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Department of Anatomy, Shimane Medical University, Izumo 693-8501, Japan
Address all correspondence and requests for reprints to: Dr. Hiroki Otani, Department of Anatomy, Shimane Medical University, Izumo 693-8501, Japan. E-mail: hotani{at}shimane-med.ac.jp
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Introduction |
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To extend this technique to developmental endocrinology research, we established a method of coupling it with microinjection: exo utero transplantation of hormone-secreting cells into mouse embryos. Using this exo utero cell transplantation, the ectopic hormone-secreting tumor was induced in the embryo and caused corresponding endocrine consequences.
We initiated a series of our works on transplanting AtT-20 cells, an ACTH-secreting tumor cell line (9, 10), and analyzing changes in the fetal adrenal because we were particularly interested in the ontogenesis of the neuro-immuno-endocrine network and its possible relationship with systemic mammalian development. The hypothalamo-pituitary-adrenal (HPA) axis is a main part of the network, and the adrenal plays the pivotal role in the system. Secondly, experimental research on the fetal mouse adrenal has lagged behind that on the other species (11, 12, 13, 14, 15, 16, 17, 18) due to the difficulties of the direct approaches to such small fetuses. For example, whether and by what mechanisms ACTH or other POMC peptides regulate the mouse fetal adrenal remain unclear.
We therefore, using the exo utero system, transplanted AtT-20 cells into mouse embryos on E14, induced ectopic corticotropic tumors in embryos, and examined the corresponding cellular responses of the fetal adrenal.
We have demonstrated in a separate study using exo utero microinjection that the mouse fetal adrenal was apparently reactive to a single administration of ACTH-(1–24) from E14 to E15 (unpublished observations). The present study was designed to investigate further the reaction of the fetal mouse adrenal to continuous stimulation with overexpressed ACTH during mid- to late gestation.
The major mineralocorticoid and glucocorticoid of the adult rodent adrenal are aldosterone secreted from the zona gomerulosa and corticosterone secreted from the zona fasciculata and also zona reticularis. Aldosterone synthase cytochrome P450 (P450aldo) and cytochrome P45011ß (P45011ß) are the synthesizing enzymes for aldosterone and corticosterone, respectively. Although their localization has been examined in adult and fetal rat adrenals (19, 20, 21, 22), their expression in mouse embryos remains unknown. We therefore analyzed their localization in the mouse fetal adrenal and the possible changes after the AtT-20 cell transplantation.
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Materials and Methods |
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Preparation of AtT-20 cells
AtT-20 mouse anterior pituitary corticotropic tumor cells
(American Type Culture Collection, Rockville, MD) were grown in Ham’s
F-10 medium (ICN Biomedicals, Costa Mesa, CA) supplemented with 15%
horse serum and 2.5% FBS, pH 7.2, at 37 C in an atmosphere of 5%
CO2 in air at 100% humidity. The cells were used for RIA
and microinjection when the cell concentration was approximately
105 cells/ml.
ACTH secreted from the AtT-20 cells in the cultured medium was measured using RIA (see below) before transplantation. The AtT-20 cells secreted ACTH at 6018–8920 pg/105 cells·3 h in the medium.
Cell suspensions for transplantation was prepared as following. AtT-20 cells were collected with 0.01% EDTA in Ca2+/Mg2+-free Ham’s F-10 solution and concentrated to 3 x 106 cells/ml in Ham’s F-10 solution containing 1.2 mg/ml BSA.
Exo utero cell transplantation
Exo utero manipulation was performed as previously
described (2, 3). The pregnant mice on E14 were anesthetized with 70
mg/kg BW pentobarbital before the operation. After the abdominal wall
of the dam was incised, the developing embryos inside their
extraembryonic membranes were exposed by incising the uterine wall. In
each dam, three embryos on the left and three on the right side of the
uterine horns were designated as the experimental and control groups,
respectively, and all others were removed. The embryonic limb-bud was
selected as the target site of transplantation because of the
accessibility of an accurate location for microinjection and easy
examination of the results.
Under a microscope (OME-NA, Olympus, Tokyo, Japan) and with a 40- to 50-µm diameter pipette, 1.5 µl AtT-20 cell suspension were injected into the forelimb-buds of the experimental embryos; the same volume of Ham’s F-10 solution containing BSA was injected into those of the controls. A small amount of carbon ink (1:500 dilution) was added to each of the above liquids to confirm the injected site.
After suture of the abdominal wall and recovery on a plate warmed at 37 C, the dams were returned to the mouse room and kept until E16 or E18. The survival rate of the embryos after cell transplantation was greater than 85%. The mortality rate was almost the same as that of the controls and so was unlikely to be due to the effects of cell transplantation. Only living embryos were harvested.
On E16 or E18, the embryos were removed after the dams were anesthetized by ether and killed. Upon measurement of body weight and crown-rump length of embryos, bleeding was performed, and the organs were dissected for tissue preparations.
Immunohistochemical assay for transplanted AtT-20 cells
After excision, fixation in 4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4) overnight, and being processed
and embedded in paraffin, the limb-buds were serially cut into 5-µm
thick sections. Selected sections with carbon ink (injected marker)
were stained with hematoxylin and eosin (HE).
After examination under the light microscope, sections adjacent to those with putative tumor masses were stained immunohistochemically with an antiporcine ACTH antibody [a gift from Dr. Kinji Inoue, Faculty of Science, Saitama University, Urawa, Japan, and characterized previously (23)] using the standard avidin-biotin-peroxidase complex method. For further analysis, we used only embryos in which the tumors were confirmed.
Bleeding and RIA for ACTH and corticosterone
For ACTH assay, blood was collected at 1400 h on E16 and
E18. Under the microscope, embryos were bled by cervical veins and/or
heart puncture with a micropipette coated with EDTA. Because only a
limited amount of blood could come from a single embryo, the blood was
pooled from not less than eight embryos from several different litters
to minimize the artificial error and variation. Pooled blood was
immediately centrifuged. Plasma was frozen at -80 C until assay. RIA
of plasma ACTH was performed with a kit from Nichols Institute
Diagnostics (San Juan Capistrano, CA). The sensitivity of the assay and
the coefficient of variance were 1 pg/ml and 2.0% (n = 5),
respectively.
For corticosterone assay, blood was collected on E18 using the same method described above but with a micropipette without EDTA coating. Serum corticosterone was determined by RIA using a specific antibody raised against corticosterone-3-carboxy-methyl-oxime-BSA (Medical System Service, Kanagawa, Japan). The sensitivity and coefficient of variance were 0.01 ng/ml and 8.3% (n = 10), respectively.
Light microscopic and morphometric analyses
The left adrenal glands from embryos with transplanted tumor
cells (on E16, n = 12; on E18, n = 6) and controls (on E16,
n = 14; on E18, n = 7) were collected and fixed in Bouin’s
solution overnight, and then embedded in paraffin. Serial sections (5
µm) stained with HE were observed under a light microscope.
Morphometric analyses were performed with the investigator blinded to the groups. The sections were scanned with a CCD digital camera (Olympus CS520 MD, Tokyo Electric Industry, Tokyo, Japan). The analysis was made with NIH image analysis software in a Macintosh computer. Every fourth section of each gland was measured for areas of the total gland and each of the inner and outer zones of the cortical area (see Results for detailed description). Volumes of the gland and each zone were calculated from area x distance between sections. Concerning the measurement of the medullary area, it was designed to be measured along the outermost boundary of the concentrated part of the medullary tissue that includes some penetrating or interlocking strands of the cortical cell, as the immigration of the sympatho-chromaffin elements to the center of the gland is ongoing on E16, and the concentrated medullary tissue has no exclusive boundary from the cortical tissue even on E18. Cell density (number of nuclei per area) and average cell size were measured in five different regions of a defined area in the outer zone (area = 20–30 x 102 µm2), inner zone (area = 30–50 x 102 µm2), and medullary zone (area = 5–10 x 102 µm2) in five sections from each gland of control and AtT-20 group embryos.
Immunocytochemical detection for cell proliferation
Dams were given ip injection of 50 mg/kg BW
5-bromo-2-deoxyuridine (BrdU; Sigma Chemical Co., St. Louis, MO) 1
h before death. The right adrenal glands of AtT-20 group (n = 4 on
E16; n = 4 on E18) and controls (n = 4 on E16; and n = 5
on E18) were prepared in the same way as the procedure for ACTH
immunohistochemistry (see above). All of the glands were serially
sectioned at 5 µm. The staining was carried out with a monoclonal
anti-BrdU antibody (Dakopatts, Glostrup, Denmark) using the
avidin-biotin-peroxidase complex method (2).
To estimate the labeling index (the percentage of labeled cortical cells), three to five sections of each gland were selected at 50-µm intervals throughout the length covering all transverse sections containing the center of the medullary tissue. The nuclei were counted in the inner and outer zones of the cortex areas from the photomicrographs (x100). The labeling index was calculated from the ratio of BrdU-labeled nuclei to the total cell number.
Analysis of expression of corticosteroid-synthesizing enzymes
The expression and localizations of the
corticosteroid-synthesizing enzymes, cytochrome P450aldo and cytochrome
P45011ß, were examined immunohistochemically with the specific
antibodies for P450aldo (rabbit antirat P450aldo) and P45011ß (rabbit
antirat P45011ß; provided by Drs. Yuzuru Ishimura and Fumiko Mitani,
Department of Biochemistry, Keio University School of Medicine, Tokyo,
Japan) (19, 20, 21). Sections from the right adrenal glands were prepared
as described above for ACTH and BrdU immunocytochemistry. The
transverse sections containing the central medullary tissue were
selected and immunohistochemically stained with the improved method of
immunogold-silver staining (24) using the secondary antibody conjugated
with gold (Amersham International, Aylesbury, UK) and the silver
enhancing kit (British BioCell International, Cardiff, UK) and observed
under a MICROPHOTO-FXA microscope (Nikon, Tokyo, Japan). Only darkfield
micrographs are shown in the results, although brightfield examination
was also performed to correlate the expression pattern with findings
obtained by HE staining. The adult rat and mouse adrenals were first
examined using this method, and the expression pattern was confirmed to
be essentially the same as that in the adult rat reported previously
(19, 20, 21) (see Results) and was the same between these two
species.
Electron microscopic observation
The right adrenal glands (n = 2, for each of AtT-20 and
control groups on both E16 and E18) were dissected and fixed by
immersion in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4) and rinsed in 0.1 M
phosphate buffer containing 5% sucrose. The specimens were postfixed
in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and
embedded in epoxy resin. Ultrathin sections (70 nm) were double stained
with uranyl acetate and lead citrate and examined with a JEOL JEM-1200
EX electron microscopy (JEOL, Peabody, MA) operating at 80 kV.
Statistical analysis
The data from each of the groups were averaged. All data are
shown as the mean ± SEM. Data were statistically
analyzed with the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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, A and B). Tumor cells on both E16 and
E18 intensely immunoreacted to the ACTH antibody, although the reaction
on E18 was stronger than that on E16, suggesting the higher secretory
activity on E18 (see below).
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]. These findings indicated that
exo utero transplantation of AtT-20 cells induced productive
ectopic ACTH-secreting tumors in mouse embryos.
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for E16 and
Fig. 3E for E18). The outer zone just below the gland capsule consisted
of cell masses arranged in a glomerular-like appearance (Fig. 3, A and
E). The cells were small and compact with dark nuclei (Fig. 3, A and
E). In contrast, the inner zone, which was located adjacent to the
outer zone and met the medulla directly without any boundary connective
tissue, was composed of larger eosinophilic cells (Fig. 3, A and E).
The inner zone cells were arranged in a somewhat columnar- or
fascicular-like fashion, although not universally (Fig. 3, A and E).
Occasionally, cells of the inner zone extended or penetrated into the
medullary tissue (Fig. 3, B and F; see below). The inner zone consisted
of approximately 60% of the cortex on E16 and took approximately 70%
of that on E18 (immediately before term; Fig. 3, A and E; see below).
In the inner zone, however, we could not observe the corresponding
layers of the zona fasciculata and reticularis in adults (Fig. 3, A and
E). On E16, the immigration of chromaffin cells was still ongoing (Fig. 3A). In the cortical zone, widely dispersed islands of the medullary
cells were converging toward the center of the gland, although most
parts of the medullary tissue seemed already concentrated at the center
of the gland. On E18, its centralization had almost finished, and the
loose architecture of the medullary area in the center had formed (Fig. 3E). In our experiments, the sex differences in either normal
morphology or the posttransplantation changes were not found during
this period (data not shown).
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). On E18, besides the above-mentioned events, the
cortical cells showed a more finely vacuolated and foamy appearance
described for the zona fasciculata in adults (Fig. 3G). The cortical
cells markedly extended into the medulla to form penetrating or
interlocking strands in the medullary area (Fig. 3, G and H, see
below). Consequently, the medullary tissue demonstrated a pronounced
compact structure after transplantation (Fig. 3G) compared with the
loose one in the controls (Fig. 3, G vs. E). The volume of
the adrenal gland was significantly larger in AtT-20 cell-treated
embryos (Fig. 4A). This was due to a
volume increase in the inner zone (Fig. 4A). There was no significant
change in volume in the outer zone after cell transplantation (Fig. 4A). Accordingly, the volume proportion of the inner zone to the outer
zone increased significantly after cell transplantation (Fig. 4B). In
the inner zone, cell number per area (i.e. cell density) was
significantly less (Fig. 4C), whereas cell size was significantly
larger in the AtT-20 cell group than those in the controls (Fig. 4D).
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). Interestingly, the
labeling index in the outer zone was approximately 3- to 5-fold higher
than that in the inner zone on both E16 and E18 (Table 2; see
Discussion).
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Immunohistochemistry for steroidogenic enzymes
Weak expression of cytochrome P45011ß in the normal fetal inner
zone. When the fetal mouse adrenal was examined with the specific
antibodies for P45011ß and P450aldo (Fig. 3, I and J, shows
expression patterns in the adult mouse adrenal), the immunoreactivity
to P45011ß was predominantly observed in the cytoplasm of the
cortical cells in the layer corresponding to the inner zone (Fig. 3B
for E16 and Fig. 3F for E18). In controls, the intensity of
immunoreactivity was weak on both E16 and E18, albeit significantly
higher than background (Fig. 3, B and F). The staining was not
homogeneous in the cytoplasm and was more intense in cells near the
medullary area than in those close to the outer zone, so that the
boundary of the P45011ß-positive zone was not clear, especially on
E16, although it appeared to roughly correspond to the border between
the inner and outer zones by HE staining (Fig. 3, B and F,
vs. Fig. 3, A and E). Compared with those on E16, the
P45011ß-positive cells on E18 were more homogeneously distributed and
made up a greater proportion of the cortical area, corresponding to the
measurement results (Fig. 4A and Fig. 3, F vs. B). On both
E16 and E18, some immunopositive cells were scattered in the medullary
area, although they were few in number (Fig. 3, B and F). P450aldo was
not expressed in the adrenal cortex on either E16 or E18 (data not
shown; see below).
Strong induction of P45011ß and increase in serum corticosterone
after AtT-20 cell transplantation. After AtT-20 cell
transplantation, the staining became dramatically increased and
homogeneous compared with that in the controls on both E16 and E18
(Fig. 3, D and H). The thickness of the immunopositive zone increased,
with a sharp border that corresponds well with that between the inner
and outer zones by HE (Fig. 3, D and H vs. C and G). The
positive cells represented a fascicular-like arrangement in particular
on E18 (Fig. 3H). It was noticed that the positive cells extended into
the medullary area and were interlaced with the negative cells, which
was more obvious on E18 (Fig. 3H). In most of the positive cells,
the expression was throughout the cytoplasm, and the expression area
was significantly larger than that of the controls (Fig. 3, D and H
vs. B and F), which is compatible with the measured increase
in cell size (Fig. 4D). P450aldo was not induced in the adrenal cortex
on either E16 or E18 (Fig. 3K).
The level of corticosterone in the pooled sera after transplantation on
E18 was 422.5 ng/ml, approximately 4-fold higher than the control value
(130.0 ng/ml; Fig. 2B), which is in the same range with the previously
reported normal perinatal level (126 ± 5 ng/ml) (25).
Electron microscopy
Normal cortical cells on E16 and E18. The cells of the outer
zone on E16 were characterized by the elongated mitochondria with
vesiculo-lamellated or shelf-like cristae and less abundant smooth
endoplasmic reticulum (SER) and lipid droplets (data not shown). The
cells in the inner zone had nuclei similar to those in the outer zone,
but with relatively larger cytoplasm. They were characterized by the
scattered SER and numerous spherical mitochondria (Fig. 5A). The cristae in the mitochondria were
either flat lamellar or tubular or vesicular (Fig. 5A). Lipid droplets
were diffusely found, and some of them existed in clumps (Fig. 5A). On
E18, the organelles of the cell were generally more developed (data not
shown).
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). Cristae in the mitochondria were homogeneously
vesicular (Fig. 5B). The lipid droplets were sparse and closer to the
mitochondria (arrows in Fig. 5B). The tendency for changes
on E18 was similar to that on E16 (data not shown). |
Discussion |
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The changes in the adrenal in the present study occurred predominantly in the inner zone of the gland. The increase in gland volume appeared to be due to hypertrophy rather than hyperplasia of the adrenocortical cells in the inner zone. The present BrdU labeling analysis showed that ACTH did not stimulate proliferation of mouse adrenocortical cells in vivo at this stage. The mitotic response of the adrenal cortex to pituitary peptides during late gestation has been controversial. Recent studies on rat fetal (preterm stage) adrenal cells in primary culture has shown that ACTH has a biphasic effect on cell division, with an initial lag phase followed by a mitotic response (26, 27). However, in vivo data do not necessarily agree with this in vitro effect. Recent in vivo studies on adult rats suggested no proliferative effect of ACTH on the adrenal (28, 29), although a study on adult female rats suggested a stimulatory effect on the proliferative activity only after 4-day treatment (30). In fetal in vivo research, it was recently shown that the metyrapone-induced increase in ACTH did not alter the total cell number of the adrenal cortex in the fetal primate (13). Our data, the only in vivo data from fetal mice, showed that ACTH as well as possibly other peptides from the tumor cells (see below) had no mitogenic effect on the mid- to late gestation fetal mouse adrenal even after 4-day stimulation. This discrepancy from the results of the former studies may be attributed to the difference in the experimental systems. The adrenal in vivo may respond to a cocktail of signals in addition to the overexpressed ACTH, which is much different from the conditions of isolated primary culture. Further, the cellular mitotic response to signals could differ significantly depending on the developmental stage of the tissue.
P45011ß and P450aldo form corticosterone and aldosterone, respectively, and are expressed in a zone-specific manner in adult rats (19, 20) and mice (this report). The present study showed that E16 and E18 mouse adrenal expressed P45011ß weakly in the inner zone, whereas P450aldo was expressed in neither the inner nor outer zone, corresponding to the findings in rats (21). One of the most prominent findings in the present study is that AtT-20 cell transplantation caused the dramatic increase in the expression level of P45011ß in the inner zone cells, which became hypertrophic and arranged in a fascicular fashion, whereas no induction of P450aldo was observed in either the inner or outer zone. These findings and an increased serum corticosterone level on E18 after AtT-20 cell transplantation indicate that the inner zone is functional to synthesize corticosterone and responds well to ACTH at midgestation. Interestingly, the outer zone expressed neither P45011ß nor P450aldo. Recently, a novel cell layer that did not contain any steroidogenic enzymes was found between the zona glomerulosa and zona fasciculata of the adult rat adrenal cortex (20). Based on BrdU incorporation and pulse-chase studies, this cell layer was proposed to serve as the progenitor cell zone for the steroidogenic zones of the adrenal cortex (20). The present study has shown that the mouse fetal outer zone not only lacks steroidogenic enzymes, but also has a 3–5 times higher BrdU labeling index. This ratio is comparable to that between the newly identified cell layer and steroidogenic zones in adult rats (20), although the absolute BrdU labeling indexes are much higher in fetal stages, in particular on E16. These findings suggest that the mouse fetal outer zone also represents the progenitor zone or the origin of the adult progenitor cell layer.
By electron microscopic observation, active forms of the mitochondria and SER after cell transplantation were found in the adrenocortical cells of the inner zone. Enzymes of cortical steroid biosynthesis and elaboration are located in the SER and mitochondria (31, 32). At the cellular level, ACTH is known to stimulate several steps of the process of the steroid synthesis (33, 34). Cholesterol is the main precursor of adrenal corticosteroids and is mostly contained in the lipid droplets; cholesterol must move from the lipid droplets to the mitochondria to enter the steroidogenic pathway (31, 33). Therefore, the observed change in distribution of the lipid droplets and their closer contact with the mitochondria can be explained by the stimulation with ACTH of intracellular transport and utilization of stored cholesterol (31, 32, 33, 34).
The present electron microscopic findings together with the mature cell arrangement and cell hypertrophy in the inner zone determined by light microscopy, the induction of steroidogenic enzymes in the inner zone determined by immunohistochemistry, and the increase in the corticosterone level suggest that ACTH induces adrenal hyperfunction and promotes differentiation, in addition to growth, and therefore plays a key role in the development of the mouse adrenal during late fetal life.
Another problem that deserves some comment in this study is the pronounced interlocking or penetrating strands of the presumptive cortical cells in the medullary area, in particular on E18 after transplantation. With HE staining, an expansion of the inner cortical tissue was observed. The immunohistochemistry also showed that these corresponding cells or strands expressed P45011ß. Interestingly, it was shown that these interlocking cells were ACTH dependent. Whatever its identity, its corresponding part in adults is interesting. This reminded us of the X-zone in mice and the fetal zone in primates, both of which are the innermost cortical zones surrounding the medulla. It is said that the fetal zone, which developed in embryos and disappeared gradually postnatally, is present only in primates (35). The X-zone in mice developed from the fifth day after birth, although its location and fate are similar to those of the fetal zone in primates (36). Despite considerable literature on the mouse X-zone, there is still no clear description of the origin or precursor of this zone (37). Interestingly, the interlocking layer observed in the present study became pronounced and expanded after stimulation of ACTH on E18. This ACTH-dependent phenomenon is exactly characteristic of the fetal zone in primates during the prenatal period (13), and the significant response to ACTH mainly occurred in the inner zone in the present study. These two points implied that at least a part of the inner zone of the fetal mouse adrenal may be the X-zone or its precursor. A study to better understand the relation of the interlocking zone with the X-zone or with the fetal zone is currently underway.
There has not been any published experimental study on the regulation of the mouse fetal adrenal. In a separate study using exo utero microinjection of ACTH-(1–24), we have also demonstrated that ACTH regulates the adrenal development and differentiation of fetal mice from E14 to E15 (unpublished observations). The present study showed that fetal mouse adrenal reacted to continuous ACTH treatment from E16 to term. Taken together, the cytological response of the mouse fetal adrenal is largely consistent with those of sheep, primates (baboons), and other rodents, such as rats (10, 15, 16, 17). This suggests that mice, with their advantage of an established genetic basis, are a promising model to study the mammalian fetal HPA axis using the methods of molecular genetics.
As a cloned mouse pituitary tumor cell line that derived from a single cell, AtT-20 cells is known to mainly produce ACTH. However, AtT-20 cells also secrete a variety of POMC products, including ß-endorphin and ß-lipoprotein, as well as non-POMC peptides, albeit in relatively minor amounts (10, 38). Therefore, we cannot formally rule out the possible effects of these peptides. However, our separate study in which a single dose of ACTH-(1–24) was given to fetal mice demonstrated basically similar adrenal changes as the ones reported here, including adrenocortical hypertrophy and ultrastructural differentiation consistent with an increased capacity for steroidogenesis (unpublished observations). The effects observed in the present study can therefore be basically attributed to ACTH. To confirm this, further analyses of the serum levels of other AtT-20-derived peptides in the present system as well as single dose injection of these peptides are currently underway.
As experimental studies of the fetal adrenal demand direct approaches to fetuses and are difficult in embryos of small species, its history is relatively new, and the understanding is limited, especially in such small species of animals as mice. Inducing a corticotropic tumor by taking advantage of the exo utero system and immunohistochemical detection is a preferable method to study the regulation of the fetal adrenal in mice. Our study demonstrated that induction of ectopic corticotropic tumors by exo utero cell transplantation makes possible a new approach to the study of the roles of ACTH during the ontogeny of the HPA axis in postimplantation embryos of small species of rodents. The difficulty in approaching the embryos directly in utero for surgery has delayed studies on fetal endocrine problems, particularly in vivo, compared with those in other research fields. Extending exo utero cell transplantation to other hormone-secreting cell lines or to functionally or genetically modified cells should lead to new perspectives for understanding the roles of other endocrine organs and hormones in embryogenesis. Furthermore, the approach of transplanting these functional cells into embryos should be valuable to experimental interventions for congenital or genetic endocrine disorders. These include the experimental treatment of hormone insufficiency before birth or organ-specific gene therapy for congenital endocrine diseases.
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Acknowledgments |
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Received December 9, 1997.
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