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Distribution of the mRNAs encoding the thyrotropin-releasing hormone (TRH) precursor and three TRH receptors in the brain and pituitary of Xenopus laevis: effect of background color adaptation on TRH and TRH receptor gene expression.
Bidaud I
,
Galas L
,
Bulant M
,
Jenks BG
,
Ouwens DT
,
Jégou S
,
Ladram A
,
Roubos EW
,
Tonon MC
,
Nicolas P
,
Vaudry H
.
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In amphibians, thyrotropin-releasing hormone (TRH) is a potent stimulator of alpha-melanotropin (alpha-MSH) secretion, so TRH plays a major role in the neuroendocrine regulation of skin-color adaptation. We have recently cloned a third type of TRH receptor in Xenopus laevis (xTRHR3) that has not yet been characterized in any other vertebrate species. In the present study, we have examined the distribution of the mRNAs encoding proTRH and the three receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the frog CNS and pituitary, and we have investigated the effect of background color adaptation on the expression of these mRNAs. A good correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs, including the novel receptor subtype xTRHR3, were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was expressed selectively in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe. Adaptation of frog skin to background illumination had no effect on the expression of proTRH and xTRHRs in the brain. In contrast, adaptation of the animals to a white background provoked an 18-fold increase in xTRHR3 mRNA concentration in the intermediate lobe of the pituitary. These data demonstrate that, in amphibians, the effect of TRH on alpha-MSH secretion is mediated through the novel receptor subtype xTRHR3.
Fig. 1. In situ hybridization histochemistry showing the distribution
of TRH precursor mRNA in the brain and pituitary of Xenopus
laevis. A–F: Frontal brain sections from white-adapted frogs were
hybridized with an antisense TRH precursor riboprobe and exposed
onto Hyperfilm max for 8 days. The anatomical structures, identified
by microscopic analysis, are designated on the right hemisections.
G: A control section incubated with a sense riboprobe (right hemisection)
is compared with a consecutive section hybridized with the
antisense probe (left hemisection). The rostrocaudal levels of the
sections are indicated on the schematic representation of the brain.
Abbreviations as in Table 1. Scale bars 1 mm.
Fig. 2. Darkfield photomicrographs illustrating the localization of
TRH precursor mRNA hybridization signal in selected regions of the
brain of white-adapted Xenopus laevis. Brain sections were dipped
into Kodak NTB-2 liquid emulsion and exposed for 2 weeks. A: In the
medial septum (ms), a dense accumulation of silver grains was seen
over numerous cell bodies. B: In the anterior preoptic area (Poa), a
strong hybridization signal was visualized in cells lining the ventricle.
C: The highest density of silver grains was observed in the magnocellular
preoptic nucleus (Mg) and the suprachiasmatic nucleus (SC),
with dense accumulation of proTRH mRNA signal over cell bodies.
D: Control section, consecutive to that in C, incubated with a sense
riboprobe. E: In the ventral hypothalamic nucleus (VH), many cells
were densely covered by silver grains. F: In the mesencephalic tectum
(tect), scattered cells were strongly labeled. G: In the nucleus reticularis
medius (Rm), a moderate density of silver grains was seen over
a few cells. Scale bar 100 m.
Fig. 3. In situ hybridization histochemistry showing the distribution
of xTRHR1 mRNA in the brain and pituitary of Xenopus laevis.
A–G: Frontal brain sections from white-adapted frogs were hybridized
with an antisense xTRHR1 receptor riboprobe and exposed onto Hyperfilm
max for 2 weeks. H: A control section incubated with a sense
riboprobe (right hemisection) is compared with a consecutive section
hybridized with the antisense probe (left hemisection). See legend to
Figure 1 for other designations. Scale bars 1 mm.
Fig. 4. Darkfield photomicrographs illustrating the localization of
xTRHR1 mRNA hybridization signal in selected regions of the brain of
white-adapted Xenopus laevis. Brain sections were dipped into Kodak
NTB-2 liquid emulsion and exposed for 1 month. A: In the internal
granular layer of the olfactory bulb (igl), labeled cells were observed
along the lateralventricle (lv). B: A moderate hybridization signal
was seen in the striatum (Str) especially in the accumbens nucleus
(Acc). C: Control section, consecutive to that in B, incubated with a
sense riboprobe. D: The highest density of silver grains was observed
in the anterior preoptic nucleus (Poa), with a dense concentration of
xTRHR1 mRNA signal over cell bodies lining the ventricle. E: In the
nucleus of the paraventricular organ (Npv) and in the ventral hypothalamic
nucleus (VH), many cells showed a moderate density of
silver grains. F: A weak hybridization signal was detected in the
dorsal part of nucleus princeps of the trigeminal nerve (Vpr). 3v, Third
ventricle. Scale bar 100 m.
Fig. 5. In situ hybridization histochemistry showing the distribution
of xTRHR2 mRNA in the brain and pituitary of white-adapted
Xenopus laevis. A–F: Frontal brain sections from white-adapted frogs
were hybridized with an antisense xTRHR2 receptor riboprobe and
exposed onto Hyperfilm max for 2 weeks. G: A control section incubated
with a sense riboprobe (right hemisection) is compared with a
consecutive section hybridized with the antisense probe (left hemisection).
See legend to Figure 1 for other designations. Scale bars
1 mm.
Fig. 6. Darkfield photomicrographs illustrating the localization of
xTRHR2 mRNA hybridization signal in selected regions of the brain of
white-adapted Xenopus laevis. Brain sections were dipped into Kodak
NTB-2 liquid emulsion and exposed for 1 month. A: A moderate
hybridization signal was seen over cells scattered throughout the
medialpallium (mp). B: In the medialseptum (ms), many cells were
moderately labeled, although a few scattered cells showed strong
labeling. C: In the pars medialis (Apm) and pars lateralis (Apl) of the
amygdala, a high and moderate density of xTRHR2 mRNA, respectively,
was seen over several cell bodies. D: The highest concentration
of silver grains was observed in the dorsal part of the habenula (Hd),
whereas moderate labeling was seen in the ventral part (Hv). E: Control
section, consecutive to that in D, incubated with a sense riboprobe.
F: In the suprachiasmatic nucleus (SC), several cells were
strongly labeled. G: In the nucleus of the oculomotor nerve (III), a few
cells showed a moderate density of silver grains. lv, Lateralventricle.
Scale bar 100 m.
Fig. 7. In situ hybridization histochemistry showing the distribution
of xTRHR3 mRNA in the brain and pituitary of Xenopus laevis.
A–F: Frontal brain sections from white-adapted frogs were hybridized
with an antisense xTRHR3 receptor riboprobe and exposed onto Hyperfilm
max for 2 weeks. G: A control section incubated with a sense
riboprobe (right hemisection) is compared with a consecutive section
hybridized with the antisense probe (left hemisection). See legend to
Figure 1 for other designations. Scale bars 1 mm.
Fig. 8. Darkfield photomicrographs illustrating the localization of
xTRHR3 mRNA hybridization signal in selected regions of the brain of
white-adapted Xenopus laevis. Brain sections were dipped into Kodak
NTB-2 liquid emulsion and exposed for 1 month. A: A dense accumulation
of silver grains was seen throughout the internal granular layer
of the olfactory bulb (igl). B: In the anterior preoptic area (Poa), a
moderate hybridization signal was visualized around the ventricle.
C: In the suprachiasmatic nucleus (SC), intense labeling was observed
throughout the nucleus. D: Control section, consecutive to that in C,
incubated with a sense riboprobe. E: In the magnocellular preoptic
nucleus (Mg), a moderate hybridization signal was found over a number
of cell bodies. F: In the ventral hypothalamic nucleus (VH), a
moderate density of silver grains was seen in the area lining the
ventricule. lv, Lateralventricle. Scale bar 100 um.
Fig. 9. Darkfield photomicrographs illustrating the localization of
TRH precursor and TRH receptor mRNAs in the pituitary of whiteadapted
Xenopus laevis. A: A moderate density of proTRH mRNA was
observed in the distal lobe (pd), but no hybridization signal was seen
in the intermediate lobe (pi). B: xTRHR1 mRNA was not present in
the distal lobe or in the intermediate lobe of the pituitary. C: Moderate
expression of xTRHR2 mRNA was seen in the distal lobe but not
in the intermediate lobe. D: xTRHR3 mRNA was virtually absent in
the distal lobe, although a strong hybridization signal was observed in
the intermediate lobe. Scale bar 20 um.
Fig. 10. Quantitative analysis of proTRH and xTRHRs gene expression
during background adaptation. A: Expression of proTRH
mRNA in the anterior preoptic area (Poa) and the magnocellular
nucleus (Mg) in white- and black-adapted Xenopus laevis (open bars
and solid bars, respectively). B: Expression of xTRHR1, xTRHR2 and
xTRHR3 mRNAs in the anterior preoptic area in white- and blackadapted
Xenopus laevis. C: Expression of proTRH and xTRHR2 mRNAs
in the pars distalis (pd) and xTRHR3 mRNA in the pars intermedia
(pi) of the pituitary in white- and black-adapted frogs. Results
are expressed as the mean optical density (O.D.) SEM. The O.D.
values were obtained from three white- and three black-adapted animals.
Statistical significance was determined with Student’s t-test
(***P 0.001; n.s., not statistically significant).
Fig. 11. RT-PCR analysis of xTRHR3 and GAPDH mRNAs in the
neurointermediate lobe of Xenopus laevis. Total RNA was reverse
transcribed, and the cDNAs obtained were amplified by PCR using
specific primers for xTRHR3 and GAPDH to generate products of 110
and 230 bp, respectively. The experiment was performed on tissues
from black (B)- or white (W)-adapted frogs. M, molecular weight
markers.
