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Previous Article | Next Article 
Molecular and Cellular Biology, January 2001, p. 663-677, Vol. 21, No. 2
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.2.663-677.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Loss of Orphan Receptor Germ Cell Nuclear Factor Function Results
in Ectopic Development of the Tail Bud and a Novel Posterior
Truncation
Arthur C.-K.
Chung,
Deborah
Katz,
Fred A.
Pereira,
Kathy J.
Jackson,
Francesco J.
DeMayo,
Austin J.
Cooney,* and
Bert W.
O'Malley
Department of Molecular and Cellular Biology,
Baylor College of Medicine, Houston, Texas 77030
Received 30 August 2000/Returned for modification 13 October
2000/Accepted 26 October 2000
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ABSTRACT |
The dynamic embryonic expression of germ cell nuclear factor
(GCNF), an orphan nuclear receptor, suggests that it may play an
important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the
GCNF gene in mice. Germ line mutation of the
GCNF gene proves that the orphan nuclear receptor is
essential for embryonic survival and normal development.
GCNF
/ embryos cannot survive beyond 10.5 days
postcoitum (dpc), probably due to cardiovascular failure. Prior to
death, GCNF/ embryos suffer significant defects in
posterior development. Unlike GCNF+/+ embryos,
GCNF/ embryos do not turn and remain in a lordotic
position, the majority of the neural tube remains open, and the hindgut
fails to close. GCNF/ embryos also suffer serious
defects in trunk development, specifically in somitogenesis, which
terminates by 8.75 dpc. The maximum number of somites in
GCNF/ embryos is 13 instead of 25 as in the
GCNF+/+ embryos. Interestingly, the tailbud of
GCNF/ embryos develops ectopically outside the yolk
sac. Indeed, alterations in expression of multiple marker genes were
identified in the posterior of GCNF/ embryos, including
the primitive streak, the node, and the presomitic mesoderm. These
results suggest that GCNF is required for maintenance of somitogenesis
and posterior development and is essential for embryonic survival.
These results suggest that GCNF regulates a novel and critical
developmental pathway involved in normal anteroposterior development.
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INTRODUCTION |
Germ cell nuclear factor (GCNF; also
called NR6A1) is an orphan member of the nuclear receptor gene
superfamily (12, 15). The nuclear receptor gene
superfamily includes a group of ligand-dependent transcription factors
that bind to steroids and other lipophilic molecules, such as retinoic
acid, which function to regulate many types of differentiation,
homeostasis, and developmental processes (11, 23, 38). In
addition, several members of this superfamily are orphan receptors for
which ligands have yet to be identified (23, 56). Homologs
of mouse GCNF have been cloned from several other species, including
human, Xenopus, and zebrafish (9, 30).
GCNF is an orphan nuclear receptor that specifically binds to a novel
nuclear receptor response element known as a DR0 (5, 12, 16,
70), a direct repeat of the estrogen receptor half-site sequence
(AGGTCA) with no nucleotides between the half-sites. Binding
to this element appears to be evolutionarily conserved, since
Xenopus GCNF has the same DNA binding specificity as mouse GCNF (30). Unlike many other orphan receptors, GCNF does
not heterodimerize with the retinoid X receptor; rather, it binds to
DR0 elements as homodimers (5, 8, 16, 70).
We and others have shown that GCNF is a transcriptional repressor
(5, 16). Like thyroid hormone receptor and chicken ovalbumin upstream promoter transcription factor, this repressor function was localized to the ligand binding domain, which is transferable to the Gal4 DNA binding domain (DBD) (4,
16-18); thus, in the absence of a ligand, GCNF represses target
genes. Among the candidate GCNF-responsive genes that have been
identified are two mouse genes, protamine 1 and protamine 2, which
contain DR0 elements within 400 bp of the transcriptional start site.
GCNF was initially described to be predominantly expressed in germ
cells of the adult mouse (12, 26, 34, 74) and human (1, 31, 35); further analysis revealed expression in
embryonic carcinoma cells as well (24, 35). In the mouse,
GCNF expression is detected in the embryonic ectoderm at 6.5 days
postcoitum (dpc) by section in situ hybridization (62);
after gastrulation at 7.5 dpc, it is expressed in all three germ
layers. GCNF expression is strong in the developing nervous system by
8.5 dpc but decreases significantly after 9.5 dpc. Studies of
Xenopus embryos have shown that the GCNF gene is
expressed between the gastrula and mid-neurula stages (19, 30,
57). Because of these significant patterns of expression during
gastrula and neurula stages of embryonic development and in specific
organs of the adult, GCNF may regulate multiple developmental
processes, particularly during embryonic development. Indeed, using a
dominant negative GCNF transcript, normal frog embryonic development
was disrupted (19, 30, 57). GCNF has also been shown to be
involved in midbrain-hindbrain development in Xenopus
embryos (19, 30, 57).
Recently, the targeted mutation of nuclear receptor genes in embryonic
stem (ES) cells has demonstrated that many orphan receptors are
essential for embryonic development (13, 27, 32, 33, 36, 37, 42,
47, 48, 51, 61, 74). To date, little is known of the role of the
nuclear receptors during the postgastrulation and neurulation stages.
Based on its embryonic expression pattern, GCNF is likely to play a key
regulatory role during this critical developmental period.
In this paper we describe the dynamic expression of GCNF in
postgastrulation and neurulation mouse embryos, showing that it is
regulated differently from the Xenopus GCNF expression. In addition, we describe the results of a germ line mutation of the GCNF gene. Interestingly, the GCNF null mutation
results in embryonic lethality, probably due to cardiovascular
complications. Prior to death, the GCNF/ embryos stall
in development, with open neural tubes, failure to turn, and an absence
of posterior ventral structures. GCNF/ embryos also
show a halt in somitogenesis after 13 somites, leading to a posterior
truncation. Interestingly, the tailbud and the posterior of the embryo
develop ectopically outside the yolk sac, a phenotype not previously
reported for any germ line mutation. We propose a mechanism to account
for the ectopic development of the tailbud. The posterior defects and
failure to turn may be explained in part by the deregulation of genes
required for normal somitogenesis and mesodermal development. These
data suggest that the GCNF signaling pathway is required during
postgastrulation and neurulation stages of mouse development.
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MATERIALS AND METHODS |
Construction of the GCNF targeting vector.
Genomic clones
containing GCNF sequences were obtained from a mouse genomic
library (129Sv) in the Lambda FixII vector (Stratagene) by using
GCNF cDNA as a probe (data not shown). The targeting vector
was constructed from two clones that overlapped with the exon encoding
the DBD. A 4-kb ApaI/EcoRV fragment of the
5'-most clone upstream of the DBD exon was subcloned into pBluescript KS (Stratagene). This plasmid was ligated with the 3-kb
ApaI/Acc65I fragment of a clone 3' downstream of
the DBD exon. After partial ApaI digestion, the resulting
vector was linearized between the two fragments, and
ApaI/XhoI adapters were ligated in place. The herpes simplex virus (HSV) tk gene from pSP72 (Promega),
lacking all sites of the polylinker except BamHI, was
created by digestion with XhoI, followed by filling-in of
the ends using the Klenow fragment of DNA polymerase I and further
digestion with BamHI and calf intestinal phosphatase
treatment. The two homologous arms, cut with BamHI and
EcoRV, were then ligated into pSP72 containing HSV
tk (39), thus placing the HSV tk
gene downstream of the 3' arm. The resulting vector was cut with
XhoI, treated with calf intestinal phosphatase, and ligated
with the 1.6-kb XhoI fragment of PGKneo (58),
thus adding the neo (neomycin resistance) gene between the
two homologous sequences. Restriction analysis was used to determine
the orientation in which the neo gene was inserted.
Generation of GCNF null heterozygous mice.
A clone with the
neo gene in the same orientation as the homologous sequence
was selected for targeting. This plasmid was linearized at a unique
XmnI site in the ampicillin resistance gene and
electroporated (Gene Pulsar; Bio-Rad) into AB 1.2 ES cells
(41) (see Fig. 2A). ES cells were maintained on STO
fibroblasts to retain their undifferentiated phenotype
(49). G418 and FIAU [1-(2'-deoxy-2'-fluoro-1-
-D-arabinofuranosyl-5-iodo)uracil]
(59, 60) drug selection began 2 days after electroporation
and continued for approximately 8 days, when 768 ES cell colonies were
picked and grown individually in 96-well plates.
The colonies were screened by Southern analysis after digestion with
enzymes Bsp106I and Acc65I, which produced a
21-kb band from the wild-type allele and a 9-kb band for the targeted
allele when a 700-bp SacI/AccI fragment, derived
from sequences 5' of the homologous arms, was used as a probe (see Fig.
2B). Additionally, the neo gene was used with the same
restriction enzyme digestion strategy to confirm that the correct
recombination event had occurred and there was only a single insertion
site. In this case, the targeted allele produced a 5.5-kb band.
Hybridizations and washes were performed as previously described
(34).
ES cell screening revealed six correctly recombined clones, which were
expanded and injected into C57BL/6 blastocysts to produce chimeric
mice. Four clones produced chimeric males that demonstrated germ line
transmission of the targeted allele. The embryos analyzed are the
F2 and F3 progeny from a mixed 129/C57 genetic
background. Similar phenotypes were obtained with homozygous mutant
embryos from the F1 generation of mice in an inbred 129Sv background.
Genotyping and identification of GCNF/
embryos.
DNA was extracted from tail samples, tissues samples
scraped from serial sections, or embryos. Genotypes of weaned mice or embryos were determined by Southern analysis using the strategy described above or by PCR analysis of DNA samples. Two separate sets of
primers were used. The primer set for the mutant allele (5'TCGATGCGATGTTTCGCTT3' and 5'ATATGGGATCGGCCATTGA3')
was derived from the sequence in the neo gene; the
primer set for the wild-type allele (5'CAGTGCTGACTTATCCATG3'
and 5'TTCCTGTTCATGCCCATCT3') was from sequences within
the DBD exon deleted in the mutated allele. PCR of the wild-type allele
produced a band of 239 bp, while PCR of the mutant allele produced a
band of 416 bp. In each of 30 cycles of PCR, DNA was denatured at
95°C for 3 min, primers were annealed at 58°C for 1 min, and
extension was carried on at 72°C for 1 min.
In situ hybridization and histological analysis.
Section in
situ hybridization of normal GCNF was performed with a
33P-labeled riboprobe of either the full-length
GCNF cDNA or the DBD cDNA as described previously
(34). Probes used for whole-mount in situ hybridization
(48) were cRNA probes for brachyury T (69),
HNF3 (2), Hoxb-13 (72), lunatic fringe
(29), mCer-1 (7), myogenin (54),
nodal (14), Otx2 (40), paraxis
(10), RALDH-2 (44), and Wnt-3a
(63). For histological analysis, the embryos were embedded
in 3% agarose, dehydrated, processed for paraffin embedding, sectioned
at 7 µm, and stained with hematoxylin and eosin.
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RESULTS |
Embryonic GCNF expression.
Using whole-mount in situ
hybridization, we examined expression of the GCNF gene in
mouse embryos between 7.5 and 10.5 dpc. A 3' untranslated fragment of
the murine GCNF cDNA was used to generate the antisense
probe to ensure specificity and lack of cross-reaction with other
members of the nuclear receptor superfamily. GCNF
transcripts were detected as early as 7.5 dpc in the anterior neuroepithelium of the head fold and throughout the primitive streak in
the posterior (Fig. 1A). At 8.5 dpc, GCNF
expression was stronger in the posterior of the embryo than in the
anterior (Fig. 1B). This result is consistent with the pattern of
expression found by Susens et al. for tissue sections
(62). Section in situ hybridization analysis also showed
that GCNF expression became significant in the posterior proliferating
neuroepithelium and in the underlying mesoderm of the primitive streak
by 8.5 dpc (62). GCNF expression was up-regulated again in
the anterior at 8.75 dpc (Fig. 1C) and down-regulated in the
neuroepithelium by 9.5 dpc. GCNF expression remained abundant in the
anterior but significantly decreased in the posterior (Fig. 1D).
Finally at 10.5 dpc, GCNF expression was markedly reduced, as
determined by either whole-mount or section in situ (not shown). Thus,
the dynamic expression of GCNF during the postgastrulation and
neurulation stages suggests that GCNF may be involved in several
different processes during early embryonic development.
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FIG. 1.
GCNF expression during postgastrulation and neurulation
stages. (A to C) Whole mount in situ hybridization of GCNF at 7.5 (A),
8.5 (B), 8.75 (C), and 9.5 (D) dpc.
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Targeted deletion of GCNF by homologous
recombination.
To determine the physiological role of GCNF in the
mouse, a targeted mutation of the GCNF gene was undertaken
using homologous recombination in ES cells. Genomic clones from the
GCNF locus were isolated from a mouse 129Sv library by
hybridization with the full-length mouse GCNF cDNA. The
initial screen produced three partial but overlapping clones that
covered approximately 40 kb of genomic sequence (Fig.
2A). The 5'-most cDNA sequence in these genomic clones was that of the DBD exon. Unlike most other nuclear receptor genes, both zinc fingers are encoded in a single exon.
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FIG. 2.
Targeted mutation of the GCNF gene. (A) Gene
targeting strategy. Approximately 40 kb of the GCNF locus is
shown, with exons depicted as solid boxes. The targeting vector with
homologous arms, neo gene, and tk gene are shown
relative to the genomic structure. The recombined locus is missing the
DBD exon. (B) Southern analysis to genotype ES cell clones and embryos,
using a 5' probe upstream of the targeting vector. Digestion with
Bsp106I and Acc65I yielded a 21-kb band for the
wild-type allele and a 9-kb band for the mutant allele. The presence of
a single integration was determined by using the neo probe
on the same digests to produce a 5.5-kb band. (C) PCR strategy used for
genotyping produced a 239-bp wild-type DBD exon band and a 416-bp
neo gene band. MW, molecular weight markers.
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To construct the targeting vector (see Materials and Methods),
sequences surrounding the zinc finger exon were subcloned to serve as
the homologous upstream (4 kb) and downstream (3 kb) arms (Fig. 2A).
The six positive ES cell clones identified (targeting efficiency of 1 to 2%) were injected into C57BL/6 blastocysts, and two produced male
chimeric mice that transmitted the mutant allele to their offspring.
The heterozygous offspring of chimeric males and C57BL/6 females were
mated to produce mice homozygous for the mutant GCNF allele.
At weaning, these progeny were screened to determine their genotype by
PCR analysis (Fig. 2C). None of over 500 mice screened were homozygous
for the mutant allele (Table 1),
indicating that the absence of a wild-type GCNF allele
caused neonatal or perinatal lethality.
To precisely determine when homozygous GCNF mutants die, heterozygous
females from timed matings with heterozygous males were sacrificed at
various days postcoitum, and the embryos were genotyped by PCR
analyses. At 7.5, 8.5, and 9.5 dpc, the ratios of wild-type, heterozygous, and homozygous mutant embryos were similar to the expected Mendelian ratios (Table 1). By 10.5 dpc, though, there were
fewer homozygous mutants than predicted. At 11.5 dpc, the homozygous
mutant tissues that could be genotyped were remnants of resorbing
embryos. Therefore, lethality due to the lack of an intact
GCNF gene occurred at midgestation, around 10.5 dpc.
To ensure that embryonic lethality was due to the targeted mutation of
GCNF leading to a loss of gene expression, we evaluated expression at 7.5 dpc by section in situ hybridization using an antisense probe to the DBD. GCNF was strongly expressed in all three
germ layers of the wild-type epiblast of GCNF+/+ embryos
(Fig. 3A), as well as in the
extraembryonic tissue of the ectoplacental cone. GCNF/
embryos, as determined by PCR of tissue scraped from serial sections, showed no positive hybridization signal, indicating that the
GCNF gene had been functionally disrupted (Fig. 3B).
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FIG. 3.
Analysis of GCNF expression and gross morphology of
GCNF/ embryos. GCNF is expressed in all
three germ layers (gl) and in the extraembryonic tissues of the
ectoplacental cone (epc) in GCNF+/+ embryos (A). No
positive signal was detected with the same DBD cDNA probe in
GCNF/ embryos, indicating that GCNF/
embryos do not express any GCNF transcript (B). (C) Gross
morphology of a 10.5-dpc GCNF+/+ embryo. (D)
GCNF/ embryo at 10.5 dpc, showing severe trunk and
posterior defects, open neural tube, failure of axis rotation, and
distended pericardium.
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Unlike wild-type embryos at 10.5 dpc (Fig. 3C), the
GCNF/ embryos were severely malformed, showing
trunk and posterior defects, open neural tubes, failure of axis
rotation and hindgut closure, and distended pericardia (Fig. 3D). The
GCNF/ embryos died probably as a result of
cardiovascular distress resulting from the defects in trunk and
posterior development. Similar phenotypes were observed in homozygous
GCNF/ embryos in two independent lines of founder mice.
Gross morphologic abnormalities in GCNF/
embryos.
At 8.25 dpc, no gross morphologic differences between
GCNF+/+ and GCNF/ embryos were evident
(data not shown); by 8.5 dpc, however, GCNF/ embryos
were distinguishable from GCNF+/+ embryos by several
morphologic criteria. One difference was the curvature of the spine
within the yolk sac, which was much wider in GCNF/
embryos than in GCNF+/+ embryos at 8.5 dpc (Fig. 4A and
B). In addition, the allantois, which was
often enlarged, was not always attached to the chorion. The lack of
proper chorioallantoic development probably also contributed to
embryonic lethality. Also at 8.5 dpc, a small protrusion of tissue
(Fig. 4B and D) was seen at the base of the allantois of GCNF/ embryos, which continued to develop outside the
yolk sac (Fig. 4F and J). Unlike GCNF+/+ embryos that
turned (Fig. 4G and K), GCNF/ embryos never turned and
remained in a lordotic position with open neural tubes (Fig. 4H and L).
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FIG. 4.
Gross morphology of GCNF/ embryos. (A
and B) GCNF+/+ (A) and GCNF/ (B) embryos
within the yolk sac at 8.5 dpc. Double-headed arrows indicate the
extent of the curvature of the ventral body. (C and D)
GCNF+/+ (C) and GCNF/ (D) embryos outside
the yolk sac at 8.5 dpc. A small protrusion of tissue was first seen at
the base of allantois of the GCNF/ embryo (B and D).
The brackets (B, D, F, and J) indicate a presumptive tailbud protruding
from the yolk sac of a GCNF/ embryo. (E and F)
GCNF+/+ (E) and GCNF/ (F) embryos within
the yolk sac at 8.75 dpc. (G and H) GCNF+/+ (G) and
GCNF/ (H) embryos outside the yolk sac at 8.75 dpc.
Unlike GCNF+/+ embryos, this GCNF/ embryo
does not turn and remains in a lordotic position. The tailbud lies
perpendicular to the anteroposterior body axis (H). (I and J)
GCNF+/+ (I) and GCNF/ (J) embryos within
the yolk sac at 9.5 dpc. (K and L) GCNF+/+ (K) and
GCNF/ (L) embryos outside the yolk sac at 9.5 dpc. The
GCNF/ embryo remains in a lordotic position with an
opened neural tube.
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At 9.5 dpc the GCNF/ embryos were clearly
developmentally malformed, with a significant reduction of trunk and
posterior structures. No more than 13 somites were observed in
GCNF/ embryos, instead of the 20 to 25 somites present
in GCNF+/+ embryos at 9.5 dpc (Fig. 4I to L). The posterior
of the GCNF/ embryos, including some somites, protruded
outside the yolk sac. When 9.5-dpc GCNF/ embryos were
compared to 8.75-dpc GCNF+/+ embryos with a similar somite
number, the continued growth of anterior neural tissue and tailbud was
clear. The gross morphology of mutant embryos suggested that growth had
continued beyond 8.5 dpc at the extreme anterior and posterior ends but
not in the intervening trunk region. Thus, development of
GCNF/ embryos was not completely halted.
Posterior morphogenetic defects in GCNF/
embryos.
Analysis of sagittal sections of GCNF+/+
embryos showed the position of yolk sac attachment around the node
(Fig. 5A); however, the yolk sac was
attached at the base of allantois in the GCNF/ embryos
at 8.5 dpc (Fig. 5B). In addition, the neural epithelium formed a large
invagination within the primitive streak, a phenomenon also seen in
cross sections (Fig. 5B and D).
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FIG. 5.
Posterior histologic defects in GCNF/
embryos. (A and B) Sagittal sections of GCNF+/+ (A) and
GCNF/ (B) embryos at 8.5 dpc. Arrows indicate positions
of yolk sac attachment to the embryos. Note that in the
GCNF/ embryo, the position of attachment is at the base
of the allantois (al). Arrowheads indicate the large invagination
formed by the posterior neural epithelium (ne) in the
GCNF/ embryo. Brackets (B and D) indicate the
region of the tailbud (tb). (C and D) Cross sections of
GCNF+/+ (C) and GCNF/ (D) embryos at 8.5 dpc. Both embryos contain open neural tubes at the anterior and
posterior ends. The arrowhead indicates the large invagination formed
by posterior neural epithelium (ne) in the GCNF/
embryo. (E and F) Dorsal views of GCNF+/+ (E) and
GCNF/ (F) tailbuds at 9.5 dpc. Arrows indicate the
presence in the GCNF+/+ tailbud (tb) but absence in the
GCNF/ tailbud of a posterior neuropore. (G and H) Cross
sections of tailbuds of GCNF+/+ (G) and
GCNF/ (H) embryos. The GCNF+/+ embryo
has an open neural tube (ne) on the dorsal side of the tailbud and a
hindgut (hg) located centrally, whereas the GCNF/
embryo has a closed neural tube (ne) in the middle of the tailbud. No
hindgut is seen, but the notochord (nt) lies ventrally to the neural
tube.
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At 9.5 dpc the posterior neuropore was still open in
GCNF+/+ embryos (Fig. 5E), and the neural tube was closed.
In addition, cross sections of 9.5-dpc GCNF+/+ embryos also
showed the hindgut situated in the middle of the tailbud (Fig. 5G). In
contrast, no posterior neuropore was observed in the tailbuds of
GCNF/ embryos (Fig. 5F), and the neural tube remained
open (Fig. 4L). Closure of the neural tube, however, was observed in
the middle of the ectopic tailbud (Fig. 5H); in addition, ventral
structures such as hindgut and ventral body wall were poorly developed
in the GCNF/ embryos. It is important to note
that the notochord was present throughout the length of the neural
tube (Fig. 5H) of GCNF/ embryos. In summary, the
tissues in the primitive streak and the GCNF/
tailbud were histologically disorganized, suggesting that loss of GCNF
function disrupted normal posterior development in the embryo.
Altered expression of anteroposterior genes in
GCNF/ embryos.
To examine more closely the extent
of the observed defects and to determine the molecular defects in
GCNF/ embryos, we probed mutant embryos with genes that
serve as markers for different structures involved in development at
this time. The first set of marker genes examined the gross development
of the GCNF/ embryos to determine which tissues were present.
First, the homeobox gene Otx2 was used to analyze anterior
development in GCNF/ embryos (55).
Otx2 was strongly expressed in the anterior neural folds,
with a sharp limit at the presumptive midbrain and hindbrain junction
in GCNF+/+ and GCNF/ embryos by 8.5 dpc
(Fig. 6A). This result implied that
anterior neural development had been initiated in GCNF/
embryos.
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FIG. 6.
Expression of anteroposterior marker genes in
GCNF+/+ and GCNF/ embryos. (A) Strong
Otx2 expression in GCNF+/+ (left) and
GCNF/ (right) embryos. (B) Detection of the
HNF3 probe throughout the notochord of both
GCNF+/+ (left) and GCNF/ (right) embryos.
Expression is also seen in the foregut (fg), (liver) (li), and hindgut
(hg) of the GCNF+/+ embryo. In contrast, only foregut
expression is seen in GCNF/ embryos. al, allantois; tb,
tailbud. (C) Hoxb-13 expression in the posterior end of both
GCNF+/+ (left) and GCNF/ (right) embryos.
(D) Wnt-3a expression in the extreme posterior end of the
tailbud of both GCNF+/+ (left) and GCNF/
(right) embryos. (E and F) GCNF/ embryo in the yolk sac
before (E) and after (F) hybridization with the brachyury T probe. (F)
Brachyury T expression in the tailbud (tb), which protrudes outside the
yolk sac. The high level of signal in the anterior portions of the
embryo is due to trapping within the yolk sac.
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Since some posterior structures developed poorly in
GCNF/ embryos, HNF3 was used as a marker
to analyze the development of the midline and ventral tissues.
HNF3 is expressed in the node, the floor plate, the
developing foregut, and the hindgut at 8.5 dpc (3, 53) and
in the primordial liver at 9.5 dpc (3, 43) (Fig. 6B). In
GCNF/ embryos, HNF3 expression was
clearly seen in the midline, the foregut, and in the tailbud; however,
there was no expression in posterior presumptive hindgut tissues. This
suggests that there is a lack of hindgut development in
GCNF/ embryos. Interestingly, there is also a loss of
anterior HNF3 expression, suggesting that the anterior
neural development is not entirely normal.
As a marker of midgestation posterior development, we used
Hoxb-13 because it is involved in patterning along the
anteroposterior axis of the mouse (10, 72).
Hoxb-13 is not expressed, though, until 9.0 dpc, after
turning is complete. In GCNF+/+ 9.5-dpc embryos (Fig. 6C),
expression of Hoxb-13 extends anteriorly from the tailbud.
Hoxb-13 was expressed in GCNF/ embryos, even
though the embryos had not turned (Fig. 6C). Interestingly, this
suggests that Hoxb-13 expression may be independent of
turning. Expression of Hoxb-13 was more limited in the
GCNF/ embryos, though, possibly due to the lack of a
hindgut and malformation of structures anterior to the tailbud. The
presence of Hoxb-13 indicated that certain aspects of
posterior development in the GCNF/ embryos had
progressed beyond 8.75 dpc, even though others, such as somitogenesis,
had not.
We analyzed the expression of Wnt-3a, which is expressed
in the tailbud, to ascertain that the extreme caudal portion of
the GCNF/ embryos is indeed the tailbud. A mutation of
Wnt-3a produces mice that are truncated in tissues posterior
to the forelimbs (63). In GCNF+/+ embryos,
Wnt-3a was expressed in the most posterior region, the tailbud (Fig. 6D). Interestingly, the GCNF/ embryos at
9.5 dpc had Wnt-3a expression at the tip of the presumed tailbud (Fig. 6D), consistent with the fact that the lack of GCNF did
not result in a loss of tailbud development and truncation of all
caudal tissues.
Since GCNF/ embryos have a reduced number of
somites, we examined development of the presomitic mesoderm (PSM).
Analysis of the PSM marker brachyury T (68) on embryos
within their yolk sacs showed the presence of PSM in the ectopic
tailbud (Fig. 6E and F). This result is consistent with the development
of some somites outside the yolk sac.
In summary, the expression patterns of these marker genes in the
anterior and posterior of GCNF/ embryos were similar to
those for wild-type embryos, indicating that certain aspects of
development of the mutant embryos had progressed beyond 8.75 dpc, even
though others, such as somitogenesis, had stalled. The defects in the
trunk of GCNF/ embryos were not the result of a
truncation of the entire posterior and might be due to molecular
defects in the primitive streak, the tailbud, or the node.
Altered differentiation of PSM in GCNF/
embryos.
At 9.5 dpc, the maximum number of somites in
GCNF/ embryos was 13, similar to the somite number of
GCNF+/+ embryos at 8.75 dpc. This suggests that GCNF might
affect somitogenesis after 8.75 dpc. Having demonstrated the presence
of PSM in the GCNF/ embryos (Fig. 6F), we proceeded to
determine the underlying molecular mechanism that resulted in a halt in
somitogenesis in GCNF/ embryos.
We first analyzed the extent of somite differentiation using
myogenin, a late myotome muscle marker (54). In
GCNF+/+ embryos, myogenin was expressed in all
differentiating somites by 9.5 dpc (Fig. 7A). In GCNF/
embryos, myogenin was expressed in the anterior seven
differentiating somites at 9.5 dpc but not in the posterior six somites
(Fig. 7B). Thus,
although somite differentiation had initiated in the GCNF/ embryos, it may have been delayed.
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FIG. 7.
Expression of marker genes of somitogenesis. Whole-mount
in situ hybridization was performed on GCNF+/+ (A, C, E, G,
I, and K) and GCNF/ (B, D, F, H, J, and L) embryos. (A
and B) Expression of myogenin in both GCNF+/+
(A) and GCNF/ is (B) only in the first seven
differentiating somites in GCNF/ embryos (arrowheads in
panel B). Paraxis is expressed in the somites of 8.5-dpc
GCNF+/+ (C) and GCNF/ (D) embryos, in
somites of a GCNF+/+ embryo at 9.5 dpc (E), and ectopically
at the posterior end (arrow) of the tailbud in the
GCNF/ embryo at 9.5 dpc (F). (G) mCer-1
expression in the two newly formed somites (arrows) of a
GCNF+/+ embryo. (H) Reduction of mCer-1
expression (arrow) in a GCNF/ embryo at 9.5 dpc. (I) Expression of lunatic
fringe in the first two somitomeres (arrows) and a broad swath of
cells (arrowhead) in the PSM of GCNF+/+ embryos. (J) In
GCNF/ embryos at 9.5 dpc, lunatic fringe is
expressed with equal intensity in the first two somitomeres (arrows) of
the PSM. Only a transient, faint expression of lunatic
fringe is found at the posterior end of the PSM (arrowhead). (K
and L) RALDH-2 expression in cervical mesenchyme, caudal
somites, and the cloacal region of a GCNF+/+ embryo (K) and
ectopic expression of RALDH-2 at the posterior end (arrow)
of the tailbud in a GCNF/ embryo at 9.5 dpc (L).
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We next used paraxis as a marker gene for the formation and
epithelialization of somites (10). At 8.5 dpc, we detected
paraxis expression in both newly formed and preexisting
somites of GCNF+/+ and GCNF/ embryos (Fig.
7C and D). Thus, formation of these early anterior somites was not
affected by the loss of GCNF expression. However, the intersomitic
boundaries of paraxis expression were not well demarcated in
GCNF/ embryos as in the GCNF+/+ embryos.
The failure to form well-defined epithelial somites in
GCNF/ embryos suggests that early somite formation may
be affected. Surprisingly, examination of paraxis expression
in GCNF/ embryos at 9.5 dpc revealed a separate ectopic
domain in the PSM, more posterior to its normal domain (Fig. 7I and J).
This result suggested that there was an altered differentiation of the
PSM within the ectopic tailbud, which failed to remain in an
undifferentiated state.
Mouse Cerberus-related-1 (mCer-1), which belongs
to the Cerberus/Dan-related gene family (46), was then
used to analyze early somite formation. Expression of mCer-1
is evident at the onset of gastrulation in the anterior visceral
endoderm (6, 7). From the early somite stage,
mCer-1 expression was observed solely in the first and
second newly formed somites of GCNF+/+ embryos (Fig. 7G),
as previously described (7). However, mCer-1 expression was greatly reduced in the newly formed somites in GCNF/ embryos (Fig. 7H), which suggests that the
formation of new posterior somites is affected at 9.5 dpc.
Since lunatic fringe, a gene that participates in the Notch
signaling pathway during segmentation and exhibits distinct dynamic expression during early embryonic development (21, 29), is required in the PSM for the formation of somites, we determined its
expression in GCNF/ embryos. At 9.5 dpc, lunatic
fringe was expressed weakly in the first presumptive somitomere;
however, it was strongly expressed in the second presumptive somitomere
within the PSM of GCNF+/+ embryos (Fig. 7I). In the
posterior-most PSM, lunatic fringe was expressed in a broad
swathe of cells. Unlike GCNF+/+ embryos,
GCNF/ embryos did not possess this broad posterior-most
expression domain of lunatic fringe in the PSM (Fig. 7J). In
addition, the levels of lunatic fringe expression in the
first and second presumptive somitomeres were often equal. These
results suggest that there are molecular defects in the PSM present in
GCNF/ embryos which likely affect the formation of new somites.
Further evidence that differentiation of the PSM was altered in
GCNF/ embryos came from analysis of the expression of
RALDH-2, an enzyme important for the generation of retinoic acid during
early embryonic development (44). Interestingly,
RALDH-2/ embryos (45), like
GCNF/ embryos, have open neural tubes and posterior
truncations. At 9.5 dpc, RALDH-2 was expressed within the
cervical mesenchyme, newer and more caudal somites, and the cloacal
region toward the base of the allantois in GCNF+/+ embryos
(Fig. 7K). In GCNF/ embryos, RALDH-2 was
expressed in the somites similarly to GCNF+/+ embryos, with
highest levels detected in the anterior 12 somites (Fig. 7L). However,
an unexpected ectopic expression of RALDH-2 was also
observed in GCNF/ embryos at the tip of the tailbud, in
a manner similar to the ectopic expression of paraxis at
this stage.
In summary, GCNF is not required for the early initiation,
epithelialization, and differentiation stages of somitogenesis since
genes such as myogenin and paraxis were
appropriately expressed up to 8.75 dpc. However, GCNF is required for
the continued development of somites since in its absence, there was an
altered differentiation of the PSM with ectopic expression of
paraxis and RALDH-2 and a reduction of
lunatic fringe and mCer-1. Inappropriate somite differentiation and altered potential of the PSM eventually led to a
halt in somitogenesis.
Persistent expression of nodal and brachyury T in nodes and PSM of
GCNF/ embryos.
Since the mouse node is also
crucial for mesoderm formation and recruitment of cells required for
somitogenesis, the next step was to determine whether the node was
functioning appropriately in GCNF/ embryos. The node,
which is located at the anterior end of the primitive streak, is
crucial for organizing and patterning the midline axis of the
developing embryo (14). Expression of nodal, a
transforming growth factor family member, is required for mesoderm
formation and axial rotation (14). In GCNF+/+
embryos, nodal was expressed around the node at the onset of gastrulation and during the early somite stages (Fig.
8A); however, its expression was
down-regulated after 9.0 dpc (Fig. 8C), as expected (67).
At 8.25 dpc, nodal was appropriately expressed at the node in
GCNF/ embryos (Fig. 8B); however, its expression was
not down-regulated even by 9.5 dpc (Fig. 8D). This persistent and
disorganized expression of nodal in the node is consistent with the
disorganization and altered differentiation of the PSM that resulted in
the halt in somite formation at later somite stages. This suggests that
the loss of GCNF function and ectopic development of the tailbud led to
a disruption of the normal function of the node.
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FIG. 8.
Expression of nodal and brachyury T in the node and PSM.
Whole-mount in situ hybridization was performed on GCNF+/+
(A, C, E, G, and I) and GCNF/ (B, D, F, H, and J)
embryos. By 8.25 dpc, nodal was expressed at the node (arrow) in both
GCNF+/+ (A) and GCNF/ (B) embryos. Nodal
expression (arrow) decreased in GCNF+/+ embryos at 9.5 dpc
(C), and persistent and disorganized expression (arrow) was found at
the node of GCNF/ embryos at 9.5 dpc (D). By 8.5 dpc,
brachyury T was expressed in the node (arrow) and the primitive streak
of both GCNF+/+ (E) and GCNF/ (F) embryos.
tb, tailbud. By 8.75 dpc, brachyury T is restricted to the PSM in
GCNF+/+ embryos (G) but is still expressed in the node
region (arrow) of GCNF/ embryos (H). Additional
expression domain (arrowheads) is observed between the base of the
allantois and the tip of the tailbud (tb). Like in embryos at 8.75 dpc,
brachyury T expression is observed in the PSM and notochord at the
posterior end of GCNF+/+ embryos at 9.5 dpc (I); in
addition to expression in the node (arrow), brachyury T has a V-shape
expression pattern in the tailbud (tb) of the GCNF/
embryo at 9.5 dpc (J).
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Since the development and differentiation of the node and PSM were
altered, the next question was whether the primitive streak was normal
in GCNF/ embryos. Brachyury T expression is found in
the primitive streak at 7.0 to 7.5 dpc and persists in the PSM as long
as new somitic mesoderm is being produced (68). At 8.5 dpc, brachyury T was expressed in the node and primitive streak in
GCNF+/+ embryos (Fig. 8E) and in GCNF/
embryos (Fig. 8F); however, the expression domain was broader in the
PSM of GCNF/ embryos than in the PSM of
GCNF+/+ embryos. At 8.75 dpc, the brachyury T expression
domain was significantly different in GCNF/ embryos
compared to GCNF+/+ embryos, although it was still
expressed in the node region (Fig. 8H). Brachyury T was expressed in a
V-shaped domain in GCNF/ embryos, compared to the
linear domain in GCNF+/+ embryos (Fig. 8H). At 9.5 dpc, the
expression of brachyury T persisted in the tailbud of
GCNF/ embryos (Fig. 8J) in a more exaggerated V shape,
while it was down-regulated in the PSM of GCNF+/+ embryos
(Fig. 8I). The persistent, disorganized brachyury T expression in the
tailbud of GCNF/ embryos further suggests that loss of
GCNF function resulted in an altered differentiation of the PSM.
| |
DISCUSSION |
Germ line mutation of the GCNF gene proves that the
encoded orphan nuclear receptor is essential for embryonic survival and normal development. GCNF/ embryos cannot survive beyond
10.5 dpc, probably due to cardiovascular failure. Prior to death,
GCNF/ embryos suffer significant defects in posterior
development, which are probably due to the loss of GCNF expression
within cells of the posterior. The defects observed are not due to the
neo cassette, as a GCNF knockout line with the
neo cassette deleted phenocopies the GCNF knockout described
here (data not shown). Defects in GCNF/ embryos may be
related to the retardation and/or halt in somitogenesis after 13 somites in mutant embryos and the abnormal positioning of the tailbud.
Indeed, alterations in expression of multiple marker genes were
identified in the posterior of GCNF/ embryos, including
the primitive streak, node, and PSM. These results suggest that GCNF is
required for proper anteroposterior development, somite formation, and
posterior development and that it is essential for embryonic survival.
The results of this study highlight some interesting similarities and
differences in how GCNF functions in amphibians and mammals (19,
30). First, GCNF is important for both amphibian and mammalian
embryonic survival. In Xenopus, the expression of a
dominant-negative form of GCNF results in malformations of
the head, eyes, ears, cement gland, and somites, resulting in embryonic lethality (19). Second, when wild-type GCNF is
overexpressed in Xenopus embryos, it causes
disturbances in somitogenesis and tail formation, with normal head
development (19). Finally, failure of neural tube closure
was also observed in Xenopus embryos after expression of the
dominant-negative GCNF (19). Together with the results
from our study, the findings indicate that the presence of GCNF is
critical for normal posterior development, somitogenesis, and neural
tube closure. Unlike the results of our study, expression of the
dominant-negative form of GCNF in more posterior regions of
Xenopus had no significant effect on posterior development.
Alternatively, severe posterior defects in Xenopus may have
been overlooked, as only embryos exhibiting less severe phenotypes were
analyzed (19).
The results presented here suggest that the lack of trunk development
in GCNF/ embryos is distinct from the effects of other
genes that have previously been shown to cause truncations of caudal
structures. For example, brachyury T/ embryos have
little or no notochord and have somites only up to the region of
forelimb buds, forming no trunk or tail (22, 71). The
posterior truncation is due to a failure to form mesoderm in
T/ embryos. Inactivation of the HNF3 gene
has demonstrated effects on notochord development (2, 20).
Although the expression pattern of HNF3 in
GCNF/ embryos is somewhat different from the wild-type
pattern, HNF3 is expressed in the midline structures,
such as the notochord and floor plate, of GCNF/
embryos. When Wnt-3a was mutated (63), the
resulting truncation occurred caudal to the forelimb buds.
Wnt-3a was expressed in the ectopic tailbud of
GCNF/ embryos, although forelimbs did not develop. In
contrast to Wnt3a/ embryos, which do not express
brachyury T, GCNF/ embryos do express brachyury T in
their tailbuds, indicating that these embryos possess a tailbud
structure similar to that of wild-type embryos. In addition, the
development of the GCNF/ tailbud progresses to the
expression of later-stage markers such as Hoxb-13. Hoxb-13 is not
expressed in the normal tailbud until 9.0 dpc; thus, its appearance in
GCNF/ embryos implies that the tailbud not only is
present but has also sustained some degree of normal development. Other
mouse models, such as paraxis, lunatic fringe,
and Mesp2, have less severe posterior defects (10, 52,
73). Taken together, these data suggest that GCNF may regulate a
unique signaling pathway distinct from the brachyury T, HNF3,
Wnt-3a, and Hoxb-13 pathways.
The initiation and differentiation of the first seven to nine somites
are not affected by the absence of GCNF. Previous fate mapping studies
suggest that by the late streak stage, the bulk of prospective cranial
mesoderm has already exited the streak (64, 66). The cells
in the anterior segment of the primitive streak at this stage are
mainly destined for the first 6 to 10 somites and the PSM of the
early-somite-stage embryo, a finding which was confirmed by ablation
experiments (65). Thus, GCNF may not be required for the
initiation of primary body formation or participation in somite
formation during the early stages of development. The expression of
paraxis at 8.5 dpc in these somites reinforces this
speculation. A transition in cellular recruitment to the paraxial
mesoderm from the primitive streak to the tailbud mesenchyme occurs at
the stage of posterior neuropore closure at the end of the neural stage
(65, 66). Since GCNF may be required in the PSM and/or
tailbud, somitogenesis is affected after this stage in
GCNF/ embryos. Ectopic expression of paraxis
and RALDH-2 in the posterior of GCNF/
embryos indicates there is inappropriate differentiation within the
PSM, ultimately leading to the halt in somitogenesis and the posterior
truncation. The altered differentiation of the PSM is probably due to
the loss of GCNF within cells of the posterior; however, the ectopic
location of the tailbud also likely contributes to the phenotype.
The tailbud develops ectopically outside the yolk sac after the late
streak stage, which could be due to the failure of the embryos to turn
and envelop correctly in their embryonic membranes. The failure of
mutant embryos to turn has been described for many gene knockout
models, yet the positioning of the tailbud outside the yolk sac
suggests that this is a novel phenotype related to the loss of GCNF
function and not merely due to a failure of the GCNF mutant embryos to
turn (25, 28, 45, 50). The halt in somitogenesis does not
occur until after the posterior of the GCNF/ embryo
develops outside the yolk sac. Therefore, in addition to the loss of
GCNF function contributing to the halt in somitogenesis and the
posterior truncation, the ectopic location of the tailbud may play a
role as well.
The exact mechanism that results in the formation of this protrusion
remains to be investigated. Here we propose a model that accounts for
its formation based on the data presented here (Fig. 9). In both
GCNF+/+ and GCNF/ embryos, as the somites,
which express paraxis, are generated from the primitive streak, the
neural plate forms at 8.0 dpc (Fig. 9A and B). As GCNF+/+
embryos continue to grow at 8.5 dpc, the node, which expresses nodal,
regresses caudally (Fig. 9C), with a resulting lengthening of the
notochord that continues to induce the neural plate to form the neural
folds. The edges of the neural plate begin to elevate, forming the
neural groove. In GCNF/ embryos, a neural groove has
been observed histologically, but neural epithelium abnormally
invaginates within the primitive streak region by 8.5 dpc (Fig. 9D;
Fig. 5B and D). This invagination then pushes the primitive streak away
from the original direction of posterior regression.
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FIG. 9.
Model of ectopic tailbud formation in
GCNF/ embryos. At 8.0 dpc in both GCNF+/+
(A) and GCNF/ (B) embryos, the neural plate arises when
the somites are generated from the primitive streak. Arrows indicate
the direction of node regression. The orange area represents brachyury T expression in the primitive streak and PSM. (C)
In the 8.5-dpc GCNF+/+ embryos, the node, which expresses
nodal (yellow), moves caudally (arrow), with a resultant lengthening of
the notochord, which continues to induce the neural plate to rise to
form the neural fold. The edges of the neural plate begin to elevate,
forming the neural groove. (D) In GCNF/ embryos the
node does not regress posteriorly, and invagination of neural
epithelium occurs within the primitive streak region by 8.5 dpc. This
invagination then pushes the primitive streak away from the original
direction of regression (arrow). Arrowheads (C and D) indicate
positions of yolk sac attachment to the embryos. (E) By 9.5 dpc, the
posterior neuropore (PNP) in GCNF+/+ in GCNF+/+
embryos is closed by zipping up the neural grove caudally (arrow). (F)
In 9.5-dpc GCNF/ embryos, the neural tube elongates
inside the tailbud as the tailbud pushes out of the yolk sac (arrow).
Paraxis expression in somites and PSM is shown in blue. P, posterior;
P*, new posterior.
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A factor contributing to ectopic development of the tailbud is the
altered location of the posterior site of attachment of the yolk sac.
The yolk sac is normally attached around the node, anterior to the
primitive streak in GCNF+/+ embryos (Fig. 9C). In
GCNF/ embryos, however, the yolk sac is attached more
caudally, at the posterior of the primitive streak close to the
allantois (Fig. 9D). Consequently, the resulting PSM and
undifferentiated somites are pushed out of the yolk sac by the
invagination of the neuroepithelium and growth of the posterior. At 9.5 dpc, the posterior neuropore in GCNF+/+ embryos is closed
by "zipping up" the neural groove caudally (Fig. 9E). In contrast,
no posterior neuropore was observed in GCNF/ embryos.
In addition nodal expression is down-regulated in wild-type embryos at
9.5 dpc (Fig. 9C and E); however, the disorganized and persistent
expression of nodal (Fig. 9F) is indicative of altered function of the
node, the posterior organizer, which in turn may lead to the
abnormalities observed in the posterior of GCNF/
embryos. The neural tube elongates within the tailbud as it pushes through the yolk sac (Fig. 9F) and forms a new posterior that continues
to produce somites until the abnormal differentiation observed in the
PSM, i.e., ectopic expression of paraxis, eventually leads
to a halt in somitogenesis.
In summary, inactivation of the GCNF gene leads to embryonic
lethality with major disruption of normal anteroposterior development. GCNF is likely to be a receptor for a novel ligand signaling pathway that is involved in regulating various aspects of embryonic development and normal anteroposterior development. Further work will be required to elucidate the GCNF signaling pathway.
| |
ACKNOWLEDGMENTS |
The first two authors contributed equally to this work.
We thank R. R. Behringer, K. Mahon, and R. L. Johnson for
many helpful suggestions on the manuscript. We also thank A. Bradley for providing the HSVtk plasmid and S. Aizawa, D. L. Ang, R. Beddington, M. Buckingham, P. Chambon, R. P. Harvey, N. Heintz,
A. P. McMahon, R. Nusse, E. N. Olson, E. J. Robertson,
and T. F. Vogt for providing plasmids to generate RNA probes for
in situ hybridization.
This work was supported by NIH grant DK57743 to A.J.C.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6250. Fax: (713) 790-1275. E-mail: acooney{at}bcm.tmc.edu.
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Top
Abstract
Introduction
