(Received for publication, July 12, 1995; and in revised form, September 28, 1995)
From the
The ``winged helix'' or ``forkhead''
transcription factors comprise a large gene family whose members are
defined by a common 100-amino acid DNA binding domain. Here we describe
the structure and expression of the mouse fkh-2 gene, which
encodes a protein of 48 kDa with high similarity to other winged helix
transcription factors within the DNA binding region, but unique
potential transactivation domains. The gene is encoded by a single exon
and is expressed in headfold stage embryos in the notochord, the
anterior neuroectoderm, and a few cells of the definite endoderm. This
expression becomes restricted to the anteriormost portions of the
invaginating foregut and the developing midbrain. From day 11.5 of
gestation onward, fkh-2 transcripts are restricted to the
midbrain and become progressively localized to the red nuclei as the
sole site of expression. The fkh-2 gene maps to chromosome 19B
and is a candidate gene for the mouse mutation mdf (muscle-deficient) which is characterized by nervous tremors and
degeneration of the hindlimb muscles. Although the expression patterns
of the fkh-2 gene and another winged helix protein,
HNF-3, are overlapping in early stages of gestation and although
the promoter of the fkh-2 gene contains a HNF-3 binding site,
we demonstrate that the activation of the fkh-2 gene is
independent of HNF-3
.
The ``winged helix'' or ``forkhead''
transcription factors comprise a large and growing gene family whose
members are defined by a common 100-amino acid DNA binding domain. The
determination of the crystal structure of the DNA binding domain of
HNF-3 (
)revealed that DNA recognition is mediated by a
variant of the helix-turn-helix motif which contains two loops or
``wings'' at the COOH-terminal side of the helix-turn-helix
(Clark et al., 1993). The winged helix proteins bind DNA as a
monomer and make base-specific contacts by the
helix H3 and loop
W2 which bind in the major groove of the DNA. A 20-amino acid region
NH
-terminal to helix 3 also has been shown to be important
in the determination of binding site specificity (Overdier et
al., 1994).
Since its discovery as a region of homology between
the rat HNF-3 (Lai et al., 1991) and the Drosophila gene forkhead (Weigel and Jäckle,
1990), the forkhead motif has been found in more than 40 genes in
species ranging from yeast to man (reviewed in Lai et
al.(1993)). Analyses of expression patterns as well as gain or
loss of function mutations of one of them have implicated these genes
in pattern formation during embryogenesis. The HNF-3 gene
product, for example, is normally found in the node, notochord,
endoderm, and central nervous system during early stages of mouse
development (Ang et al., 1993; Sasaki and Hogan, 1993;
Monaghan et al., 1993). Ectopic expression of HNF-3
in
the midbrain and hindbrain of transgenic mice leads to changes in the
expression of floorplate-specific genes and to abnormal neural
patterning (Sasaki and Hogan, 1994). Loss of function of the mouse HNF-3
gene through targeted mutagenesis results in severe
defects in midline development, specifically the absence of the
notochord and floorplate (Ang and Rossant, 1994; Weinstein et
al., 1994). The expression patterns of two other members of the
winged helix gene family, BF-1 and BF-2, define
adjacent domains in the developing forebrain and suggest a role for the
winged helix proteins in the establishment of positional identity along
the anterioposterioir axis of the neuroepithelium (Tao and Lai, 1992;
Hatini et al., 1994).
We have previously isolated nine
members of the winged helix family from mice (HNF-3, -
, and -
(Kaestner et al., 1994)
and fkh-1 to fkh-6 (Kaestner et al., 1993)),
which exhibit specific and diverse patterns of expression in adult
tissues. Here we report the detailed characterization of one of these
genes, fkh-2, whose expression pattern suggests that it plays
an important role in the regionalization of the foregut, notochord, and
midbrain of mouse embryos.
Figure 1:
Physical map of the mouse fkh-2 gene. The restriction map of the gene is shown together
with the extent of the phage clones from which it was derived.
The exon is shown as the large box, the translated region as a black box, and the forkhead DNA binding domain as a hatched box. Two of the fkh-2 cDNAs are indicated as thin lines. The boxes labeled A, B,
and C are probes referred to in the text. Abbreviations: E, EcoRI; S, SalI; N, NotI; P, PstI; Nc, NcoI; Sm, SmaI; X, XbaI; Xh, XhoI.
Figure 4: Mapping of the fkh-2 gene to mouse chromosome 19B by fluorescence in situ hybridization. Detection of the hybridized probe via fluorescein isothiocyanate revealed highly specific signals as indicated by the arrows in the left panel showing a complete metaphase spread. A simultaneously hybridized probe specific for 19C-D (see ``Materials and Methods'') was detected via rhodamine (see arrowheads). Panels on the right side present additional chromosome 19 homologs from other metaphase spreads to illustrate the subchromosomal localization on chromosome 19B.
Linkage analysis was
carried out using the DNA samples of the European Backcross (EUCIB)
kindly provided by the European Backcross Collaborative Group (The
European Backcross Collaborative Group, 1994). A 786-base pair fragment
of the fkh-2 gene was amplified with primers A
(CCATGGACCTCTGGACTATCTAGTTG) and B (CTTGGTCCCTCCTCTTCACACCC) in a
polymerase chain reaction buffer containing 1.5 mM MgCl with 35 cycles of 94 °C (30 s), 67 °C (60
s), and 72 °C (120 s). The polymerase chain reaction products were
digested with CfoI and analyzed on a 1.5% agarose gel after
staining of the DNA with ethidium bromide. The Mus musculus allele produces a band of 786 base pairs, while the Mus
spretus allele results in two bands of 466 and 320 base pairs.
Figure 5: Expression of the fkh-2 mRNA during mouse embryogenesis. 20 µg of total RNA from the developmental stages indicated (10 µg for day 8.5) were analyzed by RNase protection as described under ``Materials and Methods.'' The antisense probes and protected fragments (arrows) are labeled. The band for TBP indicates that equal amounts of RNA were hybridized in all cases. The autoradiograph was exposed for 96 h.
Figure 2: Nucleotide and translated amino acid sequence of the mouse fkh-2 gene. Shown is the sequence of the proximal promoter with the potential binding sites of the transcription factors indicated, followed by that of the composite cDNA sequences and the corresponding amino acid sequence of the longest open reading frame. The composite cDNAs extend from position 604 to 2931. The start site of transcription is denoted by an arrow at position 586. The translation initiation codon at position 1062 matches 4 out of 7 bases of the Kozak consensus (Kozak, 1987). The potential polyadenylation signal centered around position 2930 is indicated in italics. The sequence of the 100-amino acid forkhead DNA binding domain as well as domains A and B are underlined.
Figure 3:
In vitro translation of fkh-2 mRNA. In vitro synthesized mRNAs for the genes
indicated were translated in vitro in the presence of
[S]methionine and the reaction products
separated by SDS-polyacrylamide gel electrophoresis. The auroradiograph
was exposed for 16 h.
In order to determine the
chromosomal localization of the fkh-2 gene, the entire
G2 phage was used as a probe for fluorescence in situ hybridization to mouse metaphase chromosome spreads. Specific
probe signals were detected on mouse chromosome 19, as revealed by
4`,6-diamidino-2-phenylindole banding. In order to confirm the
chromosome assignment, a differently labeled probe targeting chromosome
19C-D was simultaneously hybridized and detected (see Fig. 4).
Of the 30 evaluated metaphase spreads, 57% showed signals on both
homologs and 27% on one homolog of chromosome 19 in region B. Since no
additional signal doublets were found on other chromosomal regions,
these experiments reveal localization of the fkh-2 gene within
19B, a region to which the mdf (muscle deficient)
locus is assigned (Lyon and Searle, 1989). mdf is defined by a
mutation which leads to a reduction in the mass of hindlimb muscles and
to nervous tremors (Womack et al., 1980; see
``Discussion''). In order to confirm this chromosomal
assignment and to better estimate the proximity of fkh-2 and mdf, we also performed linkage analysis using the DNA
collection of the European backcross (The European Backcross
Collaborative Group, 1994; see ``Materials and Methods'').
Analysis of DNAs from 1500 recombinants assigned fkh-2 to
chromosome 19 at position 17.5 ± 0.3 centimorgans (95%
confidence level), in close agreement to the data obtained from the in situ hybridization.
In order to precisely define the cellular localization of the fkh-2 mRNA in midgestation embryos, in situ hybridization studies were performed on embyos starting at day 6.5 p.c. of gestation. The fkh-2 gene is first expressed on day 7.5 p.c. in the ectoderm of the headfold (Fig. 6, A and B, and 7A) and is one of the earliest known markers of the prospective neuroectoderm. Transcripts are absent from the tip of the headfold but extend in the ectoderm approximately halfway to the node (Fig. 6, A and B). Expression is absent in the ectoderm adjacent to the primitive streak and in the extraembryonic tissues. fkh-2 transcripts are also detected in the notochord, weakly in the node and in endoderm cells anterior to the node (Fig. 6, A and B). These endoderm cells will subsequently invaginate to form the foregut. From day 8 to 8.5 p.c. (Fig. 7, B-E), fkh-2 expression becomes restricted in the neuroectoderm to the developing posterior diencephalon and to the midbrain region. Transcripts are also evident in the anterior tip of the foregut endoderm, extending from the oral plate to the otic vesicle, suggesting that the foregut is already regionalized at this stage. In addition transcripts are detected in the notochord anterior to the otic vesicle (data not shown). The expression outside the neurectoderm disappears between day 9 and 11.5 p.c. As the neural folds rise and fuse, fkh-2 mRNA becomes restricted to ventral regions of the neural tube excluding the prospective floorplate (Fig. 8D and data not shown). While the activation of the fkh-2 gene in the notochord and foregut is only transient, its expression in the neuroectoderm becomes progressively localized to the midbrain ( Fig. 7and Fig. 8). Between day 11 and 15 p.c. the neuroepithelium differentiates into four regions: the ventricular, subventricular, intermediate mantle, and mantle layers. On day 11.5 p.c. fkh-2 transcripts are localized to a subpopulation of intermediate mantle and mantle cells in the mesencephalon and metencephalon extending from the mesencephalic flexure to the region of the future pons (Fig. 8, A and B). In this region the anlage of the mesencephalic nuclei are born. Transverse sections through the midbrain on day 12.5 p.c. reveal labeling in two areas ventral of the cerebral aqueduct and excluding the floorplate (Fig. 8D). During further development, fkh-2 expression becomes restricted to a subpopulation of cells in the area of the red nuclei, most clearly seen in transverse sections of day 15.5 p.c. embryos (Fig. 8E). fkh-2 gene activity is maintained in the red nuclei until birth (Fig. 8F).
Figure 6:
Expression of the fkh-2 gene in
day 7.5 p.c. mouse embryos. Whole mount in situ hybridization
of day 7.5 p.c. wild type (A and B) or HNF-3 mutant (C) embryos with an antisense riboprobe to fkh-2. Lateral (A) and frontal (B) view of a
headfold stage embryo demonstrating fkh-2 expression in the
neuroectoderm of the headfold and in the notochord. Lateral view (C) of a HNF-3
homozygous mutant embryo shows fkh-2 expression in the anterior neurectoderm. Magnification
10. Abbreviations: n, node; nc, notochord; ne, neurectoderm, hf,
headfold.
Figure 7: Expression of the fkh-2 gene in mouse embryos from day 7.5 until 8.5 p.c. fkh-2 mRNA is initially detected in the neuroectoderm on day 7.5 p.c. in a transverse section (A). Subsequently expression becomes localized to the diencephalon and regions of the midbrain (B, C, and E, day 8.5 p.c.). Labeling in the anterior tip of the foregut of day 8.5 p.c. embryos is demonstrated under higher magnification in E. Staining of the notochord is evident in a transverse section of day 8.5 p.c. embryo (D). Scale bars represent 100 µm in B and C and 50 µm in A, D, and E. Abbreviations: d, diencephalon; en, endoderm; Fg, foregut; mb, midbrain; n, node; nc, notochord; ne, neuroectoderm.
Figure 8: fkh-2 transcripts during brain development from day 11.5 p.c. to postnatal day 1. Sagittal (A-C), coronal (D and F), and horizontal (E) sections through mouse embryos from day 11.5 (A and B), 12.5 (C and D), 15.5 (E) p.c. and postnatal day 1 (F) hybridized with the fkh-2 probe. fkh-2 transcripts, indicated by arrows, are localized to the intermediate mantle and mantle layers in the mesencephalon and metencephalon (A and B). On day 12.5 p.c., a sagittal section shows fkh-2 expression in the mesencephalon (C). Arrowheads in C indicate the plane of section for the coronal section shown in D. Labeling is confined to two areas ventral to the cerebral aqueduct and excluding the floorplate. Activation of the fkh-2 gene in the developing red nuclei is demonstrated in the horizontal section of a day 15.5 p.c. embryo (E). Expression in the red nuclei is maintained until after birth (F). Scale bars represent 400 µm in A, C, and E, 200 µm in B and F, and 100 µm in D. Abbreviations: Aq, aqueduct; Cb, future cerebellum; d, diencephalon, im, intermediate mantle layer; m, mantle layer; M, mesencephalon; MF, mesencephalic flexure; P, future pons; r, red nuclei; RP, Rathke's pouch; sv, subventricular zone; v, ventricular zone; 4, fourth ventricle.
Characterization of cDNA and genomic clones of the mouse fkh-2 gene have identified the gene as a winged helix protein with unique structural domains. The fkh-2 gene is the first rodent forkhead homolog described to date which consists of only one exon and which contains both acidic and proline-rich domains of potential transcriptional activation function. Acidic domains have been shown to be potent transcriptional activators, for example in the yeast transcription factor GCN4 (Hope et al., 1988), and it seems possible that the acidic domain A of fkh-2 is such a functional module. Domain B, located just COOH-terminal of the forkhead domain, is rich in proline and alanine. For the transcription factor CTF/NF1 (Mermod et al., 1989) a proline-rich domain has been shown to function in transactivation of target genes. In this context it is noteworthy that an amino-terminal proline-rich domain of the winged helix protein MNF has been shown to transactivate in a MNF/Gal4-fusion protein (Bassel-Duby et al., 1994). We therefore suggest that one or both of these domains of the fkh-2 gene might play a role in transcriptional activation or repression.
The fkh-2 gene appears to be the ortholog of the rat HFH-6 gene (Clevidence et al., 1993). This conclusion is supported by the almost identical amino acid sequence of the DNA binding domain and the recent finding that the HFH-6 gene in the mouse maps to chromosome 19 (Avraham et al., 1995) close to the position identified for fkh-2.
The fkh-2 gene is expressed in the developing notochord, foregut and midbrain. fkh-2 is initially expressed in headfold stage embryos in the notochord, in anterior portions of the definite endoderm and in the ectoderm, anterior to the node but excluding the tip. In the foregut fkh-2 transcripts are restricted to regions anterior to the otic vesicle between day 8 and day 9 p.c. It is tempting to speculate that fkh-2 is involved in establishing this anterior-posterior specification of the foregut.
In the neuroepithelium fkh-2 is activated in headfold stage embryos in a defined region anterior to the node but excluding the tip. This region of the ectoderm is fated to become di- and mesencephalon (Tam, 1989). The continued expression of the fkh-2 gene in these same regions suggests that this gene may be involved in their development and determination. It is interesting to note that another winged helix protein, BF-1, is localized to very specific regions of the brain, i.e. the developing telencephalon, and is thought to be required for the formation of the cerebral hemispheres (Tao and Lai, 1992). The expression patterns of these winged helix genes may be reflecting progressive differentiation of the neuroepithelium.
Subsequently, fkh-2 transcripts are localized to the anlage of the red nuclei. In the adult, the rubrospinal tract originates in the magnocellular portion of the red nucleus in the midbrain and terminates contralaterally in the spinal gray matter. This tract forms the major lateral descending pathway from the brain stem. It leads from the red nuclei through the medulla to the dorsal part of the lateral column of the spinal cord. The lateral pathways function in controlling the distal muscles used in a variety of fine movements. In humans, lesion of the red nuclei in the tegmental or central midbrain syndrome leads to hemichorea, a condition characterized by tremors and involuntary movements of the contralateral limbs. It will be interesting to investigate whether mutations of the fkh-2 gene are detrimental to motor control. Deletion of fkh-2 function through gene targeting is currently under way in our laboratory to address this question.
The close linkage of fkh-2 with mdf suggests that this mutation might be caused by a defect in fkh-2 function. mdf homozygous mice are first distinguishable from their litter mates at 5-6 weeks of age. These mice have a waddling gait and by 12 weeks can progress only by pulling themselves with their forelimbs. The mass of the sartorius, vastus lateralis, and rectus femoris is markedly reduced. It is important to emphasize that these muscles are controlled by impulses from the lateral pathway. In addition many homozygotes exhibit a nervous tremor (Womack et al., 1980). Experiments to investigate whether the mdf mutation is caused by an alteration of the fkh-2 locus are currently under way.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X86368[GenBank].