Expression of the somatolactin  gene during zebrafish
embryonic development
Mauricio Lopeza, Gabriela Nicac, Patrick Motteb, Joseph A. Martiala, Matthias
Hammerschmidtc and Marc Mullera,*
a
Laboratory for Molecular Biology and Genetic Engineering, Institut de Chimie, Bât. B6,
Université of Liège, B-4000 Liège (Sart-Tilman), Belgium
b
Laboratory of Plant Cellular Biology, Bât. B22, University of Liège, B-4000 Liège (Sart
Tilman) , Belgium, and Center for Assistance in Technology of Microscopy (CATM), Bât.
B6, Université of Liège, B-4000 Liège (Sart-Tilman), Belgium
c
Max-Planck-Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany
* Correspondence: M. Muller
LBMGG, Inst. de Chimie, B6, (Sart-Tilman)
Université of Liège, B-4000 Liège, Belgium
Tel: +32 4 366 4437
Fax: +32 4 366 2968
E-mail: m.muller@ulg.ac.be
Keywords: somatolactin, pituitary, zebrafish, differentiation, Pit-1, Aal
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Abstract
Somatolactin (Sl) is a pituitary hormone closely related to prolactin (Prl) and growth hormone
that was until now only found in various fish species. We isolated the cDNA coding for
zebrafish Sl and we identified the gene encoding this hormone. We also obtained a 1 kb
genomic fragment corresponding to the sl upstream promoter region. Furthermore, the sl
expression pattern was examined during zebrafish embryogenesis using whole-mount in situ
hybridization. Sl mRNA is first detected in a single cell at the anterior border of the neural
plate starting at 23 hours post fertilization (hpf). Sl-expressing cells also express the
transcription factor pit1 and are located close to prl-expressing cells. Using combined
fluorescent in situ hybridization, we show that sl- and prl-expressing cells are clearly distinct
at 29 hpf. Starting at 30 hpf, the number of sl positive cells increases and their location
becomes more clearly distinct from lactotrope cells, in a more posterior position. At later
stages (48 hpf), sl expression was observed posterior to growth hormone expression, again in
a distinct cell type. We show that zebrafish mutants aal, as well as mutants in the pit1 gene,
are deficient in sl expression.
In conclusion, sl expression defines a new, additional cell type in zebrafish pituitary that
depends on pit1 and aal for its differentiation.
3
Present in all vertebrates, the various functions of the pituitary gland in diverse physiological
processes are mediated by specific hormones, each of which is produced and secreted by
specific cells that constitute the mature organ (Dasen and Rosenfeld, 1999). In mammals, the
complex network of morphogen gradients and the activation of a cascade of transcriptional
regulators ultimately directing the differentiation of the specific cell types in the mature gland
has been extensively investigated (Scully and Rosenfeld, 2002). In contrast, until now, few
studies directly addressed pituitary organogenesis during embryo development in teleosts. In
zebrafish, expression of the early pituitary marker lhx3 (lim3) was shown at the anterior
border of the neural plate as soon as 22 hours post fertilization (hpf) (Glasgow et al., 1997).
At this stage, pituitary precursor cells appear to be located in two laterally symmetrical
placodes in the anterior neural ridge, which later join to form the mature gland (Herzog et al.,
2003; Sbrogna et al., 2003). The requirement of Shh signaling for pituitary formation and cell
differentiation in zebrafish was shown (Herzog et al., 2003; Sbrogna et al., 2003); and several
zebrafish mutants deficient in pituitary formation have been described (Herzog et al., 2004a;
Herzog et al., 2004b; Nica et al., 2004). In the pit1 mutant, a defect in the Pit1 (Pou1f1)
transcription factor leads to the absence of cells expressing prl, gh and tsh(Herzog et al.,
2004b; Nica et al., 2004). In contrast, the aal mutant expresses prl but is deficient in gh,
tshand pituitary pomc (Herzog et al., 2004b).
In teleosts, an additional hormone was found to be secreted by specific cells present only in
the anterior pituitary from fishes (Kaneko, 1996). First described in 1990 (Ono et al., 1990),
this hormone was called somatolactin (Sl) due to its high sequence similarity to the pituitary
hormones prolactin (Prl) and growth hormone (Gh) (Forsyth and Wallis, 2002). Sl was since
described in many different fish species such as sole (Pendon et al., 1994), goldfish (Cheng et
al., 1997), eel (May et al., 1997), chum salmon (Takayama et al., 1991) or rainbow trout
(Yang et al., 1997). To date, no clear function could be assigned to the Sl hormone in the
various teleost species where it was extensively studied (Kaneko, 1996). However, a medaka
(Orizyas latipes) mutant deficient in proliferation and morphogenesis of chromatophore
pigment cells was recently shown to be mutated in the sl gene (Fukamachi et al., 2004).
Recently, the presence of two closely related sl genes was reported in zebrafish (Zhu et al.,
2004) and their differential expression in the adult pituitary was illustrated . Sl, more closely
related to sl genes described in many other fish species, is expressed in the pars intermedia as
expected, while the additional sl gene is expressed closer to the pars distalis. Here we
describe the cloning of a zebrafish sl cDNA, analysis of the sl gene including 1 kb of
4
promoter sequence and we present the detailed expression pattern of sl mRNA in wild type
and mutant zebrafish embryos.
1. Results and discussion
1.1.
Isolation and characterization of the zebrafish somatolactin  cDNA
In order to obtain zebrafish sequences corresponding to the somatolactin gene, the predicted
amino acid sequence (accession: U72940) for goldfish (Carassius auratus) somatolactin
(Cheng et al., 1997) was used to search the genomic sequence database from the ongoing
zebrafish genomic project (http://www.ensembl.org/). Three separate genomic regions were
identified, each presenting a high similarity to parts of the goldfish somatolactin cDNA.
Primers directed against the putative zebrafish 5’- or 3’- coding regions were used to perform
RT-PCR reactions on mRNA from 5 days post fertilization (dpf) zebrafish embryos. The
predicted 420 bp fragment was obtained, confirming that the identified genomic sequences
give rise to one single mRNA. The fragment was cloned and sequenced (accession:
AJ867249), the deduced amino acid sequence was highly similar to the recently reported sl
cDNA (Zhu et al., 2004). Only two nucleotides differed between the two sequences, leading
to amino acid changes Val(114) to Ala and Glu(192) to Gly in our sequence. Consistent with
the fact that the carp cDNA used for primer design was classified as sl(Zhu et al., 2004),
we thus obtained a cDNA coding for zebrafish sl.
1.2.
Isolation and analysis of the sl gene promoter
Analysis of the genomic sequence upstream from the 5’-end of the obtained cDNA revealed
the presence of a putative TATA-box at 63 bp upstream from the ATG. Primers were
designed in order to amplify approximately 1 kb of genomic sequence immediately adjacent
to the ATG codon. The obtained fragment was cloned and sequenced to confirm its identity
(accession: AJ867250). Sequence analysis of the zebrafish sl promoter region revealed, in
addition to the TATA-box, the presence of several sites for the pituitary-specific transcription
factor Pit1 (Pou1f1) and for the early pituitary marker Lhx3.
1.3.
Expression pattern of sl during zebrafish embryogenesis
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An antisense RNA probe was obtained corresponding to the 420 bp sl cDNA and used for in
situ hybridization on 3dpf zebrafish embryos. A clear signal was observed, located medially
at the base of the brain in a region corresponding to the pituitary at this stage (data not
shown). No signal was observed in control experiments using a sense probe.
No sl mRNA was observed during the first stages of embryo development until the end of
somitogenesis (data not shown). The earliest, weak sl expression was detected at 23 hpf in
the anterior ventral head region, corresponding to the pituitary anlage (Fig. 1A,B). Double in
situ experiments revealed that this expression was concomitant to the first expression of the
prolactin (prl) gene and that sl mRNA is present just posterior to prl-expressing cells. In a
frontal view (Fig. 1B), it appears that sl mRNA is observed in one single cell always located
on the left side of the embryo, while several prl-expressing cells are already present in a more
medial position. Single-cell expression was maintained until 29 hpf (Fig. 1D), while
additional sl-expressing cells appeared at 30 hpf (Fig. 1C; 2A-C). At these stages, the
existence of cells expressing both prl and sl could not be clearly ruled out by double in situ
hybridization; therefore a protocol for combined fluorescent in situ hybridization was set up.
Observation from a lateral view at the confocal microscope revealed that sl- and prlproducing cells are clearly in different focal planes (Fig. 1D,E and supplementary material
S1) and confirmed that the sl-expressing cell was distinct from early lactotropes. Double
labeling with pit1 (Nica et al., 2004) at 30 hpf revealed that sl expression occurs in the
posterior region of the Pit1 expression domain (Fig. 2A-C). At 33 hpf, sl mRNA is detected
in several cells intermingled with posterior early lactotropes, while at 35 (Fig. 1F-H) and 48
hpf (Fig. 1I-K) the sl expression domain is clearly posterior to the prl domain.
In order to determine the position of sl-producing cells relative to gh-secreting somatotropes,
we searched the zebrafish genome for gh genomic sequences using the known partial cDNA
sequences (accession AY286447.1) (Herzog et al., 2003). Using a specific primer pair
covering the presumed transcribed region, an RT-PCR reaction performed on mRNA from 5
dpf zebrafish embryos yielded a 735 bp fragment containing the entire zebrafish Gh coding
region and 103 bp of 5' and 3' non-coding region. (accession AJ937858). This highly specific
probe was used to perform double in situ hybridization experiments for gh and sl expression.
At 48 hpf, gh expression was observed in the medial zone of the pituitary anlage, as expected
(Herzog et al., 2003), while sl mRNA was detected in a more posterior position (Fig. 2D,E).
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Combined fluorescent in situ hybridization clearly revealed that sl-expressing cells are
distinct from gh-expressing somatotropes (Fig. 2F)
In conclusion, sl is expressed in specific cells in the developing zebrafish anterior pituitary
primordium, these cells appear to belong to the pit1-expressing lineage but are clearly distinct
from lactotropes or somatotropes.
1.4.
Expression of SL in pituitary mutants
In order to gain first insights concerning the regulatory factors involved in sl expression, we
investigated two different mutants deficient in pituitary organogenesis (Herzog et al., 2004b;
Nica et al., 2004). Embryos resulting from crosses between heterozygous pit-1 or aal mutant
parents were analyzed at several stages between 24 to 48 hpf. To be able to identify the
homozygous pit1 mutants, we tested the expression of sl and prl by double in situ
hybridization. Homozygous pit-1 mutant embryos were characterized by the absence of both
prl and sl expression as compared to their heterozygous or wild type siblings (Fig. 3E,F).
Similarly, homozygous aal mutant embryos (25%) lacked sl expression (Fig. 3A-D), while
prl expression was unaffected (Fig. 3A,B), indicating that both aal and pit1 genes are required
for sl expression.
In conclusion, we obtained a cDNA encoding zebrafish Sl and 1 kb of the upstream
regulatory region of the sl gene. Sl was first expressed at 23 hpf, concomitant with the
expression of prl, at the anterior border of the neural tube in a single cell. Only at around
30hpf do we observe several sl-expressing cells intermingled with lactotrope cells in the
anterior pituitary primordium. Combined fluorescent in situ hybridization enabled us to
exclude a possible co-localization of sl and prl-expressing cells. In 48 hpf embryos, the
expression domain of sl is posterior to that of prl and distinct from and posterior to that of
gh, consistent with its described expression close to the pars distalis in the adult (Zhu et al.,
2004). Analysis of the promoter region revealed the presence of several binding sites for the
pituitary-specific transcription factor Pit1 and double in situ hybridization revealed the colocalization of sl and pit1 expression. Pit1 was previously shown to activate trout and chum
salmon sl promoter/reporter constructs in transient expression experiments in cultured cells
(Ono et al., 1994; Yamada et al., 1993). We clearly show by analyzing pituitary gland mutants
that sl expression requires Pit-1 and the Aal factor. Thus, sl-expressing cells represent a
7
new, distinct pituitary cell type depending on the presence of Pit1 and Aal, similar to gh and
tsh-expressing cells (Herzog et al., 2004b). Although prl and sl start to be expressed at
similar stages of development, their genes are clearly differently regulated, as prl does not
require Aal for expression. Further work will be needed to understand the differentiation of
these three different cell types secreting the closely related pituitary hormones Prl, Gh and
Sl.
2. Materiels and methods
sl an gh cDNAs were obtained by RT-PCR on adult zebrafish brain RNA. We designed the
primers
(SLfor1:
5’-GTCAAACAGGACCTTCCTTTTGCG-3’;
SLfor2:
5’-
GTGTGGTGGTTCTGGTCTGC-3’ and SLrev1: 5’-GAGAAACGTCTGAATCTTGTG-3’)
according to the SL coding exons identified in the zebrafish genome database and primers
(GHfor:
5'-ACCAACCTTCAATCAAGAACG-3'
and
GHrev:
5'-
TGGTCTTAGATTTGCAGAAAAGG-3') covering the predicted transcribed region of the
zebrafish gh gene. Similarly, the sequence corresponding to the sl promoter was obtained by
PCR
on
zebrafish
genomic
DNA
TGGCAAATAATGTTGCTTGTTTTAAGC-3’
using
the
and
primers
SLpromfor:
Slpromrev:
5’5’-
GCGCAAAAGGAAGGTCCTGTTTGAC-3’. The cDNAs for zebrafish prl and pit1 were
previously described (Herzog et al., 2003; Nica et al., 2004).
Whole-mount in situ hybridizations were performed as described (Close et al., 2002;
Hauptmann and Gerster, 1994). For combined fluorescent in situ hybridization, the
digoxygenin-labeled sl probe was recognized by a anti-DIG antibody coupled to alkaline
phosphatase and revealed with Fast-Red (Roche, Mannheim, Germany) in 0.1 M Tris-HCl,
pH=8.2 until a clear signal appeared. The embryos were rinsed three times in PBST (0,8%
NaCl; 0,02% KCl; 0,1% Tween20, 20 mM phosphate; pH=7.3) and the fluorescein-labeled
prl (or gh) probe was recognized using a rabbit anti-fluorescein antibody (Molecular Probes,
Eugene.USA) in PBST, 6% BSA for 2 hrs at 23°C. After rinsing three times in PBST, a
second anti-rabbit antibody coupled to peroxydase (diluted 100-fold) was added; and the
green fluorescent signal was revealed by adding an Alexa488-labelled thyramid substrate
(Molecular Probes, Eugene, USA) according to the manufacturer's instructions.
For confocal analysis, images were acquired using a Leica TCS SP2 inverted confocal laser
microscope (Leica Microsystems, Heidelberg, Germany) equipped with one argon and two
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helium-neon lasers. Digitized images were acquired using a 10x (NA 0.4) or 63x (NA1.4)
Plan-Apo water-immersion objectives at 1024 x 1024 pixel resolution. The diameter of the
pinhole was set up equal to the Airy unit. Series of optical sections were carried out to analyse
the spatial distribution of fluorescence, and for each embryo, they were recorded with a Z-step
ranging between 1.0 and 3.0 µm. Images were acquired under identical conditions and we
ensured that the maximal fluorescence signal was not saturating the photomultiplier tubes
(PMT). For multicolor imaging, Alexa488 was visualized by using an excitation wavelength
of 488 nm and the emission light was dispersed and recorded at 500 to 535 nm. FastRed was
detected by using an excitation wavelength of 543 nm and the 488/543 dichroic mirror, and
the FastRed emission was dispersed and recorded at 595 to 650 nm. The acquisition was set
up to avoid any crosstalk of the two fluorescence emissions. Captured images were exported
as TIFF format files and further processed using Photoshop.
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Acknowledgements
This work is supported by the "Fonds de la Recherche Fondamentale Collective"; 2.4555.99,
the SSTC; PAI: P5/35 and the University of Liège; GAME project, and by FRFC grant
2.4542.00 and the Special Fund for Research to P.M. M.L. held a fellowship from the CGRI
and M.M. is a "Chercheur Qualifié du F.N.R.S."
10
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Fig. 1. Sl expression pattern during embryogenesis. WT zebrafish embryos at 23 hpf (A,B),
29 hpf (D,E), 30 hpf (C), 35 hpf (F-H) and 48 hpf (I-K). (A,C,H,J,K) lateral view, anterior to
the left; (B,F,G,I) frontal view, dorsal to the top; (D,E) lateral view, ventral to the top, anterior
to the left; corresponding to the orientation in supplementary material S1. The scale bars in
each picture correspond to 40 µm. Picture (B) corresponds to an assembly of two pictures: the
same embryo focused in the medial part on prl and sl signals, assembled with a picture of a
more anterior plane with eyes in focus to be able to spot the morphology in comparison with
the red and blue label. The arrows point at the sl label. Prl (in red) is expressed anterior to
sl(in blue) at 23 hpf (A), in several cells (B), while sl is detected only in one cell located
on the left side of the embryo (B). At 30 hpf, sl- and prl-expressing cells are intermingled
(C). (D) Prl and sl are expressed in different cells. Merger of red and green fluorescence in
confocal microscopy: A 29 hpf embryo was labeled by combined fluorescent in situ
hybridization: red fluorescence for sl and green for prl. (E) classical microscopy picture
showing the orientation of the embryo represented in (D) and in supplementary material S1,
lateral view, ventral to the top, anterior to the left. Note the red sl label. The square outlines
the enlarged portion in (D) and S1. (F-K) The blue label corresponds to sl, the red label to
prl. Sl expression increases at 35 hpf (F-H) through 48 hpf (I-K) and the expressing cells
move posterior relative to the prl domain.
Fig. 2. Sl expression pattern during embryogenesis. WT zebrafish embryos at 30 hpf (A-C)
and 48 hpf (D-F). (A-D) Lateral view, anterior to the left. (E-F) ventral view, anterior to the
top. Scale bars represent 40 µm. (A-C) Sl is expressed in several cells also expressing pit1.
The blue label corresponds to sl, red to pit1. (B) same emryo as in (A) after the red label was
washed out. (D-F) Gh and sl are expressed in different cells. (D,E) sl label is in blue and
Gh was revealed by using Fast Red, for visualization by classical microscopy (D) and by
fluorescence (E). Gh (in red) is expressed anterior to sl(in blue) (F) The same embryos were
analyzed by combined fluorescent in situ hybridization: red fluorescence for sl and green for
gh. Gh-expressing cells are clearly anterior to sl-expressing ones.
Fig. 3. Expression of sl requires Pit1 and Aal. In situ hybridization of wt and mutant 35 hpf
embryos. The red label corresponds to prl and blue to sl. The scale bar in (A) represents 40
13
µm (A) wt sibling expressing prl and sl (B) Aal homozygous mutant expressing only prl;
(C) and (D) the same embryos as in (A) and (B) are shown, after the red label was washed
out; (E) wt sibling and (F) Pit-1 mutant deficient in prl and sl. (A-F) Lateral view, anterior
to the left.
S1. Movie displaying successive optical sections assembled from the embryo shown in Fig.
1D,E; lateral view, ventral to the top, anterior to the left, moving from left to right.
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Figure 1
15
Figure 2
16
Figure 3