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Division of Neurophysiology (C.M., L.M., N.B., D.F.C., A.K.S., K.E.M., I.C.A.F.R.), National Institute for Medical Research Mill Hill, London NW7 1AA, United Kingdom; Department of Human Anatomy and Genetics (H.C.), University of Oxford, Oxford OX1 3QX, United Kingdom; and INSERM U-469 (L.C., X.B., P.M.), Montpellier 34094, Cedex 5, France
Address all correspondence and requests for reprints to: Professor Iain C. A. F. Robinson, Division of Neurophysiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. E-mail: irobins{at}nimr.mrc.ac.uk
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Introduction |
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One way to achieve this is to use the intrinsically fluorescent reporter molecule, green fluorescent protein (GFP) (3), which when expressed from cell-specific promoters in transgenic animals, can identify specific cell types in situ (4, 5, 6, 7) and provides a fluorescent tag for their isolation and analysis, using fluorescence-activated cell sorting (FACS) techniques (8, 9). Because GFP fluorescence is often unaffected by fusion to other sequences, intracellular distribution and secretion events can also be visualized by tagging GFP with sequences that target it to different subcellular compartments (10, 11, 12).
In this study, we have targeted enhanced GFP (eGFP) to the secretory vesicles of pituitary GH cells in transgenic mice. By combining RIA with fluorescence and immuno-electronmicroscopic imaging of eGFP and performing calcium imaging in pituitary slices in situ (13), secretory processes may be now be studied in GH cell populations, at the single GH cell level and even at a subcellular level of resolution. Some of these results have been presented in preliminary form (14).
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Materials and Methods |
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).
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).
Construction of a GH-eGFP cosmid for generating transgenic
animals
A 40 kb (K2B) cosmid (15), containing the locus
control region (LCR) for the human GH gene was a generous gift from
Professor Nancy Cooke (Pennsylvania University). After reversing the
orientation of the insert of this cosmid (B2K), a unique
MluI site was introduced upstream of the coding region of
the hGH gene by PCR site-directed mutagenesis to alter the sequence at
-326 bp from 5'-CCACGT-3' to 5'-ACGCGT-3'. The
hGH gene sequences of this cosmid (cosGH.M) could then be excised as a
single MluI fragment (Fig. 1c) and replaced with the
MluI-linked GH-eGFP sequence to give cosGH-eGFP. The final
cosmid thus contained an approximately 40 kb insert containing the LCR,
5'and 3'untranslated sequences for the hGH gene driving expression of
the GH-eGFP fusion protein described above in p48GH-eGFP. Note that
intact hGH cannot be generated from this construct.
Cell cultures and production of stable GC cell lines
Reagents were from Sigma unless otherwise stated.
GC cells (17) were maintained in a complete medium
consisting of D-MEM, 15% horse serum, 2.5% FCS (PAA,
Weiner Strasse, Austria), 2 mM L-glutamine,
supplemented with penicillin, streptomycin and amphotericin. GC cells
(200,000 in a 60-mm culture dish) were transfected with 2 µg plasmid
DNA using Lipofectamine, (Life Technologies, Inc.)
following the protocols supplied by the manufacturer. Stably
transfected cells were selected for neomycin resistance by addition of
G-418, 250 µg/ml, for 21 days. Strongly eGFP-positive cells were
readily apparent under low power fluorescence microscopy.
Generation of transgenic animals
DNA of the cosGH-eGFP construct was digested with
NotI, the 40-kb insert purified by ultracentrifugation in a
5–20% salt gradient (18), and brought to a concentration
of 1–5 ng/µl with 0.5 mM EDTA, 1
mM Tris-HCl, pH 7.5. Transgenic mice were
generated by pronuclear microinjection of fertilized oocytes of
superovulated (CBa/Ca x C57Bl/10) mice followed by oviductal
transfer into pseudopregnant recipients (19).
DNA and RNA analysis of transgenic animals
Genomic DNA from tail biopsies was analyzed for transgene DNA by
standard Southern and PCR procedures. A PCR assay for the first intron
of hGH sequence present in the transgene was developed, using exonic
primers: forward: 5'-ACCACTCAGGGTCCTGTGGACAG.3' reverse:
5'-CCTCTTGAAGCCAGGGCAGGCAGAGCAGGC.3'), which amplified across the
intron. Thirty cycles of amplification were performed under the
following conditions: 94 C for 1 min, 60 C for 30 sec, and 72 C for 90
sec per cycle.
Total RNA from pituitaries was isolated by using the Trizol reagent as described by the manufacturer (Life Technologies, Inc.). For Northern analysis, RNA was electrophoresed in a 1.2% agarose gel containing 8% formaldehyde, blotted onto a N+ membrane (Amersham Pharmacia Biotech) and hybridized at 45 C in 5xSSC, 5x Denhardt’s solution, 50 mM phosphate buffer, pH 6.5, 0.1% SDS, salmon sperm DNA (250 mg/ml) and 50% formamide. Membranes were washed with 0.1 x SSC and 0.1% SDS at 65 C. A 700-bp XmaI-NotI fragment of pEGFP-N3 vector was radiolabeled by random priming (Prime-a Gene, Promega Corp.) and used as a hybridization probe for eGFP sequences.
Immunocytochemistry
Mouse pituitaries were fixed in 4% paraformaldehyde for 12
h, washed in acetone for 2 h, and embedded in paraffin wax. Tissue
sections (6 µm) were dewaxed in histoclear (National Diagnostics,
GA), taken through 100%, 70%, and 30% acetone for 20 sec each, and
then washed in distilled water. After incubation in a blocking solution
(20% normal goat serum, 5% BSA in Tris/HCl saline buffer) for 30 min
at room temperature, they were exposed to a monkey anti-rGH serum
(NIDDK, 1:2000 dilution) overnight at 4 C. Sections were washed and
then incubated with biotinylated goat antihuman antiserum (NIDDK, 1:200
dilution) for 30 min at room temperature. After washing, sections were
incubated with TRITC-avidin (Sigma, 1:1000) for 30 min at
room temperature. Finally, DAPI (Molecular Probes, Inc., 1
µg/ml) was added for 2 min to stain cell nuclei.
Electron microscopy
After initial fixation (2.5% glutaraldehyde in phosphate buffer
for 2 h then 0.25% overnight), pituitary segments were postfixed
in osmium tetroxide (1% wt/vol in 0.1 M phosphate buffer)
stained with uranyl acetate (2% wt/vol in distilled water), dehydrated
through increasing concentrations of ethanol (70–100%) and embedded
in LR Gold (London Resin Co., Reading, UK) or Spurr resin. Ultrathin
sections (50–80nm) were incubated at room temperature with a
polyclonal anti-GFP (1:300) followed by Protein A linked to 15 nm gold
(British Biocell, Cardiff, UK). Primary antibody incubations were for
2 h and secondary antibody incubations for 1 h and all
antisera were diluted in 0.1 M phosphate buffer containing
0.1% egg albumin. For control sections, the primary antibody was
replaced by an unrelated polyclonal antibody. After immunolabeling,
sections were lightly counterstained with lead citrate and uranyl
acetate and examined with a transmission electron microscope (JEM-1010,
JEOL, Peabody, MA).
FACS analysis
Ten pituitaries from GH-eGFP transgenic mice were gently minced
and then treated with collagenase (0.1 mg/ml) for 15 min at 37 C. DNase
(50 µg/ml) was added and incubated for a further 45 min. Dispersed
cells were pelleted by centrifugation and resuspended in FACS buffer
(10 g NaCl, 0.25 g KCl, 1.37 g
Na2HPO4, 0.25 g
KH2PO4, 1 g BSA, per
liter, pH 7.3), layered onto 4% BSA in FACS buffer in a 15-ml tube and
centrifuged for 5 min at 100 x g to remove cell
debris. Cells were gently resuspended in 0.5 ml of FACS buffer and
analyzed on a FACS Star Plus machine (Becton-Dickinson and Co., San Jose, CA) with WinMDI software, using the FITC
channel to gate for eGFP fluorescence. Aliquots of the starting cell
suspension, and cell pools sorted by eGFP fluorescence intensity were
collected and assayed for mouse GH (mGH) content.
GH release studies
Freshly dissected pituitary glands were placed in 2 ml Eagle’s
medium without glutamine, rinsed several times and then incubated for
2 h at 37 C with medium changed every 30 min. Following this
washout period, the pituitaries were incubated in 0.5 ml aliquots of
medium and exposed to 1 µg/ml hGRF 1–29
NH2(Bachem, Inc.), and after a
further 90-min recovery period, to 5 µg/ml hGRF 1–29
NH2. The medium was collected and assayed for GH
and eGFP contents by RIA (see below).
RIA
Mouse GH in pituitary or cell extracts was assayed by RIA as
previously described for the rat (20), using mouse
reagents kindly provided by NIDDK (Bethesda, MD). For eGFP a new RIA
was developed as follows: recombinant eGFP (CLONTECH Laboratories, Inc.), 5 µg, was radioiodinated with
NaI125 using the Iodogen method as previously
described (21), and purified by Sephadex G75
chromatography. For assay, 100 µl of iodinated eGFP (5–7000 cpm)
were mixed with 100 µl of tissue extract or standards (0.01–10 ng)
of recombinant eGFP and 100 µl polyclonal antibody against GFP
(Molecular Probes, Inc., Eugene, OR) at a dilution
of 1:500,000 for 16 h at room temperature. Bound and free
fractions were separated by addition of 2 vol 18% polyethylene glycol,
followed after 30 min by centrifugation. Radioactivity in the pellets
was determined by 
counting. The assay sensitivity was 10 pg
eGFP.
Cytosolic calcium imaging of pituitary tissue
The procedure was essentially as previously described (13, 22), but adapted for the mouse. Briefly, anterior pituitary
slices (150 µm) were prepared from 7- to 9-week-old male mice.
Because of the small size of the mouse pituitary gland, the tissue was
immobilized within a droplet of ultra-low temperature gelling agarose
(type IX-A, Sigma, St. Louis, MO) before cutting with a
vibrating blade microtome (Leica Corp. VT 1000S,
Leica Corp., Nussloch, Germany). Before recording, slices
were incubated with Ringer’s saline supplemented with essential amino
acids for 1–8 h in a humidified incubator (5%
CO2-95% O2). For
fluorometric calcium recordings, slices were loaded with the
membrane-permeable form of fura-2 (fura-2 AM, Molecular Probes, Inc.). An FITC cube was used to demarcate the boundaries of
eGFP-positive cells and thus generate a map of GH cells within each
field. A combination of a 380-nm excitation filter, a 430-nm dichroic
mirror, and a 480-nm barrier filter (Nikon, Paris, France)
was then used to monitor fura-2 emission. Only cells showing both eGFP
and fura-2 emission fluorescences were recorded. Fluorescent images
were taken with an intensified cooled charge-coupled device camera
(PentaMAX Gen Iv; Princeton Instruments, Trenton, NJ).
Camera acquisition rate was 20–100 msec per frame, and each pixel was
digitized at 12 bits. Images were acquired with Metafluor (Universal
Imaging Corp., West Chester, PA), and analyzed with Igor Pro 3.16
software (Wavemetrics, Inc., Lake Oswego, OR).
[Ca2+]i changes were
expressed as an –F/F0 ratio, where
F0 was the minimum fluorescence intensity
measured after off-line correction of the basal level and data
inversion (13).
Data analysis
Unless otherwise stated, data are shown as mean ±
SEM. Differences between groups were analyzed by Student’s
t test, with P < 0.05 taken as
significant.
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Results |
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, a and b) were
transfected into the GH-producing GC cell lines, and several stable
lines were established. Expression of eGFP in these cells was examined
by confocal microscopy. Both constructs produced brightly fluorescent
cells, but with a markedly different distribution of fluorescence (Fig. 2). The shorter construct, expressing
eGFP with only 8 amino acids of the GH signal peptide showed a
relatively uniform distribution of fluorescence throughout the cells,
whereas the longer construct expressing the entire signal peptide and
the first 22 residues of hGH fused to eGFP gave a punctate distribution
of fluorescence, consistent with a granular targeting of this
product.
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Northern analysis of pituitary RNA with an eGFP probe showed a single
abundant transcript of the expected size in the transgenic but not
wild-type mice (Fig. 3, A and B). RIA
showed that eGFP-immunoreactive protein was readily detectable in
extracts of pituitary glands from transgenic but not wild-type animals
(Fig. 3C). No eGFP expression was detected in other tissues examined
such as brain, kidney, spleen (not shown). Measurements of pituitary
mGH content in transgenic and wild-type mice showed a significant
reduction in GH stores in both male and female transgenic animals
compared with wild-type littermates, but this did not affect their
growth rates (Table 1), and the
transgenic animals appeared phenotypically normal.
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). As was observed in GC cells
transfected with the same construct, individual pituitary GH cells from
GH-eGFP transgenic mice showed a punctate distribution of eGFP
fluorescence when examined by scanning confocal microscopy (Fig. 4B). The cells showing eGFP fluorescence were compared with those
expressing GH, as identified by immunocytochemistry. Figure 4 shows
three-color confocal microscopy of a section of a GH-eGFP mouse
anterior pituitary (Fig. 4C) stained with an antibody to GH and
visualized with TRITC (Fig. 4D), and also stained with DAPI (Fig. 4E)
to visualize all cell nuclei. About half of the cells showed eGFP
fluorescence, and virtually all of these colocalized with GH
immunoreactivity, (Fig. 4F).
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), and no
specific labeling of any other structure was apparent.
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. Both eGFP
and GH were released in a highly parallel, dose-dependent manner in
response to this GH secretagogue.
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illustrates the results from an
experiment in which pituitaries from a group of 10 GH-eGFP mice were
isolated, the cells dissociated, and subjected to FACS. Strongly
fluorescing eGFP-containing cells could readily be separated, counted
and collected (Fig. 7A). Measurement of GH by RIA showed the strongly
fluorescent population (Fraction II) to be markedly enriched in GH
content compared with the unsorted cell suspension, whereas the
remaining cells (Fraction I) were depleted in GH (Fig. 7B).
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illustrates the
results of such an experiment in which spontaneous changes of
intracellular calcium concentration
([Ca2+]i) were recorded
from three fura-2-loaded cells identified as GH cells by their eGFP
fluorescence. Mouse GH cells showed spontaneous fast transient rises in
[Ca2+]i (time to
peak = 210 ± 29 msec, n = 24). All the GH-eGFP cells
displayed [Ca2+]i bursts,
but with different patterns. In some cells, bursts displayed a stepwise
onset followed by a high frequency spiking plateau phase (Fig. 8, cells
labeled 2 and 3) whereas in others, they showed an incremental rising
phase due to the summation of high frequency, low amplitude
[Ca2+]i transients (Fig. 8, cell labeled 1). All these patterns of
[Ca2+]i transients in
GH-eGFP cells were reversibly suppressed upon local application of a
Ringer’s saline containing 500 µM
Cd2+ ions (n = 13), suggesting that the
[Ca2+]i transients were
due to spontaneous Ca2+-dependent action
potentials (22).
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When expressed alone or with minimal N-terminal peptide extensions, eGFP pervades throughout the cytoplasm. However, targeting signals may be fused to GFP that direct localization of the fluorescent product to specific subcellular structures (10, 25, 26). In particular, GFP variants targeted to secretory vesicles have been used to follow the genesis, trafficking and regulated release from these organelles in endocrine cell lines (12, 27, 28). The hGH signal peptide (29) is sufficient to enable heterologous reporter sequences to be processed through the secretory pathway in cell cultures (30, 31). We fused eGFP sequences with those encoding the signal peptide and an additional portion of the N terminus of hGH, and found that the resulting fluorescent product was targeted to GH secretory vesicles, not only in cell lines but also in transgenic animals.
The inclusion of the additional N-terminal peptide was determined by several factors. With both constructs, the first intron of the hGH gene was included because this contains enhancer sequences that could be important for efficient transgene expression (32). This intron begins after the sequences encoding the first 3 residues of the hGH signal peptide, and to preserve the nucleotide sequence around the splice acceptor site, we also included sequences encoding the next 5 residues of the signal peptide from exon 2 before linking with eGFP sequences. We felt it unlikely that this short N-terminal octapeptide extension would alter the cytoplasmic fate of eGFP and so it proved when this was expressed in GC cells.
The construct that targeted eGFP to secretory vesicles included sequences encoding the entire hGH signal peptide and the first 22 residues of the N-terminal sequence of hGH. This was chosen as the product would include the two N-terminal histidine residues of hGH (18His and 21His), which contribute significant Zn2+ binding activity to hGH and which may be important for packaging of GH dimers and oligomers into secretory granules (33). Our data do not show whether these residues were important for granule packaging of eGFP or merely fortuitous, and a further series of constructs will be required to address this issue. One possibility is that the N-terminal GH sequences in this eGFP product interacted with rat or mouse GH sequences which facilitated copackaging in GC cells or in mouse somatotrophs. However, this cannot be the only explanation because the same product also gave granular staining when expressed in other secretory cell types (PC12 cells, unpublished results, hypothalamic GRF neurones (14)] that do not express endogenous GH.
Although minimal GH promoter sequences can express transgene reporters in somatotrophs, the intensity of expression is often low and variable. We used a much larger promoter including the entire LCR of hGH that reliably directs position-independent copy-number-dependent expression in the pituitaries of transgenic mice (15). This LCR contains several DNA elements, which are necessary for somatotroph specific expression (34, 35), so we made minimal changes to this cosmid, mutating 2 bp to generate a unique site into which the hGH-eGFP reporter could be cloned. As expected, this transgene achieved high-level specific eGFP transgene expression in pituitary GH cells, with no detectable expression in other pituitary cell types or in other tissues examined. Because a B-cell receptor subunit gene (CD79b) was recently discovered to be present within this hGH LCR (36), and thus present in our transgene, we specifically examined lymphocytes from GH-eGFP mice. No eGFP fluorescence was detected in B cells isolated from these transgenic animals, and FACS analysis showed no changes in their lymphocyte population (unpublished results).
Confocal and EM immunogold studies confirmed that the eGFP was localized in the large dense-cored granules in somatotrophs. Expression of eGFP was accompanied by a significant reduction in the total amount of GH stored in the pituitaries of transgenic animals but did not otherwise disrupt the normal morphology or function of somatotrophs. The reduced pituitary GH reserve was clearly sufficient to maintain an adequate output of GH in transgenic mice because their growth was unaffected. This reduction in GH stores could reflect competition between the GH-eGFP product and endogenous mGH for granule packaging although there was much less eGFP than mouse GH stored in the pituitary. Because eGFP RNA transcripts were abundant, we suspect that the subsequent packaging or storage mechanisms are less efficient for the GH-eGFP product than for mouse GH. The aggregation and packaging of proteins in dense-cored granules probably involves specific interfacial features of protein structure favoring oligomerization (33), and it is known that sequences in addition to the signal peptide are also required for efficient packaging of GH (37, 38, 39, 40).
The eGFP product was clearly targeted to the regulated secretory pathway because it was released in response to the specific GH secretagogue, GRF. Initial attempts to quantify this by measuring eGFP fluorescence in the media were unsuccessful due to the large dilution involved in incubation studies. However, development of a sensitive RIA for eGFP enabled us to show directly that the transgene product was secreted in response to GRF in a dose-dependent fashion, closely paralleling GH release from the same tissues.
FACS analysis and sorting of live or fixed pituitary cell types has been described previously, using antibodies to the specific hormones released (41, 42). The eGFP in transgenic pituitary cell isolates provided a strong endogenous signal for FACS sorting of live cells, and a population of strongly eGFP-positive GH cells could be isolated without the need for pretreatment of the cells with antibodies or permeabilizing agents. This provides a convenient method for rapidly estimating the number of GH producing cells in individual pituitaries, and for isolating viable populations of somatotrophs that can be studied in vitro, free from paracrine interactions with other hormone-producing cell types.
GH cells are excitable and show spontaneous [Ca2+]i transients that correlate with secretion, but the study of this is labor intensive because the individual responding cells must be identified and characterized, usually by immunocytochemistry, post hoc (13, 22). We show here that intracellular calcium can readily be monitored simultaneously in several preidentified GH cells, using dual wavelength imaging for eGFP and fura-2, and observed the rapid short-lived increases in [Ca2+]i that reflect the outcome of transient calcium entry during action potentials in these cells. Furthermore, this is the first report that mouse GH cells display spontaneous rhythmic bursts of [Ca2+]i similar to those that have recently been characterized in postimmunoidentified GH cells in rat pituitary slices (22). Previous studies have recorded from single neuronal cells identified by GFP expression (7, 24). However, multicell imaging is possible in acute pituitary slices from GH-eGFP mice, and we are using this approach to study the GH cell populations in different pituitary subregions in situ and whether they coordinate the timing of their responses to the entry or exit of secretagogues or inhibitors, to or from the glandular parenchyma.
Although not addressed in this study, the eGFP transgene product could also be used to monitor GH gene expression in vivo, assuming that the hGH LCR sequences respond to those physiological signals that regulate mouse GH. Quantitative imaging of GFP at the subcellular level in single cells is clearly possible (28), but there are some kinetic limitations to using this approach due to the time taken for newly synthesized GFP to fold into a fluorophore conformation, its half-life and its sensitivity to photobleaching. Newer variants of GFP, with a shorter half-life, or sensitive to calcium (43), membrane potential (44) or pH changes (45), are useful probes of many aspects of cell physiology. Combining these with the transgenic approach we describe opens the way for direct studies of these processes not only in single GH cells, but also in GH cell populations, following their responses to physiological signals in the whole animal.
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Acknowledgments |
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Footnotes |
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Received July 20, 2000.
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