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Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Flora de Pablo, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, C/Velázquez 144, E-28006 Madrid, Spain. E-mail: cibfp1f{at}fresno.csic.es
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Introduction |
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The list of signals preventing apoptosis, at least in vitro, includes growth factors well known for their regulatory actions in adult life that play important developmental roles as well (10, 18). Paradoxically, although insulin has been exploited over decades to maintain healthy cultures of almost all cell types (19), its recognition as a physiological growth factor is quite limited. A common interpretation of the growth effects of insulin has been that it mimics the closely related insulin-like growth factor I (IGF-I), acting through the IGF-I receptor, which is present in most tissues together with the insulin receptor (20, 21, 22, 23). Indeed, IGF-I usually shows higher potency in growth bioassays (for review, see Refs. 23 and 24) and has been found to be effective in suppressing apoptosis in a variety of serum-deprived cultured cells (25, 26). In adult vertebrates, insulin and IGF-I are synthesized as preproinsulin and prepro-IGF-I, subsequently processed, and secreted mainly as hormones by pancreas and liver, respectively. However, we have previously found by reverse transcription coupled to PCR (RT-PCR), that preproinsulin messenger RNA (mRNA) is developmentally regulated from 0.5 day (E0.5) to E2 of chick embryonic development, when prepro-IGF-I transcripts are undetectable (27). In addition, insulin has a general growth effect in the chick embryo during neurulation (27) and early organogenesis (28). Therefore, to better understand how insulin signaling affects early development, we undertook a detailed analysis of preproinsulin mRNA distribution, the effects of its protein translation product(s) on cell survival, and the receptor involved.
Here we show the wide distribution of preproinsulin transcripts by in situ hybridization from the primitive streak chick embryo, through neurulation and pancreatic organogenesis (E0.5–E2.5). Using antisense oligodeoxynucleotide (ODN)-mediated interference both in vivo and in vitro, we provide evidence that autocrine/paracrine insulin, signaling through the insulin receptor, modulates apoptotic cell death in the neurulating embryo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization
Chicken-specific RNA probes for in situ hybridization
have been previously described (30). Control probes were the sense
transcripts. The whole mount in situ hybridization protocols
for embryos with 33P-labeled probes or digoxigenin-labeled
probes have been described previously (27, 31). The embryos labeled
with digoxigenin riboprobes were visualized by the alkaline phosphatase
reaction product and photographed with a Wild-Leitz M400
photomacroscope. The embryos labeled with [33P]riboprobes
were apposed to Hyperfilm BetaMax (Amersham, Arlington Heights, IL) and
exposed for 2 months, and the autoradiographic image was scanned with
an Ektron 1412 camera (Bedford, MA) and processed with the NIH Image
software.
Synthetic oligodeoxynucleotides
To interfere with the synthesis of embryonic (pro)insulin,
15-base antisense (AS) ODNs were designed from two regions of the
chicken preproinsulin mRNA (positions -6 to 9 for ASA,
5'-GAGAGCCATGATGAG-3', and positions 164–178 for ASB,
5'-GCTCGACATCCCGTC-3') (32). To interfere with the insulin receptor, a
15-mer ODN was designed from positions 75–89 in the cloned tyrosine
kinase region of the chicken insulin receptor (IR-AS;
5'-GGTGAACGAATCGGC-3') (33). To interfere with the IGF-I receptor, the
ODN correspondent to the same region (33) was designed (IgfR-AS;
5'-AGTCAATGAGTCTGC-3'). These ODN receptors are 33.3% different. As
controls we used the sense sequences of ASA (SS; 5'-CTCATCATGGCTCTC-3')
and IgfR-AS (IgfR-SS; 5'-GCAGACTCATTGACT-3') and two 15-mer random
sequence ODNs with the same nucleotide composition as the ASA (RAN;
5'-TAACGGTAACGGAGG-3') and the IR-AS (IR-RAN; 5'-GACGGCAGTACGAGT). All
ODNs were synthesized as phosphorothioate derivatives by Oligos Etc.
(Wilsonville, OR).
The efficiency of the embryo uptake of ODNs was evaluated in stage HH10
embryos cultured (see below) in the presence of 7 x
105 cpm (
30 fmol) 33P-labeled ASB mixed with
12.5 nmol nonlabeled ASB. The ASB was labeled with
[
-33P]ATP using T4 polynucleotide kinase and a DNA
tailing kit (Promega, Madison, WI). After 8 h in culture, the
embryos were fixed in 4% (wt/vol) paraformaldehyde and 0.2% (vol/vol)
glutaraldehyde in 0.1 M phosphate buffer, pH 7.1, and
embedded in Historesin (see below). Sections, 25 µm thick, were
placed on slides and apposed to Hyperfilm BetaMax (Amersham, Arlington
Heights, IL) for 1 month.
Whole embryo cultures
Stage HH10 embryos were cultured as previously described (27)
with minor modifications. Briefly, the embryo and the extraembryonic
membranes immobilized by a nitrocellulose ring were transferred to a
well of a 24-well plate filled with 0.3 ml of a soft gel consisting of
basal medium (DMEM-Ham’s F-12 medium supplemented with 100 µg/ml
transferrin, 16 µg/ml putrescin, 6 ng/ml progesterone, 5.2 ng/ml
sodium selenite, and 50 µg/ml gentamicin; all from Sigma) containing
0.5% (wt/vol) low melting point agarose (Hispanagar, Burgos, Spain).
Where indicated, a drop of 25 µl of the corresponding ODN at an
initial concentration of 500 µM was deposited on the soft
gel before transferring the embryos. Different concentrations were
tested in pilot experiments to avoid toxicity. The wells were filled
with 0.3 ml basal medium containing 0.7% (wt/vol) methylcellulose
(Serva). Where indicated, purified chicken insulin (Litron Laboratory,
Rochester, NY) or recombinant human insulin (a gift from Eli Lilly Co.,
Indianapolis, IN) was added to the medium. Note that different
concentrations of each insulin were used because chicken insulin is
approximately 10-fold more potent than human insulin.
Metabolic radiolabeling and immunoprecipitation
Stage HH10 embryos, cultured as described above, but using
methionine- and cysteine-deficient DMEM (ICN), were supplemented with
1/10th of the basal medium and 40 µM ASA or ASB or SS
ODNs. After a 2-h incubation, 0.1 mCi [35S]cysteine (1196
Ci/mmol; ICN) was added, and the culture was continued for 4 additional
h. The pooled culture media of five embryos per treatment was subjected
to immunoprecipitation, essentially as previously described (34), with
7 µl protein A-purified antiinsulin Ig (lot 627, Department of
Pharmacology, Indiana University, Indianapolis, IN) or with antiinsulin
antiserum (Sigma). The antigen-antibody complex was then precipitated
with protein A-Sepharose suspension. The immunoprecipitated material
was analyzed by alkaline-urea PAGE and fluorography, as described
previously (34).
Oligodeoxynucleotide application in ovo
In eggs of the desired incubation time, a window was cut
laterally in the shell, and the developmental stage of the embryos was
assessed. Forty microliters of 300 µM of the indicated
ODN in the basal medium containing methylcellulose, as described above,
and 25 µg/ml cytofectin GS2888 (a gift from Gilead, Foster City, CA)
were applied over the embryo (for controls of cytofectin toxicity and
additional details, see 35 . The eggs were sealed with cello-tape
and incubated for 10 h at 38.2 C, and the embryos were processed
to quantify apoptotic cell death. No macroscopic malformations were
induced by any of the ODNs, and development progressed.
Cell dissociation and nuclear staining
At the indicated times, embryos treated in ovo or in
culture were fixed in 4% (wt/vol) paraformaldehyde in 0.1
M phosphate buffer, pH 7.1, for 1 h and washed twice
with PBS containing 3 mg/ml BSA. Embryos were then dissociated to
single cell suspension by incubation at 37 C with 50 U/ml type VII
collagenase (Sigma) and 1.5 mg/ml trypsin (Worthington Biochemical
Corp., Freehold, NJ) in PBS for 30 min, followed by passes through a
siliconized Pasteur pipette every 10 min. An aliquot of the cell
suspension (50,000 cells) was deposited on a slide using a cytospin at
700 rpm for 7 min. Cells were stained and mounted with 5 mg/ml
4',6-diamidino-2-phenylindole (DAPI; Sigma) and 1 mg/ml
o-phenylenediamine (Sigma) in glycerol-PBS (9:1).
Photographs were obtained with a Photometrics CE 200A (Photometrics,
Ltd., Tucson, AZ) CCD camera adapted to a Zeiss Axiophot microscope
(Zeiss, New York, NY).
Alternatively, cultured embryos were immediately fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.1, dehydrated in 98% ethanol and embedded in Historesin (Jung, Heraeus Kulzer, Heidelberg, Germany). Twelve-micron thick sections were stained and mounted as described for the dissociated cells. To quantify apoptotic cell death, fragmented pyknotic nuclei were assessed by epifluorescence microscopy with a Zeiss Axioscop.
End labeling of fragmented DNA
Fragmented DNA was stained either in cell suspensions, obtained
as described above, or in frozen sections of embryos from different
experimental treatments. Cells or tissue slices were deposited on
poly-L-lysine-coated slides, refixed in 4% (wt/vol)
paraformaldehyde in 0.1 M phosphate buffer, pH 7.1, for 15
min, permeated with 0.6% (wt/vol) Triton X-100 in 2 x SSPE,
acetylated, and dehydrated through graded ethanols. Preparations were
then incubated for 1 h at 37 C with 50 µl of a solution
containing biotin-16-deoxy-UTP and terminal deoxynucleotidyl
transferase, as indicated by the manufacturer (Boehringer Mannheim,
Indianapolis, IN). The reaction was terminated by a 2-h incubation in
2 x SSPE at 65 C. The end labeling with biotin-16-deoxy-UTP was
then visualized, after three washes in PBS, by incubation for 45 min at
room temperature with Cy3-streptavidin (Amersham) diluted 1:100 in 30
mg/ml BSA and 100 mM glycine in PBS. After extensive
washing in the dilution buffer and PBS, slides were counterstained with
DAPI and mounted. Photographs were obtained with a Bio-Rad MRC 1024
confocal microscope (Bio-Rad Laboratory, Richmond, CA).
DNA ladder assay
Chick embryos, either maintained in ovo or cultured,
were dissected in cold PBS, pooled by fours, and homogenized in 400
µl lysis buffer [10 mM Tris-HCl (pH 7.5), 1
mM EDTA, and 0.2% Triton X-100] for 15 min at 4 C. The
fragmented DNA was isolated from the intact nuclei as previously
described (36). The DNA was electrophoresed in a 1.5% (wt/vol) agarose
gel containing 0.1 mg/ml ethidium bromide and visualized under UV
light.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Low abundance preproinsulin mRNA was
already detectable in the primitive streak embryo (stage HH4–5; Fig. 1, A–C) throughout the embryonic area and, at a lower level, in the
extraembryonic area opaca (Fig. 1A). In the blastoderm, the signal was
slightly higher in Hensen’s node, the chick organizer, better shown
with the digoxigenin-labeled riboprobe (Fig. 1C). During early (stage
HH7–8; Fig. 1D-H) and late (stage HH10; Fig. 1I-M) neurulation,
preproinsulin mRNA was widely distributed (Fig. 1, D, F, I, and K), but
showed a rostro-caudal gradient, with higher expression in the cephalic
area. Insulin receptor mRNA showed a similar expression pattern (Fig. 1, H and M) coinciding with previously published
[125I]insulin binding distribution (21). In sections of
stage HH8–10 embryos, preproinsulin mRNA was found within each of the
three blastoderm layers, although it was more prevalent in the
neuroectoderm (results not shown).
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, N and O), a prominent level of expression of
preproinsulin mRNA was also found in the caudal part of the embryo,
coincident with the posterior neuropore and the secondary neurulation
area (sinus rhombolidalis). This caudal expression was
already present in the stage HH10 embryo (Fig. 1, I and K). Two spots
of high preproinsulin mRNA in the midpart of the stage HH15 embryo
(Fig. 1N) most likely correspond to the emergent dorsal and ventral
pancreatic buds (Ref. 37 and references therein).
Prepro-IGF-I mRNA was not detected during gastrulation (not shown) or
neurulation (Fig. 1J), in agreement with previous RT-PCR data (27),
while the signal that appeared rostrally in stage HH17 (Fig. 1P) was at
a similar level as its corresponding sense probe (result not shown). We
cannot exclude that a small population of cells may express
prepro-IGF-I mRNA in early organogenesis, not easily detectable by
in toto hybridization, because by RT-PCR there is detectable
expression in the head portion of the E3 embryo (27).
Biosynthesis of a preproinsulin gene-derived protein and its
inhibition by antisense oligodeoxynucleotide treatment of stage HH10
embryos in culture
A preproinsulin gene translation product was demonstrated in
cultured stage HH10 embryos by metabolic labeling with
[35S]cysteine and subsequent immunoprecipitation of the
radiolabeled proteins with antiinsulin Ig (Fig. 2, lane C). The nature of this product
and the effectiveness and specificity of the antisense ODN strategy
were simultaneously confirmed by comparing the effect of the
preproinsulin mRNA antisense (ASA and ASB) and sense (S) ODNs on the
biosynthesis of radiolabeled protein. The biosynthetically labeled and
secreted protein present in the medium of the control embryos, either
untreated (control) or S treated, was reduced to near undetectable in
the medium of ASA- and ASB-treated embryos (Fig. 2). This results
demonstrate that the radiolabeled immunoprecipitated protein
corresponds to a preproinsulin mRNA-derived translation product. Most
of the immunoprecipitable protein was secreted into the culture medium,
as in the embryo extracts, the antiinsulin Ig-immunoprecipitable
radioactive counts were too low for reliable detection (data not
shown). In contrast, total embryonic protein synthesis, as measured by
incorporation of [35S]cysteine into TCA-precipitable
material, was not affected by the treatments (data not shown),
confirming the specificity and absence of general toxicity of the
ODNs.
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Insulin receptor mRNA antisense oligodeoxynucleotide increases
apoptosis in the neurulating chick embryo in ovo
To understand the possible role of this widespread embryonic
preproinsulin mRNA, we focused on a process that might be occurring
broadly in the embryo, namely apoptotic cell death. Apoptosis was
visualized by the presence of cells with pyknotic nuclei, one of its
most characteristic morphological features, stained with DAPI either in
sections or in dissociated cells from the cultured embryos (Fig. 3C). In selected cases, apoptosis was
detected by end labeling of the fragmented DNA (Fig. 3, A, B, and D), a
basic feature of apoptotic cell death. Staining of nuclei with DAPI was
performed in all cases. Both techniques showed a remarkably matching
pattern (compare Fig. 3, C and D), although the end labeling was
visually more dramatic. Quantitation was usually performed in
dissociated cells (Fig. 3, C and D). In addition, cellular DNA cleavage
was visualized by the characteristic electrophoretic ladder of
nucleosome-sized DNA fragments (Fig. 3E).
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). As insulin receptors and IGF-I
receptors are both present at this embryo stage, an IGF-I receptor mRNA
antisense ODN (IgfR-AS) was also used to confirm the relevance of the
insulin signaling through the insulin receptor in apoptosis. IgfR-AS
only caused a small increase in the percentage of whole embryo
apoptotic cells, much lower than that produced by the IR-AS (Fig. 4).
Therefore, apoptotic cell death is modulated in the neurulating chick
embryo in vivo by signaling involving the embryonic insulin
receptor in either the typical or the atypical/hybrid form.
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). At all
times, apoptosis was lower when the basal medium was supplemented with
10-8 M chicken insulin. This effect was also
observed at the level of DNA cleavage, which decreased markedly after
treatment with 10-7 M human insulin (Fig. 3E).
Insulin, as the only growth factor added to the cultured embryo, was
able to prevent apoptotic death in 64% of cells that would otherwise
die after 10 h in culture (Fig. 5). This time point was chosen for
further studies.
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).
In both regions, insulin prevented 30–35% of apoptotic cell death
(note that this value is not comparable with that for whole dissociated
embryo, as only a few sections were evaluated) despite the fact that
some regional differences were found in the proportion of apoptotic
cells. In the neural tube, there were 1.4-fold more apoptotic cells in
rostral than in caudal sections. In the mesoderm, the caudal sections
showed 1.8-fold more apoptotic cells than the rostral sections (Table 1). These differences were found in embryos cultured in either basal
medium or insulin-supplemented medium, suggesting that there is a
developmental and a tissue-dependent regulation of apoptosis. End
labeling applied to sections allows characterization of the
distribution of apoptotic cells, opening the possibility of future
studies analyzing insulin dependence of defined cell populations. For
instance, at the level of the rhombencephalon, stained nuclei were
clearly predominant in the dorsal region, presumably premigratory
neural crest cells (Fig. 3A), whereas they were found scattered in the
somites (Fig. 3B).
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).
Two preproinsulin antisense ODNs (ASA or ASB), separately added to the
culture, significantly increased the percentage of apoptotic cells in
the embryos compared with that in the cultures receiving control ODN
treatments (RAN or SS; Fig. 6). The
interference with the insulin receptor mRNA by the corresponding
antisense IR-AS was slightly more effective than blockage with ASA or
ASB. The proportion of apoptosis was maximally increased to 50% over
the control value by simultaneously applying ASA and IR-AS combined
treatment (results not shown). The apoptosis induced by treatment with
ASA was reverted by the simultaneous addition of 10-8
M chicken insulin to the culture medium, a further
confirmation of the specificity of this antisense ODN effect (Fig. 6).
Thus, the endogenous (pro)insulin synthesized by the stage HH10 embryo
appears to be involved in the autocrine/paracrine modulation of cell
survival in culture by signaling through the insulin receptor.
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Discussion |
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Early embryonic preproinsulin and insulin receptor gene
expression
Insulin is the most widely used survival factor in cell culture
(19). The common interpretation of its beneficial effect is that
insulin, usually used in culture at micromolar concentrations, acts as
a mere surrogate of IGF-I. This might not be the physiological
situation at certain stages of development. Remarkably, insulin
receptors along with IGF-I receptors are widely present during
development in most tissues in many species, including mouse, rat,
chicken, Xenopus, and Drosophila (Ref. 44 and
references therein). Interestingly, concerning the ligands,
preproinsulin mRNA has been detected earlier than prepro-IGF-I mRNA in
at least two species, chicken (27) and Xenopus (45, 46). In
both species, preproinsulin transcripts were found in whole embryos by
RT-PCR during early neurulation, whereas IGF-I transcripts were
undetectable at that stage. These findings along with the expression of
preproinsulin mRNA in the developing nervous system in mouse (47) and
chicken (34) suggest an early developmental role of insulin or its
precursor, proinsulin (23). This idea is supported by the broad
distribution of preproinsulin mRNA reported here. In preliminary
localization analysis, preproinsulin transcripts were found in
ectoderm, mesoderm, and endoderm in neurulating chick embryos; these
were more abundant in the neural tube. This correlates with the finding
that insulin promoter-driven transgenes have been found to be expressed
in the mouse central nervous system (48). In fact, neural tube defects
were observed when the transgene was a toxigen for expressing cells
(49). The broad distribution pattern of preproinsulin transcripts,
which roughly coincides with the expression of the insulin receptor
mRNA and protein (21), suggests a ubiquitous autocrine/paracrine
function of (pro)insulin in early development.
Like other soluble growth factors expressed in minute amounts in early embryos (50), characterization of preproinsulin mRNA translation product(s) has turned out to be much more difficult than demonstration of its membrane receptors. Currently, the very low levels of the translated protein have precluded precise localization and full biochemical characterization. The primary translation product, proinsulin, the fully processed insulin, or even an intermediate or derivative may eventually be identified as the earliest active form in development. What is now proven in cultured stage HH10 embryos is the biosynthesis and secretion of a protein immunoprecipitated by antiinsulin Ig. Whether its molecular form is processed partially or completely, the insulin-related immunoactivity extracted from prepancreatic embryos (E2) is bioactive (37).
Endogenous (pro)insulin acting though the insulin receptor protects
from apoptosis in ovo and in a factor-deprived culture
In the neurulating chick embryo, specific interference with the
production of embryonic (pro)insulin and insulin receptor, applying
antisense ODNs both in vivo and in vitro,
provoked an increase in apoptotic cell death. When the embryo is
isolated from its egg reservoir of maternal factors, including insulin
(37) and IGF-I (51), and cultured in a chemically defined medium, there
is a progressive increase in apoptotic death. As postulated, lack of
signaling leads to apoptosis. Remarkably, chicken insulin alone at
10-8 M was able to attenuate apoptosis up to
64% in this deprivation model. Certainly, many other growth/survival
factors were absent from the culture, which makes this survival effect
even more significant. Applied in ovo, the insulin receptor
mRNA antisense ODN was much more deleterious than the IGF-I receptor
mRNA antisense ODN. This suggests that the embryonic insulin receptor
is involved in the survival signaling pathway. The fact that the IGF-I
receptor interference also has a small effect on apoptosis leaves the
possibility of hybrid or atypical receptor involvement open. Effects of
insulin in the modulation of apoptosis have been observed recently in
various cell types. Insulin at a high concentration (1 µg/ml; 0.16
µM) has been reported to protect chick embryo lens cells
from apoptosis in explant cultures (52). Similarly, a high insulin
concentration fully protects cultured rat kidney fibroblasts from
platelet-derived growth factor-stimulated apoptosis (53). In these
studies, the authors did not address the possible physiological
relevance of endogenously produced insulin. In addition, unfairly, in
reports in which the effects of insulin and IGFs are compared, high
purity recombinant IGF-I or -II, but lower purity, medium grade
insulin, are often used. In our view, a greater attention to purity,
stability, and species specificity of factors is required to draw
conclusions on the physiological effects of insulin vs.
IGFs. Certainly, IGF-I and IGF-I receptor are key regulators of embryo
growth in mice (54), whereas growth appears normal in the neonatal
insulin receptor null mutant mice (55). There may be species
differences in the relative roles of insulin and IGFs in embryo
development, and a compensatory mechanism may be more effective in some
of them. In this regard, the lack of a pancreas (56) or the lack of a
functional insulin receptor in humans (57) both cause severe neonatal
growth delay. It is also worth noting that in Drosophila,
insulin (58) and the insulin receptor (59, 60) are essential regulators
of embryo growth and nervous system development.
Recently, one of the most actively investigated aspects of apoptosis has been the role played by "death genes" vs. "survival genes" and the implication of the caspase family of proteases (11, 61). In preliminary experiments, using inhibitors of caspases, we found that these proteases are involved in early embryo apoptosis.
A number of cell processes, including cell proliferation and cell differentiation, have been shown to be stimulated by insulin and others factors of the family (for review, see Refs. 23 and 41). Underlying those effects, insulin may act as a survival growth factor and, due to a "community effect," help cells to perform any process with higher efficiency. This may explain, for instance, the proliferative/differentiative action of proinsulin, insulin, and IGF-I in chick neuroretina (34) (Díaz, B., F. de Pablo, and E. J. de la Rosa, unpublished observations). At present, however, it is unclear in most systems if these factors are specifically instructing subsets of cells to proliferate or differentiate or are exerting a prevalent protective effect through modulation of apoptotic cell death that could even overcome deprivation of other growth factors.
Cooperative and sequential actions of multiple growth factors, which are likely to be exquisitely fine-tuned in their expression, balanced by the constitutive cell death program orchestrate the precise developmental events. (Pro)insulin, at least in the avian embryo, is one of these signaling molecules that counteracts cell death.
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Acknowledgments |
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Footnotes |
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Received March 21, 1997.
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