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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].
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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.
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Corresponding Editor: David Norris