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AMER. ZOOL., 37:461-469 (1997) Developmental Endocrinology of the Dipnoan, Neoceratodus forsteri1 JEAN M. P. Joss, 2 P. SYLVIA RAJASEKAR, R. ASHNI RAJ-PRASAD, AND KlRSTY RUITENBERG School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia SYNOPSIS. The development of the pineal, pituitary and thyroid glands of the extant lungfish, Neoceratodus forsteri, are being studied both morphologically and functionally. This paper presents data from hatching to 40-52 weeks for a standardised series of lungfish, bred at Macquarie University. At hatching, the pineal comprises a single organ attached to the roof of the diencephalon, with well-developed photoreceptor, supporting and ganglion cells. The photoreceptors gradually degenerate, giving way to secretory cells which contain electron dense granules. These latter are immunoreactive to melatonin antibodies and digestable with protease. The pituitary at hatching comprises a hollow ball of cells lying beneath the infundibular region of the hypothalamus. Ultrastructurally, four cell types can be distinguished by cytoplasmic granule size after the first four weeks of development posthatching. By 20 weeks, a further three cell types are recognisable. Inununogold labelling has identified corticotropes and melanotropes at four weeks and, at 20 weeks, prolactin cells, thyrotropes and somatotropes also can be identified. The thyroid is only just apparent at hatching, containing 2-3 follicles. The numbers of follicles increases gradually, and variably between animals, with age. Iodine uptake in methimazole-treated animals did not exceed that of controls at any of the three stages tested, indicating a lack of feedback control between thyroid hormones and pituitary thyrotropes at, or before, 40 weeks of age. Thyroid hormone receptors in the liver at 40 weeks are predominantly immunoreactive to human TRac antibodies. These findings taken together suggest that, up to 40 weeks post hatching, lungfish development is equivalent to amphibian premetamorphic development. This would be consistent with lungfish neoteny, but cannot be taken as evidence for neoteny until confirmed at later stages of development. bility that the seemingly "primitive" fea- INTRODUCTION Sfish m a v b e a result of explained as larval features, e.g., presence of unrestricted notochord and absence of marginal jaw bones, ^ developmental descriptions presente d in this P a P e r f o r m t h e first P 3 * o f a m u c h lar er 8 > ongoing study which seeks to address the q u e s t l o n : I s t h e apparent direct development of recent lungfish actually the equivalent of premetamorphic stages charactenstic of an obligate neotene? Several a u t h o r s i n the P a s t (Moy-Thompson and Miles ' l911> Gardiner, 1973; Smith, 1977; Bemis, 1984) have implied paedomorphic tendencies in dipnoans. The most rigorous 1 From the Symposium Developmental Endocrinol- consideration of paedomorphosis in the ogy of Non-Mammalian Vertebrates presented at the evolution of lungfish is by Bemis (1984) wh com e d Devonian Recent molecular phylogenetic studies (Meyer, 1995; Zardoya and Meyer, 1996) are providing strong evidence that lungfish are the closest living ancestor to the land vertebrates, the coelacanth being the only other living contender. Neoceratodus forsteri is the sole representative of the family most closely resembling the post-Devonian lungfish. As such, study of its development may hold the key to appreciating the origin of such novel tetrapod features as limbs and parathyroid gland. As a starting point in my study I have chosen to explore the possi- tures of recent lun neotenv and be better cZ^tZi^lT^JZ^T^buquerque, New Mexico. 2 Email: [email protected]. ° P- fossil lungfish with the three genera of recent dipnoans, and argued that several differences such as 461 462 J. M. P. JOSS ETAL. those of median and tail fins, endochondral ossification and cell size, are best interpreted as paedomorphic. These are all morphological features. Our study is making use of the captive breeding lungfish at Macquarie University to study the development of endocrine glands that may show similar patterns of activity to those associated with neoteny in some urodele amphibians. There have been almost no developmental endocrine studies carried out on any lungfish. Some of the early developmental studies by Semon (1901), Greil (1906, 1913), Kerr (1910, 1919), and Siwe (1926) include descriptions of endocrine glands but there have been no recent studies, using more modern techniques to indicate something of the development of function of these glands. By contrast, there is a wealth of knowledge about the development of endocrine glands in the amphibians, particularly in those species undergoing the most dramatic metamorphoses. Briefly, the function of the thyroid has been shown to be central to amphibian metamorphosis, with several other endocrine glands (pituitary, hypothalamus, adrenal) also being involved as they in some way affect the function of the thyroid or its hormones. The major controllers of thyroid hormone action in developing tadpoles are the levels of circulating thyroid hormones, the activity of the hypothalamicpituitary-thyroid axis and, in the tissues, the number of thyroid receptors and the activities of the 5D and 5D' deiodinase thyroid hormone converting enzymes (Galton, 1988; Becker et ai, 1997). Where neoteny has been studied in amphibians, it has been shown to be associated with a deficiency in one or more of these parameters (Rosenkilde and Ussing, 1996). For example, neotenic ambystomatids (axolotls) show a low level of thyroid activity for most life stages. However, early larvae do display a wave of increased circulating thyroxine associated with dramatic development of the central nervous system. This increased thyroxine level does not result in metamorphosis because the 5'-deiodinase activity in the peripheral tissues necessary for conversion of thyroxine to the more active triiodothyronine is out of phase with the thyroxine surge. In other urodeles, such as Necturus, metamorphosis is prevented by few or no thyroid hormone receptors in tissues which undergo metamorphosis. Other hormones, such as prolactin from the pituitary or cortisol from the adrenal gland also may be involved in suppressing thyroid-induced metamorphosis. It is the eventual aim of this study to investigate all these endocrine aspects of development in N. forsteri in order to answer the question of neoteny in lungfish. STAGING OF DEVELOPING LUNGFISH The lungfish breeding facility at Macquarie University was set up in 1992/3 with nine adult N. forsteri. Spawning began at the end of 1993 and has been repeated in late spring each subsequent year. Eggs have been collected and reared individually under constant environmental conditions in order to establish a standard size vs. age series for the study of endocrine parameters. Figure 1 represents the standard series obtained from 160-200 developing lungfish in their first year of growth. With such a large sample size, the standard error for each data point was <0.05. The range of lengths contributing to each data point was quite wide, however, so the standard series has been useful in selecting "standard" lungfish of the mean size for any given age for studying developmental changes. The embryological and early post-hatching stages of development of N. forsteri were first described by Semon (1893) and have been revised more recently by Kemp (1982). My own study confirms the observations of Kemp that stage at hatching is quite variable, ranging, in my fish, from Kemp stage 39 (Semon, 42), a larva without median or paired fins, to Kemp stage 48 (Semon, 47), in which the yolk is almost all gone, the median fins have developed, the pectoral fin skeleton is developing and the primordium of the pelvic fin is appearing. The predominant stage at hatching is Kemp stage 45 in which the hatchling contains considerable yolk in the gut, the lower and upper lips have met and the pectoral fin is beginning to grow. Over the first four weeks, the lungfish larvae almost all reach the same stage of development and have all LUNGFISH DEVELOPING ENDOCRINE GLANDS 463 60 50 40 I" 30 20 10 Age (weeks) FIG. 1. Standard growth series, obtained from a group of 200 lungfish reared from fertilised eggs under standard conditions of temperature (22°C), lighting (normal daylight) and feeding (brine shrimp from weeks 4-12; tubifex worms to 24 weeks and thereafter fish pellets, provided essentially ad lib). The standard error for each data point (mean of 200 fish) is <0.05. begun to feed on live food (brine shrimp). Figure 2 illustrates the changes in stage of development over the first four weeks posthatching of the same 150 fish. Following first feeding, growth is slow until approximately 10 weeks of age, when the first increase in growth rate is observed over the next four weeks. Growth again becomes slow and steady until approximately 36 weeks of age when the second increase 140 120 100 Stage 39 • Stage 41-42 Stage 42 80 • Stage 43 •Stage 45 £ s 60 • Stage 45-46 •Stage 46 • Stage 48 40 • Stage 49 Stage 52 20 Age (weeks) FIG. 2. Numbers of lungfish recorded at each stage of development (Kemp, 1982) over the first four weeks posthatching. 464 J. M. P. JOSS ETAL in growth rate is observed. In the following descriptions of pineal, pituitary and thyroid development, particular note will be made of these two periods of increased growth rate. DEVELOPMENT OF THE PINEAL There have been no previous studies on the development of the pineal in lungfish. In fact, there are only two reports on pineal structure in dipnoans, and these are both on Protopterus species (Holmgren, 1959; Ueck, 1969). In Neoceratodus, at hatching the pineal comprises a single ovoid organ attached to the roof of the diencephalon, with a prominent lumen confluent with the Illrd Ventricle. The pineal is clearly a photoreceptor at this time with many typical photoreceptor cells with outer segments projecting into the lumen, interspersed with supportive cells and, in association with the bases of the photoreceptor cells, there are ganglion cells whose processes project to the habenular commisure of the diencephalon. Over the first twelve months of development, there is a gradual change in pineal morphology that does not appear to alter significantly in association with either of the increased growth phases seen in the overall development during this period. The developmental changes closely resemble those described for developing amphibians (Kelly, 1963, 1971), with the exception that the more photoreceptive partner to the pineal, the frontal organ, does not develop in lungfish. As the pineal increases in size, it gradually flattens in shape and the lumen diminishes in prominence. Associated with these changes in gross morphology is a decrease in the proportion of photoreceptor cells and an increase in the number of photoreceptors with degenerating outer segments. Dense core secretory granules become evident in the cytoplasm of the cell body of the photoreceptor cells with degenerating outer segments. By 12 months, many of the cells containing these dense core vesicles are simply parenchymal in appearance. Immunogold labelling with melatonin antibodies (CIDtech Research Incorporation) shows that these granules contain melatonin. Treatment with protease confirms that the granules also contain protein. The association between the melatonin and protein remains to be elucidated. The primary function of the pineal in the early post hatching stages of lungfish development is not fully understood. The larvae are heavily pigmented, with both epidermal and dermal melanophores. The latter do respond to melatonin and these young fish blanch in response to complete darkness in the same manner as has been described previously for larval amphibians (Bagnara, 1965). These studies are presently in preparation for publication. The behavioural studies which may indicate whether this blanching response is in fact the primary function of the pineal in hatchling lungfish remain to be carried out. DEVELOPMENT OF THE PITUITARY Griffiths (1938) described the development of the pituitary in N. forsteri at a purely structural level, including no histological description of cell types. The cell types in the adult pituitary have been defined immunocytochemically by several groups (Hansen and Hansen, 1994; Joss et al., 1990). We are now attempting to define the ontogeny of the differentiation of cell types in the pituitary of N. forsteri by immunocytochemical and immunogold techniques. At hatching, the pituitary has separated from the epithelium of the roof of the mouth and comes to lie beneath the infundibular region of the hypothalamus. It comprises a hollow ball of cells which are as yet unidentified. At four weeks, four cell types can be distinguished ultrastructurally by the size of their secretory granules. Two of these have been identified by immunogold labelling as corticotropes and melanotropes. By 20 weeks, three additional cell types can be distinguished which have been identified as prolactin cells, somatotropes, and thyrotropes. Using antibodies raised against the beta chain of lungfish luteinising hormone (isolated by H. Kawauchi) and shown to be immunoreactive with putative gonadotropes in adult lungfish pituitaries, we have not yet identified gonadotropes in the pituitary of lungfish less than 40 weeks of age. The study of development of the pituitary is preliminary at the present mo- 465 LUNGFISH DEVELOPING ENDOCRINE GLANDS 0.2 0.19 0.18 ~ 0.17 2 0-16 JSP v f 1 0.15 •5 0.14 1 0.13 0.12 0.11 0.1 10 15 20 25 30 35 40 Age (weeks) FIG. 3. Preliminary observations on the relationship between thyroid follicle cell height and age in lungfish from hatching to 40 weeks of age. Each point represents the average cell height taken from measuring the diameter of all follicles and the diameter of the follicle lumina within the thyroid of a "standard" lungfish. The cell height for each follicle was calculated by subtracting the lumen from the follicle diameters, dividing by two and then averaging for all the follicles in the gland. ment but has been included here to indicate fore, experiments were carried out to exthat the sequence of differentiation of the plore the relationship between the pituitary cell types appears to follow a similar pat- and thyroid during this period of developtern to that observed in amphibians (Oota ment. These experiments included fish from and Saga, 1991; Ogawa et al, 1995; Mir- the first period of accelerated growth (16 anda et al., 1996); i.e., corticotropes ap- weeks) for comparison. At 16 weeks of age and again at 36 pearing first, followed by prolactin cells and thyrotropes. The latter will be discussed weeks and 40 weeks of age, groups of four further in the next section on the develop- juvenile lungfish were pretreated for four days with 0.1% methimazole, a potent thyment of the thyroid. roid hormone inhibitor, (added to the water DEVELOPMENT OF THE THYROID in which they were kept). Following methAt hatching the thyroid is only apparent imazole treatment, these and four untreated in those fish which hatch at a later stage of lungfish of the same age and size range development. At stage 49, 2—3 follicles, were injected intraperitoneally with 1 |xc of comprising 9-12 cells each, make up the I125 and left for a further 24-hour period. thyroid. Thereafter, follicle number increas- After this time each fish was humanely es slowly and quite variably between indi- killed and separated into lower jaw (containing thyroid tissue), upper head, anterior viduals. body (containing liver) remaining body and Pituitary control of thyroid function tail. The total c.p.m. per mg tissue was dePreliminary observations on thyroid fol- termined and the average value for tail and licle cell height (Fig. 3) suggest that an in- upper head subtracted from the total activcrease occurred at approximately 28 weeks ity of the lower jaw to correct for backof age, immediately preceding the second ground radioiodide uptake of nonthyroidal increase in growth rate of juvenile lungfish tissue. The thyroidal activity was then exand following the appearance of TSH-im- pressed as a percentage uptake of the inmunoreactive cells in the pituitary. There- jected dose per mg of thyroid tissue. There 466 J. M. P. JOSS ETAL. 0.3 • control 0.25 •S 0MMItreated 0.2 0.15 0.05 16 36 40 Age (weeks) FIG. 4. Effect of methimazole (MMI) on thyroidal radioiodide uptake in lungfish at 16, 36 and 40 weeks of age. Values are presented as mean plus standard error of the mean. *P < 0.01 vs. MMI-treated (Student /-test). was no significant change (Student r-test) in the uptake of iodine between any of the methimazole-treated groups or the control group at 16 or 36 weeks but the thyroids of the control lungfish at 40 weeks increased uptake by approximately 50% (Fig. 4). This increase in iodine uptake is reflected in the increased follicle cell height observed at this stage. This may be due to stimulation by thyrotropin, since thyrotropes can be identified in the pituitary at this age, but the negative feedback relationship between thyroid hormones and thyrotropin is not yet established because the methimazole-treated lungfish (with decreased thyroid function) did not show greater uptake of iodine than did the controls. Thyroid hormone receptors (TR) in liver In most vertebrates examined, there are two types of thyroid hormone receptors, TRa and TRp. Looking in the amphibian, Xenopus laevis, Yaoita and Brown (1990) have shown that the expression of the genes coding for these receptors begins soon after the embryo hatches. TRa expression increases through the premetamorphic stage of development, is maximal at prometamorphosis and thereafter declines to low levels in the adult. Expression of TRp, on the other hand, is barely detectable during premetamorphosis, rises during prometamorphosis, reaches a peak at metamorphic climax and drops to 10% of its peak in the adult. Although thyroid receptors have not yet been characterised in lungfish, a study was undertaken to immunocytochemically differentiate between TRa and TRp in liver cells at the time when the thyroid was increasing in activity at 40 weeks. Antibodies specific for human TRa and TRp (Affinity Bioreagents) were employed at a dilution of 1:100 with a Vectastain ABC immunocytochemistry kit. Figure 5a, b shows that the nuclei of liver cells from 40-week-old lungfish immunostain predominantly with the antibody to human TRa. CONCLUSIONS The development of the pineal, pituitary and thyroid in the lungfish, N. forsteri, over the first twelve months post hatching, is equivalent to that of a premetamorphic amphibian. While the thyroid gland does ap- LUNGFISH DEVELOPING ENDOCRINE GLANDS 467 B FIG. 5. a) Section (7(i.m) through the liver of a 40-week-old lungfish, showing the immunocytochemical reaction to human TRa-antibody (1:100). X80. b) Adjacent section through liver of same lungfish as in (a), showing the immunocytochemical reaction to human TRfS-antibody (1:100). X80. pear to increase in activity, as judged by follicle cell height, at two distinct periods during the first year of development, neither of these was shown to correlate with a mature pituitary-thyroid axis in which the thyroid hormones are capable of influencing the secretion of thyrotropin from the pituitary. Moreover, thyroid receptors in the liv- er were predominantly of the alpha-type, characteristic of premetamorphosis in Xenopus. A persistence of premetamorphic endocrine patterns, particularly those concerning thyroid function either directly or indirectly, is typical of neotenous amphibians. Therefore, these findings taken together suggest 468 J. M. P. JOSS EJAL. that up to 40 weeks post hatching, lungfish development is equivalent to amphibian premetamorphic development. This is consistent with neoteny but cannot be taken as evidence for neoteny until confirmed at later stages of development. ACKNOWLEDGMENTS We are indebted to Greg Joss for his unstinting assistance in all aspects of the maintenance of the lungfish breeding facility. We would also like to acknowledge Nikki Beckendorf who so carefully reared, measured and staged the lungfish for the standard growth series, and the Microscopy Unit, Macquarie University for assistance with electron microscopy and photography. The work has been funded by the Australian Research Council and by Macquarie University research grants to JMPJ. REFERENCES Bagnara, J. T. 1965. Pineal regulation of body blanching in amphibian larvae. In J. Ariens Kappers (ed.), Progress in brain research 10:489-506. Elsevier, Amsterdam. Becker, K. B., K. C. Stephens, J. C. Davey, M. J. Schneider, and V. A. Galton. 1997. The type 2 and type 3 iodothyronine deiodinases play important roles in coordinating development in Rana catesbeiana tadpoles. Endocrinology 138:29892997. Bemis, W. E. 1984. Paedomorphosis and the evolution of the Dipnoi. Paleobiology 10:293-307. Galton, V. A. 1988. The role of thyroid hormone in amphibian development. Amer. Zool. 28:309318. Gardiner, B. G. 1973. Interrelationships of teleostomes. In P. H. Greenwood, R. S. Miles, and C. Patterson (eds.), Interrelationships of fishes, pp. 105-135. Academic Press, London. Griel, A. 1906. Uber die Entstehung der Kiemendarmderivate von Ceratodus forsteri. Anat. 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