1 Office of Clinical Research, National Institute of Environmental Health
Sciences, Research Triangle Park, NC 27709, USA
2 Laboratory of Signal Transduction, National Institute of Environmental Health
Sciences, Research Triangle Park, NC 27709, USA
3 Laboratory of Respiratory Biology, National Institute of Environmental Health
Sciences, Research Triangle Park, NC 27709, USA
4 Department of Medicine, Duke University Medical Center, Durham, NC 27710,
USA
5 Department of Biochemistry, Duke University Medical Center, Durham, NC 27710,
USA
6 Nina Ireland Laboratory of Developmental Neurobiology, Department of
Psychiatry, University of California, San Francisco, San Francisco, CA 94143,
USA
* Author for correspondence (e-mail: zeldin{at}niehs.nih.gov)
Accepted 10 June 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hydrocephalus, Regulatory factor X, Winged helix transcription factor, Cortex, Midline, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present studies began with the evaluation of a line of transgenic mice
in which the cardiac-specific expression of an unrelated transgene (CYP2J2)
was associated with the development of head swelling and rapid neurological
decline in young adulthood. Anatomical characterization of these mice revealed
severe congenital hydrocephalus that was nonetheless compatible with life and
fertility in some cases. This obstructive hydrocephalus appeared to be
secondary to failure of development of the subcommissural organ (SCO), a
structure that is important for the patency of the aqueduct of Sylvius and
normal cerebrospinal fluid flow in the brain
(Cifuentes et al., 1994;
Perez-Figares et al., 1998
;
Perez-Figares et al., 2001
;
Rodriguez et al., 2001
;
Rodriguez et al., 1998
;
Vio et al., 2000
).
Identification of the genomic sequences flanking the inserted transgene led to
the discovery that the transgene interfered with the expression of a novel
brain-specific isoform of the winged helix transcription factor regulatory
factor X4 (RFX4), which has been named RFX4 variant transcript 3, or
RFX4_v3.
Fetal mice completely lacking in RFX4_v3 expression exhibited severe defects in the formation of dorsal midline brain structures, and intra-uterine or perinatal death. Thus, this accidental transgene insertion led to the identification of a novel splice variant of RFX4 that is crucial for normal brain development. In addition, disruption of a single allele led to an autosomal dominant pattern of expression of congenital hydrocephalus. Given the 96% identity between the mouse and human protein products of RFX4_v3, it seems possible that abnormalities of expression or primary sequence of the human gene could result in some cases of congenital obstructive hydrocephalus, a common human birth defect.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histology
For routine histology, embryos and tissues from newborn or adult mice were
fixed in Bouin's fixative for 12-48 hours, depending on tissue size, and then
cleared in 70% (v/v) ethanol. They were then embedded in paraffin wax,
sectioned and stained with Hematoxylin/Eosin by standard methods. For
immunohistochemistry, paraffin wax-embedded sections were stained with an
antibody (Rodriguez et al.,
1984) to Reissner's fibers (RF) within the SCO, as described
previously for a different antibody
(Blackshear et al., 1996
). The
anti-RF antibody was a generous gift from Dr E. M. Rodriguez (Instituto de
Histologia y Patologia, Facultad de Medicina, Universidad Austral de Chile,
Valdivia, Chile).
Identification of the transgene insertion site
A Universal GenomeWalker Kit (Clontech, Palo Alto, CA) was used to identify
the mouse genomic sequences adjacent to the transgene insertion site. Briefly,
genomic DNA from transgenic mice was digested with DraI,
EcoRV, PvuII or StuI, and ligated to adaptors
supplied by the manufacturer. PCR amplification of 3' adjacent sequences
used the Advantage Genomic PCR Kit (Clontech), the universal adaptor primers
AP1 and AP2, and the following nested gene-specific primers:
5'-ACAACTCTGCGATGGGCTCTGCTTT-3' and
5'-CTGACCAATTTGACGGCGCTGCACA-3'. PCR products were cloned into the
pCRII vector using the TA Cloning Kit (InVitrogen/Life Technologies, Carlsbad,
CA) and sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction
Kit (Applied Biosystems, Foster City, CA). PCR amplification of 5'
adjacent sequences was similarly performed using the following nested
gene-specific primers: 5'-GGCCATTGTCACCACTCGTAA-3' and
5'-CACAAGTAAAGGCTAACGCGC-3'.
Plasmids
Plasmids containing the indicated human, mouse and zebrafish ESTs were
obtained from the IMAGE consortium. A plasmid containing the putative protein
coding region of the mouse RFX4_v3 was made by first using Superscript II
RNase H- Reverse Transcriptase (Invitrogen/LifeTechnologies,
Carlsbad, CA) to reverse transcribe total adult mouse brain RNA template. The
resulting cDNA was then subjected to two rounds of nested PCR using Platinum
Pfx DNA polymerase (Invitrogen/LifeTechnologies) and primers based on the
5' and 3' sequences of apparent mouse brain RFX4 sequences from
GenBank. The first pair of primers corresponded to bp 255-278 of Accession
Number BB873367 and to bp 100-124 of Accession Number BB379807, and the second
set of primers corresponded to bp 291-309 of Accession Number BB873367 and bp
99-78 of Accession Number BB379807. The resulting PCR product was sequenced
using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystems, Foster City, CA).
Probes corresponding to the unique 5'-ends of mouse RFX4_v1 and RFX4_v3 were constructed by PCR amplification of reverse-transcribed mouse testis RNA or brain RNA, respectively. Reverse transcription was carried out using 1 µg of total RNA, an anchored oligo (dT) primer (T18VN) and Superscript II RNase H- Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA). PCR was performed using primers based on the sequence for human RFX4_v1 (Accession Number NM_032491) or the sequence for mouse RFX4_v3 contained in the mouse brain EST Accession Number BB595996. The forward primer for RFX4_v1 was 5'-AGGTGGGAAGGCAGTTATGACAG-3' (corresponding to bases 1-23 of NM_032491) and the reverse primer was 5'-TCCGTGATATTTCTGCTTAGTGGGC-3' (bases 201-177). A second round of PCR was carried out with forward primer 5'-GGCAGTTATGACAGTTGAGAAGTAGTAG-3' (bases 10-37) and reverse primer 5'-CTGCTTAGTGGGCATCTCGAATCTATC-3' (bases 189-163). The forward primer for mouse RFX4_v3 was 5'-TTTTGACGGGTTTGGCTTTG-3' (bases 118-137 of BB595996) and the reverse primer was 5'-TTCCTCCAGTAACCCACAATGC-3' (bases 447-426). A probe corresponding to the unique 5'-end of RFX4_v2 was isolated by PCR amplification from mouse L cell genomic DNA using primers based on the sequence for human RFX4_v2 (Accession Number NM_002920). PCR was carried out using forward primer 5'-TGGAGAGGCCACAGCTGCTGG-3' (bases 1-21 of NM_002920) and reverse primer 5'-TCGAGGCCTGGTCCTGTCGC-3' (bases 159-140). A second round of PCR was performed with 5'-CACAGCTGCTGGCTTCCTGG-3' (bases 10-29) and the same reverse primer as in the first round of PCR. All three unique 5'-ends of RFX4_v1, RFX4_v2 and RFX4_v3 were sequenced using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA).
A cDNA corresponding to human RFX4_v3 was cloned by screening a human fetal brain cDNA library (Stratagene) with the insert from the human IMAGE clone # 46678 (GenBank Accession Number H10145). The resulting cDNA clone was sequenced by dideoxynucleotide techniques (see above). A plasmid (GenBank Accession Number AI657628) containing a zebrafish EST sequence that predicted a protein closely related to the N terminus of mouse and human RFX4_v3 was also obtained from the IMAGE Consortium and sequenced by dideoxynucleotide techniques.
In situ hybridization histochemistry
Embryos were dissected in PBS and fixed in 4% (w/v) paraformaldehyde/PBS at
4°C. Specimens for whole-mount in situ hybridization were gradually
dehydrated in methanol/PBS and stored in 100% methanol at -80°C. Specimens
for in situ hybridization on frozen sections were cryoprotected in 30% sucrose
and embedded in TissueTek (Sakura), and 20 µm sections were obtained using
a cryostat. Whole-mount and section in situ hybridization was performed
according to the methods of Wilkinson
(Wilkinson, 1992) and Tsuchida
et al. (Tsuchida et al.,
1994
), respectively. The probes used and their sources were as
follows: Rfx4 (this paper); Otx2 (Antonio Simeone); Bf1 (Eseng Lai); Fgf8
(Gail Martin); Msx2 (Betham Thomas); Wnt3a and Wnt7b (Andrew McMahon); Lhx2
(Heiner Westphal); Pax6 and Six3 (Peter Gruss); and Emx1, Dlx2 and Nkx2.1
(J.L.R.R.'s laboratory).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
All other non-brain tissues of the transgenic mice appeared to be histologically normal.
Identification of genomic sequences flanking the transgene
Our working hypothesis was that the transgene had interrupted the coding or
regulatory regions of an important gene, and we therefore attempted to
identify the mouse genomic sequences flanking the transgene. Using PCR based
on 5' and 3' transgene sequences, we found that there were at
least two tandem copies of the 7.9 kb transgene in genomic DNA from the
transgenic mice, indicating that the potential genomic interruption was at
least 15 kb in size; Southern analysis using a transgene-specific probe
indicated that there was only one copy of this concatenated transgene in the
mouse genome (data not shown). Using the 'GenomeWalker' technique with genomic
DNA from the transgenic mice and transgene-specific oligonucleotide primers,
we identified both the 5' and 3' flanking genomic sequences into
which the transgene had been inserted. When these sequences were compared to
the mouse genomic sequences in the GenBank trace archives, the transgene
insertion site was identified as between bp 528 and 529 in gnl|ti|13973384 and
between bp 171 and 172 in gnl|ti|84074979. The 5' and 3' flanking
sequences identified by the GenomeWalker technique were contiguous in the
normal mouse genomic sequences in the trace archives, indicating that the
transgene insertion was not accompanied by a genomic deletion, as has been
seen in some recent examples of accidental transgenic insertional mutagenesis
(Durkin et al., 2001;
Overbeek et al., 2001
).
Southern analysis using a 3' insertion site-specific probe demonstrated
the presence of single novel bands in restriction enzyme-digested DNA from the
transgenic mice, confirming a single transgene insertion site at this location
(Fig. 4A).
|
According to the mouse-human alignments, the site of the transgene insertion within the mouse genome was at a corresponding region within the human chromosomal 12 sequence that would be within the intron between exons 13 and 14 of RFX4_v1 (see below); it would not have affected the exon arrangements of RFX4_v2.
Using PCR primers based on the inserted transgene and the neighboring endogenous mouse genomic DNA, we found that the wild-type (+/+) and transgene-interrupted alleles (+/- for one allele disrupted, -/- for both alleles disrupted) could be readily distinguished in a litter of newborn mice from interbred transgenic mice (Fig. 4B).
To examine the possibility that the transgene insertion had in some way
interfered with the expression of a full-length mouse RFX4 transcript in
brain, we probed northern blots from brains of neonatal +/+, +/- and -/- mice
with a mouse brain EST cDNA clone (IMAGE # 763537, GenBank Accession Numbers
AA285775 and AI462920) that was highly related (e-124 over 284 aligned bases)
to the 3'-end of the human cDNA for RFX4_v1. Brains from the +/+ mice
expressed a prominent band of 4 kb that we will refer to as RFX4 variant
transcript 3 or RFX4_v3 (Fig.
4C; see below). Brains from the +/- mice expressed
50% of the
normal complement of this transcript, whereas the brains from the -/- mice
expressed no detectable transcript of this size
(Fig. 4C). Probing the same
blot with an actin cDNA demonstrated that gel loading was similar in the three
lanes (Fig. 4C). Similar
results were obtained in three separate experiments. There was no evidence for
the expression of a truncated mRNA in the brain samples from either the +/- or
-/- mice (data not shown). These studies confirmed that an mRNA species of
4 kb that was recognized by a probe derived from putative mouse 3'
RFX4_v1 sequences was decreased in amount in brains of the +/- mice, and
absent from the brains of the -/- mice. These data suggested that the
insertion of the transgene interfered with the expression of the putative
brain RFX4_v3 transcript.
Using the same probe to examine the tissue-specific and developmental expression of this RFX4 transcript, we found high-level expression of a slightly smaller transcript in normal adult testis, and lower level expression of a considerably smaller transcript in liver (Fig. 4D). The largest species, corresponding to the apparent brain-specific transcript labeled RFX4_v3 in Fig. 4D, was the only one detected in whole embryos early in development (Fig. 4E). These data suggested that an apparently brain-specific isoform of RFX4 in the adult was highly expressed in the whole embryo during early development, initially appearing between embryonic day (E) 7.5 and 9.5 (Fig. 4E).
Identification of the RFX4_v3 transcripts and proteins
Using primers based on mouse brain EST sequences that contained internal
sequences highly related to the human RFX4 cDNAs in GenBank, we used PCR and
an adult mouse brain cDNA library to generate a 3 kb plasmid insert that
was then sequenced. This cDNA has been designated RFX4 transcript variant 3
(RFX4_v3), and the mouse sequence has been deposited in GenBank (Accession
Number AY102010). When this sequence was merged with all available 5'
and 3' mouse ESTs from GenBank, the resulting transcript was 3952 bp,
closely approximating the transcript size seen on northern blots. While this
paper was under revision, a cDNA sequence was deposited in GenBank on 5
December 2002 (GenBank Accession Number AK034131.1) that was 3535 bp in
length; over this length, it was more than 99% identical to the putative
RFX4_v3 full-length transcript described above, and included the entire
putative protein coding region. This cDNA was isolated from an adult male
mouse diencephalon library and confirms the existence in brain of at least the
protein-coding region of our predicted full-length RFX4_v3 transcript.
Similar probes as used to generate the northern blots shown in Fig. 4 were then used to screen a human brain cDNA library, and positive inserts were sequenced. This cDNA sequence has been deposited in GenBank as human RFX4_v3 (Accession Number AY102009). The predicted unique mouse N-terminal protein sequence (see below) also was used to search the non-human, non-mouse ESTs in GenBank, and a zebrafish EST clone (Accession Number AI657628) with a nearly identical predicted N-terminal protein sequence was obtained from the IMAGE consortium and sequenced. This sequence is referred to as zebrafish RFX4_v3, and the complete insert cDNA sequence has been assigned accession number AY102011.
An alignment of these three predicted amino acid sequences is shown in
Fig. 5. There was 96% amino
acid identity between the predicted mouse and human proteins, and 83% amino
acid identity between the predicted human and zebrafish proteins. The
alignment also illustrates several of the characteristic domains of the RFX
proteins that are highly conserved in all three orthologs, i.e. the DNA
binding domain, boxes B and C, and the dimerization domain
(Morotomi-Yano et al.,
2002).
|
|
The exon pattern that corresponds to the mouse and human RFX4_v3 mRNAs and proteins is illustrated at the bottom of Fig. 6. A novel exon (purple) derived from a sequence between 480,000 and 500,000 of NT_009720.8 was used to form the first 14 amino acids at the N terminus (Fig. 6). The next four exons, 2-5, are composed of the four exons of the same number from RFX4_v2 (green); exon 1 of RFX4_v2 (green hatching) is not present in the RFX4_v3 cDNA. The middle of the RFX4_v3 cDNA and protein are formed by the 10 exons (yellow) held in common between RFX4_v1 and RFX4_v2. The C terminus of RFX4_v3 is composed of the three C-terminal exons present only in RFX4_v1 (red). Thus, the novel RFX4_v3 isoform described here is composed of a unique arrangement of 18 exons derived from almost 200 kb of human genomic sequence. One exon (the first) is unique to this sequence; exons 2-5 are shared with RFX4_v2; exons 6-15 are shared with both RFX4_v1 and RFX4_v2; and exons 16-18 are shared with only RFX4_v1.
The site of transgene interruption is also illustrated in Fig. 6. The >15 kb transgene was inserted into the intron between exons 17 and 18 of RFX4_v3, within the C-terminal end of the protein coding region, and presumably interferes with splicing of the final exon and generation of an intact mature mRNA. We have found no evidence to date that a stable truncated mRNA species results from this transgene insertion.
We next designed and cloned specific cDNA probes corresponding to unique
5' sequences for each of the three RFX4 transcript variants RFX4_v1, v2
and v3. These were then used to probe northern blots of RNA from brains of
E18.5 mice as well as from adult testes, liver and brain. As shown in
Fig. 7, a probe that spanned
regions common to the RFX4_v1, v2 and v3 transcripts hybridized to two major
mRNA species in testes, a single transcript of intermediate size in liver, and
a single transcript of the largest size (4 kb) in RNA from adult brain.
This probe only hybridized to the 4 kb RNA species in brains from E18.5 mice;
the amount of hybridization of this probe decreased from the +/+ to the +/-
mouse brain, and was undetectable in brain from the -/- sample. When similar
blots were hybridized with a probe specific for v1 and v3, only the larger of
the two testes transcripts (v1) was detected, while the largest transcript
(v3) was again identified in the adult brain sample and in the brain from
E18.5 +/+ fetal mice. Again, the expression of the transcript hybridizing to
this probe decreased with decreasing allelic dosage.
|
These data indicate that the v3 transcript variant is the only form significantly expressed in the adult and fetal brain, and also confirmed it as the transcript variant expressed in the whole embryo and brain in earlier development (see Fig. 4E).
The identity of the apparently liver-specific transcript is not known, as it does not correspond to any of the three RFX4 transcript variants described above. It could represent a still unknown hypothetical 'RFX4_v4', or it could represent cross hybridization of the longer probes to another member of the RFX transcript family that is highly expressed in liver. We favor the latter possibility, as none of the shorter, specific v1-v3 probes hybridized to this species in our northern blots.
Analysis of RFX_v3 transcript expression during development
The pattern of RFX4_v3 transcript expression in mouse embryos was analyzed
using RNA in situ hybridization. The data shown are from experiments in which
a probe was used that contained sequences specific to both RFX4_v1 and v3.
RFX4_v3 RNA was found primarily in the brain where its regional expression was
highly dynamic during development. At E8.5, RFX4_v3 expression was detected in
most of the neural plate, but its expression was excluded from the presumptive
forebrain region (Fig. 8A,B).
By E9.5, most of its expression encompassed two large regions: the caudal
diencephalon/mesencephalon and the spinal cord
(Fig. 8C). The rostral limit of
the diencephalic expression approximated the zona limitans; the only
expression extending anterior of this boundary was in the caudodorsal
telencephalon (Fig. 8C).
|
Transient RFX4_v3 expression appeared in the central retina. The lateral optic stalks also exhibited RFX4_v3 expression (Fig. 8H), while the medial optic stalks showed expression at later stages (Fig. 8K).
From E12.5 to birth, the neuroepithelium and later the ependyma of most of the neural tube expressed variable levels of RFX4_v3 transcripts. For example, in the cerebral cortex, RFX4_v3 was expressed in a dorsal-to-ventral gradient (Fig. 8K). The majority of roof plate derivatives of the CNS, including most of the circumventricular organs, had turned off RXF4_v3 expression by this stage (for example, the epiphysis, and the choroid plexus of the lateral and fourth ventricles in Fig. 8L,M). A striking exception to this pattern was the expression of RFX4_v3 in the region of the developing SCO found in the caudal diencephalon, where there was strong expression from E14.5 to birth (Fig. 9C,E-G).
|
Phenotype of RFX4_v3-deficient mice
Surviving transgenic mice, which we will now refer to as RFX4_v3 +/- mice,
were interbred to generate -/- mice. Ten pregnant +/- mice were allowed to
carry to term and deliver; the average litter size of these pregnancies was
5.3±0.6, which was significantly smaller than litters from a control
line 7.0±0.4 (P=0.022). Of 53 pups born, 19 (36%) were wild
type, 28 (53%) +/-, and 6 (11%) -/-, suggesting substantial intrauterine or
perinatal loss of the -/- pups. All of the -/- pups born died within 1 hour of
birth. Seven additional litters were obtained between E8 and E18. The average
size of those litters was 8.7±0.5, which was not significantly
different from control litters. Of 61 pups obtained, there were 10 (16%) +/+,
36 (59%) +/- and 15 (25%) -/-, indicating no excess intrauterine
mortality.
The brains of the -/- mice at the time of birth and at E16.5 were grossly dysmorphic (data not shown). We therefore examined the -/- mice at an earlier developmental stage, E12.5. The phenotype at this age was striking (Fig. 10). Externally, there were clear abnormalities of head appearance, although the position of the eyes, vibrissae and other facial structures appeared relatively normal (Fig. 10A). Coronal sections suggested that dorsal structures in the rostral brain were hypoplastic and lacked morphological differentiation of medial and paramedial dorsal structures. This was most striking in the forebrain and midbrain (Fig. 10B), but abnormalities persisted into the hindbrain and spinal cord. As in the hemizygotes, the anatomy of the rest of the body in the E12.5 -/- embryos was apparently normal.
|
|
In wild-type mice, Lhx2 and Emx1 are expressed in a
dorsoventral gradient in the cortical neuroepithelium. In the RFX4_v3 mutants,
Lhx2 and Emx1 expression levels were similar to those seen
in the ventral part of the normal cortex, suggesting that dorsal parts of the
cortex were missing (Fig.
11G,I). An Emx1-negative, Lhx2-positive
territory intercalated between the striatum and the prospective piriform
cortex, which develops into parts of the claustroamygdaloid complex
(Puelles et al., 2000;
Yun et al., 2001
), was
maintained in the mutants (Fig.
11G,I). Finally, Pax6 is normally detected in a
ventrodorsal gradient. In the mutants, the ventral area where expression was
strongest was detected (Fig.
11H). Thus, the most ventral subdivisions of the cortex, located
adjacent to the striatum, i.e. the piriform cortex and parts of the
claustroamygdaloid complex, seemed to be correctly specified, while the most
medial cortical subdivisions, located adjacent to the cortical hem, i.e. the
hippocampus and the neocortex, are either severely reduced, lost, or
mis-specified.
The basal ganglia are formed in mammals by the lateral ganglionic eminence,
which develops into the striatum, and the medial ganglionic eminence, which
develops into the pallidum (Marin and
Rubenstein, 2002). In the mutants, although the size of the basal
ganglia was disproportionately large compared with the cortex, it is unclear
whether or not there was an absolute increase in the sizes of the lateral and
medial ganglionic eminences. The RFX4_v3 mutants exhibited normal expression
of Dlx2 and Six3 transcription factors in the lateral and
medial ganglionic eminences (Fig.
11J,K). Expression of Otx2, Fgf8 and Six3 in the
septum, a basal ganglia-related structure, was also detected
(Fig. 11B,C,J). In addition,
the specific expression of the transcription factor Nkx2.1 in the
medial ganglionic eminence and ventral septum was apparently normal in the
mutants (Fig. 11L).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The RFX proteins belong to the winged-helix subfamily of helix-turn-helix
transcription factors, and are so named because they bind to 'X-boxes' in
target DNA sequences and regulate expression of the target genes. The X-box
consensus sequence is 5'-GTNRCC(0-3N)RGYAAC-3', where N is any
nucleotide, R is a purine and Y is a pyrimidine. Five RFX proteins have been
described in man (RFX1-RFX5), all of which contain a highly conserved DNA
binding domain near the N terminus. A structure has been determined for the
binding of this domain from RFX1 to an X-box sequence
(Gajiwala et al., 2000); this
shows that the 'wing' of this DNA binding domain is used to recognize DNA.
Members of this family have been implicated in the transcriptional regulation
of a number of important genes.
A partial sequence of a novel family member, termed RFX4, was initially
identified by Dotzlaw et al. (Dotzlaw et
al., 1992) as part of a fusion cDNA in human breast cancers, in
which the N-terminal estrogen binding domain of the estrogen receptor was
fused with the RFX DNA binding domain. More recently, two full-length RFX4
cDNAs have been described and categorized, and their relationships and
nomenclature were updated by NCBI on 26 March 2003. The new RFX4_v3 variant
described here is composed of novel exons as well as exons derived from one or
both of these two earlier variants. As illustrated in
Fig. 6, the RFX4_v3 cDNA is the
largest of the three and is composed of a unique 5' exon of
476 bp
that encodes the first 14 amino acids of RFX4_v3; this is then followed by
four exons shared only with RFX4_v2, then 10 exons shared with both RFX4_v1
and RFX4_v2, and finally three 3'-exons shared only with RFX4_v1. The
existence of this transcript in mouse brain was confirmed while this paper was
under revision by the deposition in GenBank on 5 December 2002, of a 3535 bp
cDNA isolated from adult mouse diencephalon that is 417 bp shorter but
otherwise essentially identical to the RFX4_v3 transcript described here.
However, at the time of this writing (21 March 2003), there were no human cDNA
sequences in either the GenBank nr or est collections corresponding to the
unique 5'-end of RFX4_v3, other than the sequence described here.
Our data indicate that the novel RFX4_v3 transcript is expressed in the developing central nervous system from the neural plate stages. Its early expression is dynamic, particularly in the telencephalon, where initially it is only expressed in and adjacent to the dorsal midline. Later, its expression is extinguished in the midline, and spreads as a dorsoventral gradient throughout the cortex. It is also expressed in adult brain, although its non-developmental functions and anatomical distribution in this tissue remain to be determined. We did not detect significant levels of this transcript in other organs of the adult mouse. It will be of interest in future studies to identify genes whose expression is directly affected by RFX4_v3, presumably acting as a transcription factor, as well as other transcription factors influencing the developmental expression of RFX4_v3 itself.
Disruption of both RFX4_v3 alleles severely altered early brain
morphogenesis. The reduction of Msx2, and the loss of Wnt3a,
Wnt7b and Bmp4 expression in the cortical hem, strongly suggest
that RFX4 is required either to establish or maintain the dorsal patterning
center of the telencephalon. Mice deficient in WNT signaling from the cortical
hem have defects in growth and patterning of the dorsomedial cerebral cortex
(Galceran et al., 2000;
Lee et al., 2000
). Although
these WNT-signaling mutants have hippocampal defects, they can generate a
choroid plexus. Mice deficient in BMP-signaling, through the loss of Bmpr1a
function in the telencephalon, fail to produce choroid plexus
(Hebert et al., 2002
). Given
the loss of the choroid plexus in RFX4_v3 mutants, and the loss of
Bmp4 and the reduction of Msx2 expression, we suggest that
RFX4 has a general role in regulating dorsal patterning that involves both WNT
and BMP signaling pathways.
The hypoplasia of the cerebral cortex could be entirely due to defects in
the dorsal patterning center. However, RFX4 is expressed throughout the
cortical primordium, and therefore could have a role in regulating
proliferation and differentiation of the cerebral cortex, similar to
Foxg1 (Bf1), another winged-helix gene
(Dou et al., 1999;
Hanashima et al., 2002
).
Future studies should aim to elucidate how RFX4 regulates the dorsal
patterning center and to establish its more general role within neural
progenitors.
Disruption of a single RFX4_v3 allele led to a quantitative decrease in
RFX4_v3 mRNA expression in the brain and non-communicating congenital
hydrocephalus. Hydrocephalus has generally been divided into congenital forms,
i.e. those present at birth, and acquired forms that develop after birth.
Within the spectrum of congenital hydrocephalus are non-genetic causes, such
as uterine infection, hemorrhage and meningitis, as well as genetic causes.
Congenital hydrocephalus can be subdivided further into communicating and
non-communicating forms, in which the latter is associated with stenosis of
the aqueduct of Sylvius. In one series of individuals with isolated congenital
hydrocephalus, i.e. not associated with other congenital anomalies, about 43%
were associated with aqueductal obstruction, 36% had communicating
hydrocephalus, 15% had the Dandy-Walker syndrome and about 6% had other
lesions (Burton, 1979).
Overall, isolated hydrocephalus in man occurs in
0.6 per 1000 of newborn
children (Halliday et al.,
1986
). It is thought that the X-linked form is present in 7-27% of
male cases; this form is now known to be due to abnormalities in the
L1CAM gene, and there are several overlapping neurodevelopmental
human syndromes associated with defects in this gene
(Weller and Gartner, 2001
).
Most cases of congenital hydrocephalus, however, have no known genetic cause.
The unusual finding that expression of only a single allele leads to
congenital hydrocephalus, at least in mice, means that this defect exhibits an
autosomal dominant inheritance pattern. As in mice, expression of a single
intact allele may be compatible with life and fertility in humans. We are
currently exploring the possibility that some human cases of congenital,
non-L1 hydrocephalus are due to abnormalities in the expression or sequence of
the RFX4_v3 transcript.
Hydrocephalus in these mice was associated with the apparent absence of the
SCO. Abnormalities of the SCO have been associated with hydrocephalus in many
studies, as recently reviewed
(Perez-Figares et al., 2001).
Although there has been some debate in the literature about whether the SCO
abnormalities cause or are consequences of the hydrocephalus, the overall
consensus seems to be in favor of the SCO abnormalities preceding and causing
the hydrocephalus, owing to effective stenosis of the aqueduct of Sylvius.
Examples of damage to or abnormalities of the SCO causing hydrocephalus
include radiation during fetal life
(Takeuchi and Takeuchi, 1986
),
maternal transfer of antibodies to Reissner's fibers
(Vio et al., 2000
), congenital
absence of the SCO in the MT/HokIdr strain of mice
(Takeuchi et al., 1987
),
hypoplasia of the SCO in SUMS/np mice
(Jones et al., 1987
) and
hypoplasia of the SCO in two strains of rats with congenital hydrocephalus
(Jones and Bucknall, 1988
;
Takeuchi et al., 1988
). It
therefore seems likely that, in the present case, the aplasia or hypoplasia of
the SCO seen in the RFX4_v3 hemizygous mice is the cause of the congenital
hydrocephalus, presumably by interfering with cerebrospinal fluid flow through
the rostral part of the aqueduct. It will be of interest to determine whether
any of these previously described mutants have abnormalities in the expression
or sequence of the RFX4_v3 protein. In addition, examination of downstream
gene expression at this specific anatomical site in these hemizygous mice may
lead to new insights into the formation of this 'enigmatic secretory gland of
the brain' (Schoniger et al.,
2001
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blackshear, P. J., Lai, W. S., Tuttle, J. S., Stumpo, D. J., Kennington, E., Nairn, A. C. and Sulik, K. K. (1996). Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency. Dev. Brain Res. 96,62 -75.[CrossRef][Medline]
Burton, M. K. (1979). Recurrence risks for congenital hydrocephalus. Clin. Genet. 16, 47-53.[Medline]
Cifuentes, M., Rodriguez, S., Perez, J., Grondona, J. M., Rodriguez, E. M. and Fernandez-Llebrez, P. (1994). Decreased cerebrospinal fluid flow through the central canal of the spinal cord of rats immunologically deprived of Reissner's fibre. Exp. Brain Res. 98,431 -440.[Medline]
D'Arcangelo, G., Miao, G. G., Chen, S. C., Soares, H. D., Morgan, J. I. and Curran, T. (1995). A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374,719 -723.[CrossRef][Medline]
D'Arcangelo, G., Miao, G. G. and Curran, T. (1996). Detection of the reelin breakpoint in reeler mice. Mol. Brain Res. 39,234 -236.[Medline]
Dotzlaw, H., Alkhalaf, M. and Murphy, L. C. (1992). Characterization of estrogen receptor variant mRNAs from human breast cancers. Mol. Endocrinol. 6, 773-785.[Abstract]
Dou, C. L., Li, S. and Lai, E. (1999). Dual
role of brain factor-1 in regulating growth and patterning of the cerebral
hemispheres. Cereb. Cortex
9, 543-550.
Durkin, M. E., Keck-Waggoner, C. L., Popescu, N. C. and Thorgeirsson, S. S. (2001). Integration of a c-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in Williams-Beuren syndrome. Genomics 73,20 -27.[CrossRef][Medline]
Friedman, R. A., Adir, Y., Crenshaw, E. B., Ryan, A. F. and Rosenfeld, M. G. (2000). A transgenic insertional inner ear mutation on mouse chromosome 1. Laryngoscope 110,489 -496.[Medline]
Furuta, Y., Piston, D. W. and Hogan, B. L.
(1997). Bone morphogenetic proteins (BMPs) as regulators of
dorsal forebrain development. Development
124,2203
-2212.
Gajiwala, K. S., Chen, H., Cornille, F., Roques, B. P., Reith, W., Mach, B. and Burley, S. K. (2000). Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature 403,916 -921.[CrossRef][Medline]
Galceran, J., Miyashita-Lin, E. M., Devaney, E., Rubenstein, J.
L. and Grosschedl, R. (2000). Hippocampus development
and generation of dentate gyrus granule cells is regulated by LEF1.
Development 127,469
-482.
Grove, E. A., Tole, S., Limon, J., Yip, L. and Ragsdale, C.
W. (1998). The hem of the embryonic cerebral cortex is
defined by the expression of multiple Wnt genes and is compromised in
Gli3-deficient mice. Development
125,2315
-2325.
Halliday, J., Chow, C. W., Wallace, D. and Danks, D. M. (1986). X linked hydrocephalus: a survey of a 20 year period in Victoria, Australia. J. Med. Genet. 23, 23-31.[Abstract]
Hanashima, C., Shen, L., Li, S. C. and Lai, E.
(2002). Brain factor-1 controls the proliferation and
differentiation of neocortical progenitor cells through independent
mechanisms. J. Neurosci.
22,6526
-6536.
Hebert, J. M., Mishina, Y. and McConnell, S. K. (2002) BMP signaling is required locally to pattern the dorsal telencephalic midline. Neuron. 35,1029 -1041.[Medline]
Jones, H. C. and Bucknall, R. M. (1988). Inherited prenatal hydrocephalus in the H-Tx rat: a morphological study. Neuropathol. Appl. Neurobiol. 14,263 -274.[Medline]
Jones, H. C., Dack, S. and Ellis, C. (1987). Morphological aspects of the development of hydrocephalus in a mouse mutant (SUMS/NP). Acta Neuropathol. 72,268 -276.[Medline]
Lee, S. M., Tole, S., Grove, E. and McMahon, A. P.
(2000). A local Wnt-3a signal is required for development of the
mammalian hippocampus. Development
127,457
-467.
Marin, O. and Rubenstein, J. L. (2002). Patterning, regionalization and cell differentiation in the forebrain. In Mouse Development (ed. J. Rossant and P. Tam), pp.75 -106. London: Academic Press.
Miao, G. G., Smeyne, R. J., D'Arcangelo, G., Copeland, N. G.,
Jenkins, N. A., Morgan, J. I. and Curran, T. (1994).
Isolation of an allele of reeler by insertional mutagenesis. Proc.
Natl. Acad. Sci. USA 91,11050
-11054.
Morotomi-Yano, K., Yano, K., Saito, H., Sun, Z., Iwama, A. and
Miki, Y. (2002). Human regulatory factor X 4 (RFX4) is a
testis-specific dimeric DNA-binding protein that cooperates with other human
RFX members. J. Biol. Chem.
277,836
-842.
Overbeek, P. A., Gorlov, I. P., Sutherland, R. W., Houston, J. B., Harrison, W. R., Boettger-Tong, H. L., Bishop, C. E. and Agoulnik, A. I. (2001). A transgenic insertion causing cryptorchidism in mice. Genesis 30,26 -35.[CrossRef][Medline]
Perez-Figares, J. M., Jimenez, A. J., Perez-Martin, M., Fernandez- Llebrez, P., Cifuentes, M., Riera, P., Rodriguez, S. and Rodriguez, E. M. (1998). Spontaneous congenital hydrocephalus in the mutant mouse hyh. Changes in the ventricular system and the subcommissural organ. J. Neuropathol. Exp. Neurol. 57,188 -202.[Medline]
Perez-Figares, J. M., Jimenez, A. J. and Rodriguez, E. M. (2001). Subcommissural organ, cerebrospinal fluid circulation, and hydrocephalus. Microsc. Res. Tech. 52,591 -607.[CrossRef][Medline]
Puelles, L., Kuwana, E., Puelles, E., Bulfone, A., Shimamura, K., Keleher, J., Smiga, S. and Rubenstein, J. L. (2000). Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J. Comp. Neurol. 424,409 -438.[CrossRef][Medline]
Rice, D. S. and Curran, T. (2001). Role of the reelin signaling pathway in central nervous system development. Annu. Rev. Neurosci. 24,1005 -1039.[CrossRef][Medline]
Rodriguez, E. M., Oksche, A., Hein, S., Rodriguez, S. and Yulis, R. (1984). Comparative immunocytochemical study of the subcommissural organ. Cell Tiss. Res. 237,427 -441.[Medline]
Rodriguez, E. M., Oksche, A. and Montecinos, H. (2001). Human subcommissural organ, with particular emphasis on its secretory activity during the fetal life. Microsc. Res. Tech. 52,573 -590.[CrossRef][Medline]
Rodriguez, E. M., Rodriguez, S. and Hein, S. (1998). The subcommissural organ. Microsc. Res. Tech. 41,98 -123.[CrossRef][Medline]
Schoniger, S., Wehming, S., Gonzalez, C., Schobitz, K., Rodriguez, E., Oksche, A., Yulis, C. R. and Nurnberger, F. (2001). The dispersed cell culture as model for functional studies of the subcommissural organ: preparation and characterization of the culture system. J. Neurosci. Methods 107, 47-61.[CrossRef][Medline]
Takeuchi, I. K., Kimura, R., Matsuda, M. and Shoji, R. (1987). Absence of subcommissural organ in the cerebral aqueduct of congenital hydrocephalus spontaneously occurring in MT/HokIdr mice. Acta Neuropathol. 73,320 -322.[Medline]
Takeuchi, I. K., Kimura, R. and Shoji, R. (1988). Dysplasia of subcommissural organ in congenital hydrocephalus spontaneously occurring in CWS/Idr rats. Experientia 44,338 -340.[Medline]
Takeuchi, I. K. and Takeuchi, Y. K. (1986). Congenital hydrocephalus following X-irradiation of pregnant rats on an early gestational day. Neurobehav. Toxicol. Teratol. 8, 143-150.[Medline]
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79,957 -970.[Medline]
Vio, K., Rodriguez, S., Navarrete, E. H., Perez-Figares, J. M., Jimenez, A. J. and Rodriguez, E. M. (2000). Hydrocephalus induced by immunological blockage of the subcommissural organ-Reissner's fiber (RF) complex by maternal transfer of anti-RF antibodies. Exp. Brain Res. 135, 41-52.[CrossRef][Medline]
Weller, S. and Gartner, J. (2001). Genetic and clinical aspects of X-linked hydrocephalus (L1 disease): Mutations in the L1CAM gene. Hum. Mutat. 18, 1-12.[CrossRef][Medline]
Wilkinson, D. G. (1992). Whole mount in situ hybridization on vertebrate embryos. In In Situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), pp.75 -83. Oxford: IRL Press.
Yun, K., Potter, S. and Rubenstein, J. L.
(2001). Gsh2 and Pax6 play complementary roles in dorsoventral
patterning of the mammalian telencephalon. Development
128,193
-205.