiouracil injection, indicating that the sulphated acid mucopolysaccharides of the cartilage matrix are very sensitive to lowered thyroxine levels, and Vaughan ('70) indicated ehat protein synthesis of skeletal cells was stimulated by thyroxine, probably at the level of RNA synthesis. From studies on normal and hyperthyroid rats and mice i t has been shown that thyroxine stimulates the proliferation and maturation of osteoblasts and chondroblasts and so increases the rate of osteogenesis and chondmgenesis and that the increased cartilage proliferation is followed by enhanced cartilage resorption, further increasing endochondral ossification (Levai et al., '69; Becks et al., '46; Silberberg and Silberberg, '40. ) By contrast in hypothyroid rats and in cretinism epiphyseal cartilage persists and the formation of epiphyseal centers of ossification is delayed (Levai et al., '69; Hamburger and Lynn, '64; Vaughan, '70). Thyroxine controls the mobilization of Ca by osteogenic cells and so controls the rate of remodelling of bone (Frost, '64; MacLean and Urist, '68; Vaughan, '70; Urist et al., '63). The present results extend the above findings to the embryonic chick. The cartilaginous model of the tibia was the most affected. Initially (at 10 to 12 days of incubation, i.e., 2 to 4 days post treatment with thiourea) maturation of the chondrocytes was reduced. Subsequently (to 18 days of incubation) formation of the cartilage matrix was inhibited. The fact that, in the hypothyroid embryo, the integrity of the articular cartilage was lost; that erosion of the cartilage was extensive; that there was excessive vascularization of the epiphyses; and that the distribution of matrix acid mucopolysaccharides was abnormal indicated that thyroxine normally maintains cartilage integrity, controls the rate of erosion and resorption of the cartilage model and plays a role in synthesis or deposition of acid mucopolysaccharide-protein complex into the m a t i x of the long bones of the embryonic chick. The formation of bone in the epiphysis of thiourea-treated embryos, preceded as it was by premature invasion of the epiphysis by blood vessels and the formation of 56 B. K. HALL marrow in the erosion cavity, was reminiscent of the replacement of marrow by bone in the hypothyroid dwarf (Vaughan, ' 7 0 ) , of osteopetrosis resulting from stimulating of the parafollicular cells (thyrocdcitonin) in the mouse (Marks and Walker, '69) and of the formation of medullary bone in adult laying hens and in the metatarsus as it repairs a fracture (Sturkie, '65). Evidently the normal integrity of the marrow and marrow cavity of the tibia is also dependent on normal levels of circulating thyroxine. The degree to which the tibial growth was below normal, could, at 16 and 18 days of incubation, be divided into two groups and related to the concentration of thiourea administered. After injection of 1 or 5 mg thiourea the weight of the tibia was much lower than normal than after lower concentrations of thiourea (fig. 15). A possible interpretation is that the higher doses of thiourea had a pharmacological effect in addition to the inhibition of thyroxine synthesis. Evidently eight days were required (treatment at day 8, fast effect at day 16) before this pharmacological effect was manifested, for no such dichotomy of response was observed in 14 day tibia examined. A more precise indication of this possible pharmacological effect of high doses of thiourea could be obtained by co-injection of thiourea and thyroxine to determine whether tibial development could be restored to normal. However, such an approach may not be practical as there is evidence that thiourea not only suppresses synthesis of thyroxine but also inactivates any circulating thyroxine present in the embryo (Sturkie, '65). We are currently assaying circulating thyroxine levels in the embryonic chick, both with and without added thiourea and thyroxine, to determine the feasibility of thyroxine replacement therapy. The problem is compounded by our finding (table 3 ) that exogenous thyroxine itself, at very low concentrations (100 pg) inlhibits skeletal development, although not to the same extent as does thiourea. As rhe body weight in the thyroxine-treated embryos was normal, the inhibition of skeletogenesis may have reflected negative feedback to the thyroid and a consequent lowering of the total amount of circulating thyroxine, even though additional hormone has been administered. In any event our results indicate that skeleton of the embryonic chick is sensitive to very small levels of exogenous thyroxine and that the hormone balance of the embryo must be very precise if skeletogenesis is to be normal. ACKNOWLEDGMENTS This research as been supported by the National Research Council of Canada (grant A5056). The technical assistance of Miss Joan Calder and Miss Josephine Smith is gratefully acknowledged. LITERATURE CITED Adams, A. E., and A. L. Bull 1949 The effects of anti-thyroid drugs on chick embryos. Anat. Rec., 104: 421443. Adams, A. E., and J. M. Buss 1952 The effect of a single injection of an anti-thyroid drug on hyperplasia in the thyroid of the chick embryo. Endocrinol., 50: 234-253. Barka, T., and P. J. Anderson 1965 Histochemistry, Theory, Practice and Bibliography. Harper and Row, New York. Becks, H., M. Simpson, H. Evans, R. Ray, C . Li and W. C. Asling 1946 Response to pituitary growth hormone and thyroxine of the tibias of hypophysectomized rats after long post-operative intervals. Anat. Rec., 94: 631-656. Blumenkrantz, N., and D. J. Prockop 1970 Variations in the glycosylation of the collagen synthesized by chick embryo cartilage: effects of development and several hormones. Biochim. Biophys. Acta, 208: 461466. Dorfman, A., and S. Schiller 1958 Effects of hormones on the metabolism of acid mucopolysaccharides of connective tissue. Recent Prog. Horm. Res., 14: 427453. Dratman, M. B., and H. Kuhlenbeck 1969 Interaction of thyroxine with developing skeletal tissues of the newborn rat. Anat. Rec., 163: 180. Dziewiatkowski, D.D. 1964 Effect of hormones on the turnover of polysaccharides in connective tissue. Biophys. J., 4: 215-238. Evans, H. S. 1948 Clearing and staining small vertebrates in toto for demonstration of ossification. Turtox News., 26: 4247. Fell, H. B., and E. Mellanby 1955 The biological action of thyroxine on embryonic bones grown in tissue culture. J. Physiol., 127: 427447. Frost, H. M. 1964 Dynamics of bone remodeling. In: Bone Biodynamics. H. M. Frost, ed. Little Brown and Co., Boston, pp. 335-352. Grassowicz, N. 1946 Influence of thiourea on development of the chick embryo. Proc. Soc. Exp. Biol. Med., 63: 151-152. Ham, A. W., and W. R. Harris 1950 Histological techniques for the study of bone and some notes on the staining of cartilage. In: Handbook of Microscopical Techniques. R. McC. Jones, ed. Hoeber, New York, pp. 269-284. THYROXINE AND THE CHICK TIBIA Hamburger, M., and E. Lynn 1964 The influence of temperature on skeletal maturation of hypothyroid rats. Anat. Rec., 150: 163-172. Hoch, F. L. 1962 Biochemical actions of thyroid hormones. Physiol. Rev., 42: 605-673. Kivirikko, K. I., 0. Laitnen, J. Aer and J. Halme 1967 Metabolism of collagen in experimental hyperthyroidism and hypothyroidism in the rat. Endocrinol., 80: 1051-1061. Lawson, K. 1961 The differential growth response of embryonic chick limbbone rudiments to triiodothyroxine in vitro. 1. Stage of development and organ size. J. Embryol. exp. Morph., 9: 42-51. Levai, G., F. Moricz., P. Szerze, G. Petranyi Jr. and J. Laczko 1969 The effect of thyrotropic hormone treatment on the epiphyseal cartilage of the white rat. Acta Morphol. Acad. Sci. Hung., 17: 7-15. Lison, L. 1954 Alcian Blue 8G with chlorantine fast red 5B: a technic for selective staining of mucopolysaccharides. Stain Technol., 29: 131138. McLean, F. C., and M. R. Urist 1968 Bone Fundamentals of the Physiology of Skeletal Tissues. Univexsity of Chicago Press, Chicago. Marks, S. C. Jr., and D. G. Walker 1969 The role of the parafollicular cell of the thyroid gland in the pathogenesis of congenital osteopetrosis in mice. Am. J. Anat., 126: 299-314. Melcher, A. H. 1971 In vitro effect of oxygen, hydrocortisone, and triiodothyronine on cells of Meckel's cartilage. Israel. J. Med. Sci., 7: 374-3 76. Pantin, C. F. A. 1960 Notes on Microscopical 57 Techniques for Zoologists. Cambridge University Press, Cambridge. Pawelek, J. M. 1969 Effects of thyroxine and low oxygen tension on chondrogenic expression in cell culture. Devel. Biol., 19: 52-72. Romanoff, A. L. 1960 The Avian Embryo. Structural and Functional Development. The MacMillan Company, New York. Romanoff, A. L., and H. Laufer 1956 The effect of injected thiourea on the development of some organs of the chick embryo. Endocrinol., 59: 611-619. Schajowicz, F., and R. L. Cabrini 1956 Chelating agents as histological and histochemical decalciiiers. Stain Technol., 31: 129-134. Silberberg, M.,and R. Silberberg 1940 Changes in the skeletal tissues of mice following the administration of thyroxin. Growth., 4: 305314. Somogyi, A., and K. Kovacs 1969 Der EinfluBeiniger Hormone auf die heteroplastische Knorpel-und Knochenbildung im Herzmuskel der Ratte. W. Roux' Archiv., 163: 248-258. Sturkie, P. D. 1965 Avian Physiology. Cornell University Press, Ithaca. Urist, M. R., N. S. MacDonald, M. J. Moss and W. A. Skoog 1963 Rarefying disease of the skeleton: observations dealing with aged and dead bone in patients with osteoporosis. In: Mechanisms of Hard Tissue Destruction. R. F. Sognnaes, ed., Amer. Assoc. Adv. Sci., Washington D. C., pp. 385-446. Vaughan, J. M. 1970 The Physiology of Bone. The Clarendon Press, Oxford. PLATE 1 EXPLANATION OF FIGURES 1-3 The tibiae from 12 day (fig. l), 14 day (fig. 2 ) , and 16 day (fig. 3) thiourea-treated embryos. From left to right the tibiae represent embryos treated with 5, 1.0, 0.1, 0.01 or 0.001 mg thiourea. In figure 1 a control tibia is also shown (extreme right). Alizarin Red S. X 1. 4 The proximal epiphysis of the tibia from an 18 day embryo treated with 5 mg thiourea a t eight days. Note the extensive erosion of the articular cartilage (arrow, and see figs. 5, 6). a, articular zone; p. proliferative zone; h, hypertrophic zone. Alcian Blue-Chlorantine Fast Red. x 16. 5 The articular cartilage of figure 4 a t higher magnification ( X 217). Note the mottled appearance of the intercellular matrix, with some unstained areas indicating abnormal deposition of acid mucopolysaccharide into the matrix. 6 T h e area indicated by the arrow in figure 4 at higher magnification ( x 217). Note that the cartilage matrix a t lower left is exposed to the invading red blood cells (arrows). 58 THYROXINE AND THE CHICK TIBIA B. K. Hall PLATE 1 59 PLATE 2 EXPLANATION OF FIGURES 7 The distal epiphysis of the tibia from a n 18 day embryo treated with 1 mg thiourea. The articular cartilage has broken off leaving the proliferative zone exposed (arrow). Note the extensive vascular invasion of the epiphysis. Toluidine Blue, X 15.6. 8 The junction of the proliferative zone (left) and the articular zone of the tibia of an embryo treated as that in figure 7. The proliferative zone has been disrupted whilst the articular zone remains normaI. Masson’s trichrome. x 87. 9 Hypertrophic chondrocytes from the tibia of a n untreated 18 day embryo. Note the pronounced cellular hypertrophy and the uniform staining of the matrix (cf. fig. 10). Alcian Blue-Chlorantine Fast Red. x 217. 10 Hypertrophic chondrocytes from the tibia of an 18 day embryo treated with 5 mg thiourea. Note the reduced cellular hypertrophy and uneven distribution of acid mucopolysaccharide within the matrix (cf. fig. 9). Alcian Blue-Chlorantine Fast Red. x 217. 11 The articular zone from the tibia of a n 18 day embryo treated with 0.01 mg thiourea. The articular surface is eroded (cf. fig. 4 ) and the staining of the articular cartilage more intense than elsewhere. Toluidine Blue. x 16. 12 The distal epiphysis from the same tibia as in figure 4. Note the central core of bone (see fig. 13). The arrow indicates the site of figure 14. Alcian Blue-Chlorantine Fast Red. x 16. 13 A higher magnification of the bone from figure 12. Note trabeculae of bone lined with osteoblasts. x 110. 14 The fibrous material within the central bone and its connection to the cartilage (see fig. 12 for orientation). Toluidine Blue. x 110. 60 THYROXINE AND THE CHICK TIBIA B. K. Hall PLATE 2 61 PLATE 3 EXPLANATION OF FIGURE 15 Body weight ( O - - - - O ) , tibia weight (.---a), and tibia length (A,-.-.-A) of embryos treated with thiourea of eight days of incubation and examined at 10, 12, 14, 16 and 18 days of incubation. Values plotted as percentage of control values. 62 PLATE 3 THYROXINE AND THE CHICK TIBIA B. K. Hall 100 90 - 80 70 i'. 0 60 \0-0-0 I 10 4 O/.. *I 001 *001 - 0 90 Q: - 80 - k 2 70 - 60 0 c, - 50 \ 0 - 80 9Q s - 70 - 60 - 40 50 I I I I 10 I .I I I =01 -001 10 THIOUREA I I I I I *I -01 -001 (my) 63 64 B. K. HALL Fig. 16 A diagrammatic representation of the tibia from a normal embryo (left) and fmm a thiourea-treated embryo (right). Bone, black; cartilage, hatched; marrow, unshaded. Note: the erosion of the articular cartilage, invasion of the epiphysis by marrow and blood vessels, and core of bone within the proximal epiphysis of the treated embryo.