letters to nature

Recurrent parent similarity index

Six characters (upper petal reflexing, lateral petal reflexing, pistil length, stamen length,
lateral petal width and nectar volume) for which QTLs have been mapped6,7 were
measured on two flowers from each plant. There was a significant difference between the
multivariate flower phenotypes of wild-type and mutant NILs in both the M. lewisii
(multiple analysis of variance, MANOVA, F 18.18, Wilks l 0.32, P , 0.0001) and
M. cardinalis (MANOVA, F 11.00, Wilks l 0.56, P , 0.0001) genetic backgrounds
(PROC GLM, SAS Institute). Least-squares means for each trait within each NIL
genotypic class were normalized to the difference between trait means of the two parental
species7, setting the recurrent parent trait value at 100% and the nonrecurrent parent at
0%. Lower recurrent parent similarity (RPS) values are evidence of linkage drag, whereas
values larger than 100% represent measurement error or heterosis. In the M. lewisii genetic
background, the wild-type plants had a mean RPS index across all traits of 91% (range
66108%), whereas their mutant sibs had a value of 80% (range 51103%). In the
M. cardinalis genetic background, the wild-type plants had a mean RPS index of 95%
(range 49129%), whereas their mutant sibs had a value of 80% (range 46155%).
Although mutant NILs show more linkage drag than the wild type, we judge the
difference to be small. Nectar volume, which is known from our F2 experiments to have a
marked effect on hummingbird visitation8, has RPS index values that are very close to one
another in the NILs: 105% and 103% in the M. lewisii background, and 46% and 53% in
the M. cardinalis background. This suggests that differences in nectar production between
pairs of NILs did not affect pollinator visitation patterns.

Pollinator visitation
For each of two field experiments conducted to measure pollinator visitation, 50 pink or
dark pink (YUP/___) and 50 yellow-orange or red (yup/yup) plants were drawn at random
from five BC4S1 (M. lewisii) or BC4 (M. cardinalis) NIL families. Assessments of pollinator
visitation were performed at Mather (California, USA), the site where much of the
previous work on these two species of Mimulus has been done5. Pollinator observations
were carried out from dawn to evening, with a 12 h break at midday when pollinators
were least active. Dates of observation were 1830 August 1999 for M. cardinalis NILs, and
1827 July 2000 for M. lewisii NILs. These dates correspond closely to the peak flowering
times of natural populations of the two Mimulus species. We chose to do the experiments
in different years so that pollinators were faced with a binary choice of flower phenotypes,
as would be the case for a newly arisen mutation. Plants were placed at random on a
1 m 1 m grid to produce the experimental arrays (a black bear visit reduced the total
sample size in the M. lewisii NIL array from N 100 to N 99). A pollinator visit was
counted if it appeared that the pollinator probed the flower and contacted the anthers or
stigma. Bumblebees and hummingbirds were the only pollinators observed. We observed
1,090 bumblebee visits to the M. lewisii NILs, 180 bumblebee visits to the M. cardinalis
NILs, 201 hummingbird visits to the M. lewisii NILs, and 3,738 hummingbird visits to the
M. cardinalis NILs. The number of flowers on each plant was recorded daily, along with the
number of hours spent observing. Visitation rates were calculated by dividing the total
number of pollinator visits across all days by the aggregate number of hours in which visits
could have occurred to each flower (flower-hours). For the M. lewisii NILs, both
bumblebee and hummingbird pollinator observations were carried out simultaneously,
with 47,159 flower-hours for the wild-type NILs and 138,648 flower-hours for the
mutants. For the M. cardinalis NILs, separate pollinator observation periods were
required to keep track of the large number of hummingbird visits. During the bumblebee
observation periods, there were 16,291 flower-hours for the mutant NILs and 13,556
flower-hours for the wild-type. During the hummingbird observation periods, there were
11,505 flower-hours for the mutant NILs and 9,520 flower-hours for the wild type.
Received 15 July; accepted 3 October 2003; doi:10.1038/nature02106.
1. Orr, H. A. & Coyne, J. A. The genetics of adaptation: a reassessment. Am. Nat. 140, 725742
(1992).
2. Gillham, N. W. Evolution by jumps: Francis Galton and William Bateson and the mechanism of
evolutionary change. Genetics 159, 13831392 (2001).
3. Fisher, R. A. The Genetical Theory of Natural Selection (Dover, New York, 1958).
4. Orr, H. A. The population genetics of adaptation: the distribution of factors fixed during adaptive
evolution. Evolution 52, 935949 (1998).
5. Hiesey, W. M., Nobs, M. A. & Bjorkman, O. Experimental Studies on the Nature of Species:
V. Biosystematics, Genetics, and Physiological Ecology of the Erythranthe Section of Mimulus 16
(Carnegie Inst. Wash. Publ. 628, Washington DC, 1971).
6. Bradshaw, H. D. Jr, Wilbert, S. M., Otto, K. G. & Schemske, D. W. Genetic mapping of floral traits
associated with reproductive isolation in monkeyflowers (Mimulus). Nature 376, 762765 (1995).
7. Bradshaw, H. D. Jr, Otto, K. G., Frewen, B. E., McKay, J. K. & Schemske, D. W. Quantitative trait loci
affecting differences in floral morphology between two species of monkeyflower (Mimulus). Genetics
149, 367382 (1998).
8. Schemske, D. W. & Bradshaw, H. D. Jr Pollinator preference and the evolution of floral traits in
monkeyflowers (Mimulus). Proc. Natl Acad. Sci. USA 96, 1191011915 (1999).
9. Ramsey, J., Bradshaw, H. D. Jr & Schemske, D. W. Components of reproductive isolation between the
monkeyflowers Mimulus lewisii and M. cardinalis (Phrymaceae). Evolution 57, 15201534 (2003).
10. Vickery, R. K. Jr Speciation in Mimulus, or, can a simple flower color mutant lead to species
divergence? Great Basin Nat. 55, 177180 (1995).
11. Beardsley, P. M., Yen, A. & Olmstead, R. G. AFLP phylogeny of Mimulus section Erythranthe and the
evolution of hummingbird pollination. Evolution 57, 13971410 (2003).
12. Mauricio, R. Mapping quantitative trait loci in plants: uses and caveats for evolutionary biology.
Nature Rev. Genet. 2, 370381 (2001).
13. Hodges, S. A., Whittall, J. B., Fulton, M. & Yang, J.-Y. Genetics of floral traits influencing reproductive
isolation between Aquilegia formosa and Aquilegia pubescens. Am. Nat. 159, S51S60 (2002).
14. Grant, V. Historical development of ornithophily in the western North American flora. Proc. Natl
Acad. Sci. USA 91, 1040710411 (1994).

178

Acknowledgements We thank A. Angert, K. Kay, and D. Grosenbacher for field observations of

pollinators, P. Beardsley and S. Stefanovic for field assistance, and B. Watson for genotyping. We
are grateful to F. Nicholson and the Carnegie Institution of Washington for allowing us to use the
Mather field station. Y. Sam provided helpful comments on the manuscript. This work was
supported by an award from the National Science Foundation.
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to H.D.B.
(toby@u.washington.edu).

..............................................................

Light-induced hormone conversion

of T4 to T3 regulates photoperiodic
response of gonads in birds
Takashi Yoshimura1, Shinobu Yasuo1, Miwa Watanabe1, Masayuki Iigo3,
Takashi Yamamura1, Kanjun Hirunagi2 & Shizufumi Ebihara1
1
Division of Biomodeling, Graduate School of Bioagricultural Sciences,
Nagoya University, and 2The Nagoya University Museum, Furo-cho, Chikusa-ku,
Nagoya, 464-8601, Japan
3
Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya
University, Mine-Machi, Utsunomiya, Tochigi 321-8505, Japan

.............................................................................................................................................................................

Reproduction of many temperate zone birds is under photoperiodic control. The Japanese quail is an excellent model for studying the mechanism of photoperiodic time measurement because
of its distinct and marked response to changing photoperiods.
Studies on this animal have suggested that the mediobasal
hypothalamus (MBH) is an important centre controlling photoperiodic time measurement18. Here we report that expression in
the MBH of the gene encoding type 2 iodothyronine deiodinase
(Dio2), which catalyses the intracellular deiodination of thyroxine (T4) prohormone to the active 3,5,3 0 -triiodothyronine (T3), is
induced by light in Japanese quail. Intracerebroventricular
administration of T 3 mimics the photoperiodic response,
whereas the Dio2 inhibitor iopanoic acid prevents gonadal
growth. These findings demonstrate that light-induced Dio2
expression in the MBH may be involved in the photoperiodic
response of gonads in Japanese quail.
The molecular mechanism of photoperiodic or seasonal time
measurement is not well understood in any organism studied so far.
In birds, the MBHwhich includes the nucleus hypothalamicus
posterior medialis (NHPM), the infundibular nucleus and the
median eminenceis an important centre controlling photoperiodic time measurement (Supplementary Figs 1 and 2). For example,
introduction of a lesion to the nucleus hypothalamicus posterior
medialis and/or the infundibular nucleus resulted in loss of photoperiodic response of the gonads13 even though the gonadotrophinreleasing hormone (GnRH) system of the lesioned animal had been
left intact4. Electrical stimulation of this area increases luteinizing
hormone secretion5 and induces testicular growth6. Furthermore,
c-Fos expression has been reported in these structures as a result of
photostimulation for one long day (20/4 h light/dark cycle)7,8 and
deep-brain photoreceptors are thought to be localized in the
infundibular nucleus 9. Recently, we have also observed the
expression of circadian clock genes in the MBH, and proposed
that the clock in the MBH may function as the photoperiodic
clock10. These observations indicate that all of the essential machinery for photoperiodic time measurement is localized in the MBH.
Single light pulses within the photo-inducible phase increase

2003 Nature Publishing Group

NATURE | VOL 426 | 13 NOVEMBER 2003 | www.nature.com/nature

letters to nature
serum luteinizing hormone concentration and cause photoperiodic
response of the gonads11. Therefore, it is expected that some
molecular events take place in the MBH when animals are exposed
to light within the photo-inducible phase. To identify genes that are
responsible for the regulation of photoperiodic time measurement
in birds, we performed a differential subtractive hybridization
analysis. A total of 40 8-week-old male Japanese quail were raised
under short day conditions (8/16 h light/dark cycle). From this
group, 20 animals were exposed to a 1-h light pulse at zeitgeber time
14 (ZT14), whereas the other 20 animals were kept in darkness. One
hour after the end of the light pulse (ZT16), both groups of animals
were killed and the MBH was removed (Supplementary Fig. 1).
Differential subtractive hybridization analysis was carried out and
150 clones were sequenced. Expression of all of these genes was
verified using in situ hybridization. Among them, expression of only

Figure 1 Identification of a gene that controls photoperiodic time measurement.

a, b, Representative autoradiograms for light-induced expression of Dio2 (a) and a control
animal without light (b) in the dorsal hypothalamus (arrowhead). A single 1-h light pulse
was given at ZT14, and brain was collected 1 h after the end of the light pulse. c, Lightinduced Dio2 expression in the dorsal hypothalamus was specific to the photo-inducible
phase. Values are mean ^ s.e.m. (n 5). Asterisk, P , 0.01. d, Temporal expression
profiles of the Dio2 gene under short day (SD) and long day (LD) conditions in the dorsal
hypothalamus. Values are mean ^ s.e.m. (n 35). e, f, Representative
autoradiograms of the BTH (arrowhead) of animals housed under long day (e) and short
day (f) conditions. g, Effect of the light pulse on Dio2 expression in the BTH. Values are
mean ^ s.e.m. (n 5). h, Temporal expression profiles of the Dio2 gene in the BTH in
animals kept under short day and long day conditions. Values are mean ^ s.e.m.
(n 35). Asterisk, P , 0.05.
NATURE | VOL 426 | 13 NOVEMBER 2003 | www.nature.com/nature

one gene, Dio2, was significantly induced by the light pulse within
the photo-inducible phase (ZT14) in the dorsal hypothalamus
around the paraventricular organ (PVO) as well as the lateral
hypothalamus12 (MannWhitney U-test, P , 0.01; Fig. 1ac).
(More details of localization and the schematics of quail brain can
be seen in Supplementary Fig. 2.) We then examined the effect of
light exposure at times outside the photo-inducible phase (ZT9 and
ZT21). However, light-induced expression of Dio2 was not observed
at ZT9 or ZT21 (MannWhitney U-test; P . 0.05), which indicates
that the light-induced expression of Dio2 is specific to the photoinducible phase (Fig. 1c).
We next examined the expression profiles of Dio2 in the dorsal
hypothalamus under short and long day conditions. Unexpectedly,
expression of Dio2 in the dorsal hypothalamus was almost undetectable at any time of day, whether examined under long or short day
conditions (Fig. 1d). Notably, expression of Dio2 was observed in
the infundibular nucleus and the median eminence (both of which
are collectively known as the basal tuberal hypothalamus; BTH)
(Fig. 1e, f; see also Supplementary Fig. 2). Consequently, we
examined the effect of light on the expression of Dio2 in these
regions. Although acute induction of Dio2 expression by light pulse
was not detected at any of the phases examined (MannWhitney
U-test, P . 0.05; Fig. 1g), significant differences in the level of
expression between short day (weak expression) and long day
(strong expression) conditions were observed in these regions
(two-way analysis of variance (ANOVA), F 1,41 219.917,
P , 0.0001, *P , 0.05, MannWhitney U-test; Fig. 1e, f, h). No
clear day/night difference in expression was observed in either long
day or short day conditions (one-way ANOVA: long day,
F 5,24 2.273, P 0.0795; short day, F 5,17 0.861, P 0.5269).
It has been shown that introduction of a small lesion to the nucleus
hypothalamicus posterior medialis (including the PVO) or the
infundibular nucleus, or a combination of both lesions, completely
blocks testicular development1. The expression sites of Dio2
revealed in our study are consistent with these results. Although it
remains to be determined how these two structures are related to
each other, these results suggest that expression of Dio2 in both
regions is of critical importance for the photoperiodic response of
the gonads.
The enzyme Dio2 converts T4 to T3, which is primarily responsible for thyroid hormone action. Dio2 has an essential role in the
local control of levels of T3 in the brain through mechanisms that
operate under a variety of conditions to maintain T3 concentrations
within a narrow range13. To determine the levels of T3 and T4 in the

Figure 2 Locally generated T3 acts on the BTH. a, Plasma content of T3 and T4 under long
day and short day conditions. b, c, T3 (b) and T4 (c) contents are increased in the MBH
under long day conditions, whereas they are not changed in the stratum griseum
centrale (SGC) and the cerebellum (Cb). The MBH collected from ten animals were pooled.
df, Expression of thyroid hormone receptor genes Thra (d), Thrb (e) and Rxra (f) in the BTH.

2003 Nature Publishing Group

179

letters to nature
MBH, we measured the contents of pooled MBH collected from ten
animals in each group under short day and long day conditions.
Although the plasma concentration of T3 and T4 was not significantly different between short day and long day conditions (Mann
Whitney U-test, P . 0.05; Fig. 2a), the contents of both in the MBH
were about tenfold higher in long day than in short day conditions
(Fig. 2b, c). This difference was not observed in other parts of
the brain, such as the stratum griseum centrale and the cerebellum
(Fig. 2b, c). Recently it has been reported that expression of the gene
encoding transthyretin (Ttr), which regulates thyroid hormone
uptake, increases in Siberian hamster hypothalamus under long
day conditons14. Although we could not detect expression of Ttr in
the hypothalamus, strong expression was observed in the choroid
plexus in Japanese quail; however, there was no difference in the
expression of Ttr in the choroid plexus between long day and short
day conditions (Supplementary Fig. 3). Therefore, the content of
other type(s) of thyroid hormone transporters may increase in the
MBH of Japanese quail under long day conditions that subsequently
aid the generation of T3 through increased T4 uptake.
To explore the target site that locally generated T3 acts upon,
expression of thyroid hormone receptor genes (Thra, Thrb, Thrb2,
Rxra and Rxrg, which encode thyroid hormone receptor-a, -b, -b2
and retinoid X receptor-a and -g, respectively) were examined.
Among these genes, weak expression of Thra and Thrb and strong
expression of Rxrawas observed in the BTH, whereas expression of
Thrb2 and Rxrg was undetectable (Fig. 2df). Expression of Thra,
Thrb and Rxra was observed for the entire day under both short day
and long day conditions, and did not show a difference between day
and night (data not shown). These results indicate that locally
generated T3 acts on the BTH. Although we have also examined
expression of these genes in the preoptic area, where cell bodies of
GnRH neurons are located, no signal was detectable under any
conditions (data not shown). It has been suggested that photoperiodic GnRH release could be controlled at the GnRH terminals by
glia8,15, and thyroid hormones are known to have a critical involvement in the development, plasticity and function of the central
nervous system13. Therefore, we examined the morphological
changes in the median eminence under long day and short day
conditions. Immunoelectron microscopy revealed that GnRH nerve
terminals are in close proximity to the basal lamina in birds
subjected to long day conditions (T.Ya., K.H., S.E. and T.Yo.,
unpublished observations). We also found that the GnRH terminal
was enclosed by the end feet of glia in animals subjected to short day
conditions (T.Ya., K.H., S.E. and T.Yo., unpublished observations).
These morphological changes may allow the neurons to secrete
GnRH owing to the increased access of their terminals to the

Figure 3 Intracerebroventricular T3 infusion mimics photoperiodically induced testicular

growth. a, T3 and T4 were infused under short day conditions. Testicular length was
measured before and after infusion (n 36). Different characters (a, b) indicate
significant differences (P , 0.05). b, Iopanoic acid infusion reduces testicular growth
under long day conditions (n 45). Asterisk, P , 0.05.
180

perivascular area surrounding the fenestrated portal capillaries

under long day conditions.
To assess whether T3 mediates the photoperiodic response of the
gonads, we studied the effect of intracerebroventricular infusion of
T3 on testicular growth. Vehicle and several doses of T3 and T4 were
infused into the third ventricle by an osmotic mini-pump, and the
testicular size was measured before and after infusion. T3 infusion
induced testicular growth in a dose-dependent manner, even
though the animals were kept under short day conditions (oneway ANOVA, F 5,18 4.008, P 0.0128; Fishers LSD post-hoc test,
P , 0.05), whereas T4 infusion had only a minor effect (one-way
ANOVA, F 3,16 0.32, P 0.8111; n 36; Fig. 3a). However,
significant testicular growth was not observed in the largest dose
of T3. Iopanoic acid is known to inhibit the conversion of T4 to T3
(ref. 16), thus we examined the effect of iopanoic acid infusion and
found that it reduced testicular growth under long day conditions
(n 45; MannWhitney U-test, P , 0.05; Fig. 3b).
Follett et al. have shown that peripheral injection of pharmacological doses of thyroid hormone (T4 and T3) can mimic photoperiodically induced gonadotrophin secretion and gonadal
growth17,18. In these previous studies, however, T4 was more effective
than T3 in mimicking gonadotrophin release. The findings seem to
stand in contradiction to our results. In blood, however, about 33%
and 45% of T4 is known to be converted to T3 and reverse T3 (rT3),
respectively19. Furthermore, T3 is converted to 3,3 0 -diiodothyronine
(3,3 0 -T2) in the blood. Therefore, in the studies of Follett et al., most
of the peripherally delivered T4 might be converted to T3 and act on
the central nervous system. In addition, we have shown that
iopanoic acid, an inhibitor of Dio2, reduces testicular growth
under long day conditions. Thus, our results are consistent with
the hypothesis that Dio2 is important for regulation of the photoperiodic response of the gonads. Of note, T3 infusion did not
maximize testicular size and iopanoic acid did not block testicular
growth completely. There are two possible explanations for this
finding. One is that T3 and iopanoic acid were subject to partial
inhibition or partial loss through imprecise delivery. It is impossible
to infuse at exactly the same location in all birds, and drugs may be
diffused and metabolized through the cerebrospinal fluid in some.
Variability in the effect of T3 seems to support this idea. The other
possibility is the existence of other regulatory pathways or compensatory mechanisms. This possibility is supported by previous
studies showing that after thyroidectomy, quail can still respond to
photostimulation when transferred from short day to long day
conditions, although increases in their follicle-stimulating hormone
levels and in the sizes of testes and the cloacal gland are attenuated20,21. This is in contrast with the observation that after starlings
and house sparrows are photoperiodically blind thyroidectomy22,23.
The exploration of this mechanism is important for our eventual
understanding of the complete molecular machinery of photoperiodism. Although thyroid hormone is of critical importance in the
regulation of photoperiodically induced gonadal growth, as shown
here, this hormone is also known to be involved in the regulation of
photorefractoriness (insensitivity to previously stimulatory daylength)15,24. The observation that no significant testicular growth is
induced at the highest dose of T3 (Fig. 3) suggests the possibility that
photorefractoriness occurs as a result of downregulation of thyroid
hormone activity. This hypothesis remains to be tested.
Although exogenous thyroid hormones mimic the effect of long
day conditions, their precise role remains uncertain15. The present
study has determined the target site and molecular events of T4 to T3
conversion that take place in the brain. Finally, it is also of interest to
note that multiple studies indicate that thyroid hormones are
essential for the maintenance of seasonal reproductive changes in
a number of mammals25. Thus, our present study may provide the
means for a collective understanding of the molecular mechanism
of photoperiodic time measurement in all photoperiodic
vertebrates.
A

2003 Nature Publishing Group

NATURE | VOL 426 | 13 NOVEMBER 2003 | www.nature.com/nature

letters to nature
Methods
Animals
Four-week-old male Japanese quail (Coturnix coturnix japonica) were obtained from a
dealer and kept under short day conditions (8/16 h light/dark cycle) in light-tight boxes.
The boxes were placed in a room at a temperature of 24 ^ 1 8C. Light was supplied by
fluorescent lamps with a light intensity of 200 lx measured at the head level of the birds.
Short day animals were kept under short day conditions whereas long day animals were
transferred from short to long day conditions (16/8 h light/dark) for 2 weeks. Food and
water were available ad libitum and were replenished at least twice a week. Animals were
treated in accordance with the guidelines of Nagoya University.

Differential subtractive hybridization analysis

Brain slices (3 mm) of quail were generated by mouse brain matrix (ASI), and the MBH
was punched out (3 mm diameter). Total RNA was prepared from 20 pooled MBH using
Trizol reagent (Gibco BRL). Poly(A) RNA was purified using oligotex-dt30 Super
(Takara). Differential subtractive hybridization analysis was performed according to the
manufacturers instructions (PCR-select complementary DNA subtraction Kit, Clontech).
Final PCR products were inserted into a TA cloning vector (Invitrogen) and sequenced by
an ABIPrism 373 using the Big Dye Terminator kit (ABI).

In situ hybridization
Animals were killed by decapitation and the brains were immediately removed to avoid
acute changes in gene expression. In situ hybridization was carried out as previously
described26. Antisense and sense 45-nucleotide oligonucleotide probes were labelled with
[33P]dATP (NEN) using terminal deoxyribonucleotidyl transferase (Gibco BRL). Dio2,
5 0 -GATGGTTCAGCCTCAATGAATATCAAGACGGAAATACATTCTGTA-3 0 ; Thra,
5 0 -TTGATGGAATTGCGGTGAATGGAA CAGAAGCCCAGCACCCTGGAC-3 0 ; Thrb,
5 0 -CGGAGTGAGAGAACAGAAAATGAAGCTCTAGTAAGGTGGCAGTGG-3 0 ; Thrb2,
5 0 -TGAAGTGCACCCAGCTGCTGGTAGCAATTGCTACATGCAGTCCAC-3 0 ; Rxra,
5 0 -GGCATGAGTTAAGCACCAGCGATGGACACCAAACACTTCCTGCCA-3 0 ; Rxrg,
5 0 -ATTCCCCTGTTCATGCCAGCTCCACGTCTGTGAGCCCATCATCCA-3 0 ; Ttr,
5 0 -ACAGCTACGTTAGCTGCAGGACTTCCTCTGACTGCATCCAGCACT-3 0 .
Hybridization was carried out overnight at 42 8C. After glass slides were washed, they
were air dried and apposed to Biomax-MR film (Eastman Kodak Co.) for 2 weeks with 14C
standards (American Radiolabelled Chemicals). Relative optical densities were measured
by using a computed image-analysing system (MCID Imaging Research), and were
converted into the relative radioactive value (nCi) by 14C standards. Specific hybridization
signals were obtained by subtracting background values obtained from adjacent brain
areas that did not exhibit a hybridization signal.

13. Bernal, J. Action of thyroid hormone in brain. J. Endocrinol. Invest. 25, 268288 (2002).
14. Prendergast, B. J., Mosinger, B. Jr, Kolattukudy, P. E. & Nelson, R. J. Hypothalamic gene expression in
reproductively photoresponsive and photorefractory Siberian hamsters. Proc. Natl Acad. Sci. USA 99,
1629116296 (2002).
15. Dawson, A., King, V. M., Bentley, G. E. & Ball, G. F. Photoperiodic control of seasonality in birds.
J. Biol. Rhythms 16, 365380 (2001).
16. Leonard, J. L. & Visser, T. J. in Thyroid Hormone Metabolism (ed. Hennemann, G.) 189229 (Marcel
Dekker, New York, 1986).
17. Follett, B. K. & Nicholls, T. J. Acute effect of thyroid hormones in mimicking photoperiodically
induced release of gonadotropins in Japanese quail. J. Comp. Physiol. B 157, 837843 (1988).
18. Follett, B. K., Nicholls, T. J. & Mayes, C. R. Thyroxine can mimic photoperiodically induced gonadal
growth in Japanese quail. J. Comp. Physiol. B 157, 829835 (1988).
19. Chopra, I. J. et al. Pathways of metabolism of thyroid hormones. Recent Prog. Horm. Res. 34, 521567
(1978).
20. Follett, B. K. & Nicholls, T. J. Photorefractoriness in Japanese quail: possible involvement of the
thyroid gland. J. Exp. Zool. 232, 573580 (1984).
21. Follett, B. K. & Nicholls, T. J. Influences of thyroidectomy and thyroxine replacement on
photoperiodically controlled reproduction in quail. J. Endocrinol. 107, 211221 (1985).
22. Dawson, A. Thyroidectomy progressively renders the reproductive system of starlings (Sturnus
vulgaris) unresponsive to changes in daylength. J. Endocrinol. 139, 5155 (1993).
23. Dawson, A. thyroidectomy of house sparrow (Passer domesticus) prevents photo-induced testicular
growth but not the increased hypothalamic gonadotrophin-releasing hormone. Gen. Comp.
Endocrinol. 110, 196200 (1998).
24. Dawson, A. & Thapliyal, J. P. Avian Endocrinology (eds Dawson, A. & Chaturvedi, C. M.) 141151
(Narosa, New Delhi, 2001).
25. Nicholls, T. J., Follett, B. K., Goldsmith, A. R. & Pearson, H. Possible homologies between
photorefractoriness in sheep and birds: the effect of thyroidectomy on the length of the ewes breeding
season. Reprod. Nutr. Dev. 28, 375385 (1988).
26. Yoshimura, T. et al. Molecular analysis of avian circadian clock genes. Mol. Brain Res. 78, 207215
(2000).
27. Tagawa, M. & Hirano, T. Presence of thyroxine in eggs and changes in its content during early
development of chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 68, 129135 (1987).
28. Ikuta, K., Aida, K., Okumoto, N. & Hanyu, I. Effects of sex steroids on the smoltification of masu
salmon, Oncorhynchus masou. Gen. Comp. Endocrinol. 65, 99110 (1987).

Supplementary Information accompanies the paper on www.nature.com/nature.

Quantification of T3 and T4

Acknowledgements We thank Nagoya University Radioisotope Center for use of its facilities. We
also thank K. Aida, M. Tagawa and A. Munakata for providing antiserum, and A. Nishimura for
technical assistance. This work was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN), a Grant-in-Aid for Encouragement of Young
Scientists (to T.Yo.), and a Grant-in-Aid for Scientific Research (to S.E.) from the Ministry of
Education, Science, Sports and Culture.

Thyroid hormones in the brain were extracted with ethanol as described27,28.

Concentrations of T4 and T3 in the brain and plasma were determined by
radioimmunoassay as described previously27,28.

Competing interests statement The authors declare that they have no competing financial
interests.

Intracerebroventricular infusion
Animals were raised under short day conditions to an age of 8 weeks. T3 (T-2877, Sigma)
and T4 (T-2376, Sigma) were dissolved in a solution containing 0.001 M NaOH and 0.9%
NaCl. Iopanoic acid (I 0300, Tokyo Kasei) was dissolved in 0.05 M NaOH and 0.2 M HCl
was added. Intracerebroventricular infusion was carried out using an ALZET 2002
osmotic mini-pump with a brain infusion kit according to the manufacturers instructions
(ALZET). Animals infused with iopanoic acid were transferred into long day conditions 2
days after the beginning of the 2-week infusion period. Testicular size was measured before
and 3 weeks after the beginning of infusion. Placement and patency of the canula were
verified by injecting Evans blue dye after the experiment, as suggested by the manufacturer.
Received 14 July; accepted 25 September 2003; doi:10.1038/nature02117.
1. Sharp, P. J. & Follett, B. K. The effect of hypothalamic lesions on gonadotrophin release in Japanese
quail (Coturnix coturnix japonica). Neuroendocrinology 5, 205218 (1969).
2. Davies, D. T. & Follett, B. K. The neuroendocrine control of gonadotrophin release in Japanese quail.
I. The role of the tuberal hypothalamus. Proc. R. Soc. Lond. B 191, 303315 (1975).
3. Ohta, M. & Homma, K. Detection of neural connections to the infundibular complex by partial or
complete hypothalamic deafferentation in male quail. Gen. Comp. Endocrinol. 68, 286292 (1987).
4. Juss, T. S. in Avian Endocrinology (ed. Sharp, P. J.) 4760 (Soc. Endocrinol., Bristol, 1993).
5. Konishi, H., Foster, R. G. & Follett, B. K. Evidence for a daily rhythmicity in the acute release of LH in
response to electrical stimulation in the Japanese quail. J. Comp. Physiol. A 161, 315319 (1987).
6. Ohta, M., Wada, M. & Homma, K. Induction of rapid testicular growth in quail by phasic electrical
stimulation of the hypothalamic photosensitive area. J. Comp. Physiol. A 154, 583589 (1984).
7. Meddle, S. L. & Follett, B. K. Photoperiodic activation of Fos-like immunoreactive protein in neurons
within the tuberal hypothalamus of Japanese quail. J. Comp. Physiol. A 176, 7989 (1995).
8. Meddle, S. L. & Follett, B. K. Photoperiodically driven changes in Fos expression within the basal
tuberal hypothalamus and median eminence of Japanese quail. J. Neurosci. 17, 89098918 (1997).
9. Silver, R. et al. Coexpression of opsin- and VIP-like-immunoreactivity in CSF-contacting neurons of
the avian brain. Cell Tissue Res. 253, 189198 (1988).
10. Yasuo, S., Watanabe, M., Okabayashi, N., Ebihara, S. & Yoshimura, T. Circadian clock genes and
photoperiodism: Comprehensive analysis of clock genes expression in the mediobasal hypothalamus,
the suprachiasmatic nucleus and the pineal gland of Japanese quail under various light schedules.
Endocrinology 144, 37423748 (2003).
11. Follett, B. K. & Sharp, P. J. Circadian rhythmicity in photoperiodically induced gonadotrophin release
and gonadal growth in the quail. Nature 223, 968971 (1969).
12. Kuenzel, W. J. & Masson, M. A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus) (Johns
Hopkins Univ. Press, Baltimore, 1988).

NATURE | VOL 426 | 13 NOVEMBER 2003 | www.nature.com/nature

Correspondence and requests for materials should be addressed to T.Yo.

(takashiy@agr.nagoya-u.ac.jp).

..............................................................

APL regulates vascular tissue

identity in Arabidopsis
Martin Bonke1*, Siripong Thitamadee1*, Ari Pekka Mahonen1,
Marie-Theres Hauser2 & Yka Helariutta1
1

Plant Molecular Biology Laboratory, Institute of Biotechnology, POB 56,

FIN-00014, University of Helsinki, Finland
2
Center of Applied Genetics, BOKU - University of Natural Resources and Applied
Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria
* These authors contributed equally to this work
.............................................................................................................................................................................

Vascular plants have a long-distance transport system consisting

of two tissue types with elongated cell files, phloem and xylem1.
Phloem has two basic cell types, enucleate sieve elements and
companion cells. Xylem has various lignified cell types, such as
tracheary elements, the differentiation of which involves deposition of elaborate cell wall thickenings and programmed cell
death14. Until now, little has been known about the genetic
control of phloemxylem patterning. Here we identify the
ALTERED PHLOEM DEVELOPMENT (APL) gene, which encodes
a MYB coiled-coil-type transcription factor that is required for

2003 Nature Publishing Group

181