(Received for publication, January 24, 1997, and in revised form, April 14, 1997)
From the Several genetic factors have been proven to
contribute to the specification of the metencephalic-mesencephalic
territory, a process that sets the developmental foundation for
prospective morphogenesis of the cerebellum and mesencephalon. However,
evidence stemming from genetic and developmental studies performed in
man and various model organisms suggests the contribution of many additional factors in determining the fine subdivision and
differentiation of these central nervous system regions. In man, the
cerebellar ataxias/aplasias represent a large and heterogeneous family
of genetic disorders.
Here, we describe the identification by differential screening and the
characterization of Mmot1, a new gene encoding a
DNA-binding protein strikingly similar to the helix-loop-helix factor
Ebf/Olf1. Throughout midgestation embryogenesis, Mmot1
is expressed at high levels in the metencephalon, mesencephalon, and
sensory neurons of the nasal cavity. In vitro DNA binding
data suggest some functional equivalence of Mmot1 and Ebf/Olf1,
possibly accounting for the reported lack of olfactory or neural
defects in Ebf A number of transcription factor genes regulate cell
identity in specific body regions, both in invertebrates and
vertebrates (1). In vertebrates, the Hox genes control
identity along the body axis and provide positional cues for the
developing neural tube, particularly the rhombencephalon and spinal
cord from the branchial area to the tail. Conversely, development of
the anteriormost body domain, including the metencephalic,
mesencephalic, and prosencephalic territories (2), has remained
relatively obscure in invertebrates and vertebrates alike. Indeed, the
molecular specification of compartments or subdivisions in the
vertebrate forebrain is still a matter of debate (3).
Specifically, regarding morphogenesis of the
metencephalon-mesencephalon boundary, a recent breakthrough came with
the identification of the mouse En-1 and En-2
genes, which were cloned based on their homology to the
Drosophila segment polarity gene engrailed (4). En-2 homozygous mutant mice created by homologous
recombination in embryonic stem cells are viable and exhibit a
patterning defect in the cerebellum (5, 6). In contrast,
En-1 homozygous mutant mice die at birth and show a deletion
of most of the colliculi and cerebellum (7). Likewise, the
Wnt and Pax families of genes have been
implicated in cerebellar patterning by means of genetic or
neurobiological studies (8, 9), whereas the Fgf-8 gene has
been shown to play a critical role in the induction of the isthmic
organizing center (10).
Despite these and other relevant advances, mostly based on
developmental mechanisms conserved from Drosophila to
vertebrates, our knowledge of rostral central nervous system
differentiation in general and of metencephalic-mesencephalic
specification in particular remains fragmentary to date, and many other
as yet unidentified regulatory genes may at different times play a role in various cell fate specification or terminal differentiation processes.
To help elucidate some of the molecular mechanisms underlying the fine
subdivision and differentiation of primary brain structures during
midgestation brain development, our group set out to screen for
developmentally regulated genes restricted in their spatial and
temporal expression domains within the embryonic head. This was
achieved through a modification (11)1 of a
PCR-based2 differential screening technique
named RNA fingerprinting (12, 13).
Among other embryonic central nervous system genes of regulatory
significance found in this way, we have isolated a new helix-loop-helix (HLH) transcription factor gene, Mmot1
(metencephalon-mesencephalon-olfactory transcription factor 1), differentially
expressed along the anteroposterior axis, displaying a sharp anterior
expression boundary within the diencephalon as well as a high level
specific expression in the sensory portion of the olfactory
epithelium.
HLH transcription factors are nuclear proteins that bind DNA as
homo- or heterodimers. HLH transcription factors have been subdivided
into various subfamilies (14), and their role has been recognized in
Drosophila neurogenesis and sex differentiation as well as
vertebrate myogenesis (15) and neurogenesis (16, 17). The newest
subclass of HLH proteins identified so far includes two virtually
identical, independently cloned genes: a mouse gene named
Ebf (early B-cell
factor) (18) and a rat gene named
Olf1 (olfactory-neuronal transcription
factor) (19) as well as their
Drosophila homolog (collier) (20). A specific
feature of this subfamily is that its members lack the basic domain
found upstream of the first The present paper describes the isolation, genetic characterization,
in situ expression studies, and in vitro DNA
binding properties of Mmot1, a new member of the
Ebf/Olf1-like subclass of HLH transcription factors.
Standard molecular techniques including
nucleic acid purification, restriction analysis, gel electrophoresis,
DNA ligation, cloning, subcloning, dideoxy sequencing, probe
radiolabeling, Northern and Southern analysis, RNase protection assays,
and library screening were performed according to established protocols
(22). Automated sequencing with Dyedeoxy primers or Dyedeoxyterminators was performed on an ABI 373 machine. Hybridizations of Northern, Southern, and zoo blot filters (Pall) were performed at 65 °C in 125 mM sodium phosphate (pH 7.2), 250 mM NaCl, 7%
SDS, 10% polyethylene glycol. Filters were washed at 65 °C to final
stringencies of 0.2 × SSC (1 × SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7) for 10 min. Phage plaque
hybridizations and subsequent washes were carried out under comparable
stringency conditions.
Preparations of E12.5
embryonic central nervous system samples were done as follows. Under a
dissection microscope, brain tissue was separated from surrounding
mesoderm and ectoderm. Neuroectodermal tissue spanning fourth ventricle
through midbrain was separated from prosencephalic territories. Fresh
tissue preparations from two CD1 litters were pooled and lysed in
guanidine isothiocyanate. RNA extraction was carried out on a cesium
chloride gradient (22).
Clone 203 was derived through a
modification1 of the RNA fingerprinting protocol (13)
comparing mRNAs of mouse E12.5 mesencephalon, E12.5 prosencephalon,
and postnatal mouse brain and cerebellum. RNA fingerprinting was
conducted as follows. A reverse transcription reaction was carried out
using a (dT)16 primer on total RNAs extracted by the cesium
chloride method (22) and digested with 4 IU of DNase I/µg of total
RNA. Radioactive PCR reactions were performed in duplicate from 1 µl
of each RT reaction in a 50-µl final volume with an arbitrary 12-mer
(DR34, sequence 5 Data bank searches
(GenBankTM, GenEmbl, SwissProt, and Protein Identification
Resource) were run through the BLAST server (23). Additional sequence
analysis and contig assembly was done using the MacVector program
(Oxford Molecular Group) and the Sequencher program (Gene Code Corp.),
respectively. The nucleotide sequence of the gene was deposited into
the GenBankTM data base with accession number U71189.
Genetic mapping was done on 96 DNAs
corresponding to the parentals and 94 N2 progeny of a
(C57BL/6j × SPRET/Ei)F1 × SPRET/Ei (BSS) backcross
generated and distributed by The Jackson Laboratory (Bar Harbor, ME)
(24). An MspI restriction fragment length polymorphism was
identified in an intronic sequence and amplified with primers p1 and p2
(sequences 5 Total RNAs from mouse preparations at
E12.5, P4, and adult brain and liver were used in quantitative PCR
reactions designed to work within the linear (exponential) range of
amplification. Total RNA was reverse-transcribed using a random hexamer
primer (Life Technologies, Inc.), and the quantity of cDNA
synthesized in each RT reaction was first normalized by means of PCR
amplifications with mouse glyceraldehyde-3-phosphate
dehydrogenase primers (sequences 5 Radioactive and nonradioactive in situ
hybridization (27) was carried out as follows. 7-µm paraffin serial
sections from a single embryo were displaced in 4-6 adjacent series;
two alternative series were used for each probe. Four embryos from at
least two litters were studied at 12.5 and 13.5 days of embryonic
development. Slides were deparaffinated in xylene, hydrated through an
alcohol series, treated with paraformaldehyde and proteinase K,
acetylated, and dehydrated through an ethanol series. For radioactive
in situ studies, 1 µl (3 × 106 cpm) of
Mmot1 riboprobe labeled with 35S-UTP (Amersham)
in the hybridization mix was added to each slide. For nonradioactive
in situ studies, 1 µg of Mmot1-linearized
plasmid was transcribed in vitro in the presence of 0.8 µl
of 10 mM digoxigenin-11-UTP (Boehringer Mannheim). Both
sense and antisense probes were used. Hybridization was carried out
overnight at 65 °C. Slides were washed under stringent conditions
(65 °C, 2 × SSC, 50% formamide) and treated with RNase A. Autoradiography was performed with Kodak NT/B2 emulsion. Exposure times
were 15 days. Sections were examined and photographed on dark and
bright fields using a Zeiss SV11 microscope. Nonradioactive signal was
revealed through an alkaline phosphatase-conjugated anti-digoxigenin
antibody (Boehringer Mannheim) as recommended by the manufacturer.
Immunohistochemical analysis was conducted with a monoclonal anti-PCNA
antibody (clone PC10, Boehringer Mannheim). Sections were incubated
with a peroxidase-conjugated secondary antibody, and signal was
revealed with the Vectastain ABC kit (Vector Laboratories, Inc.) as
recommended.
A Mmot1-specific 379-nt
sequence was cloned into pBluescript and used to synthesize a riboprobe
by in vitro transcription with T7 RNA polymerase and
incorporation of [ In vitro transcription,
transcript purification, and translation using rabbit reticulocyte
lysate (Promega) were done according to manufacturer recommendations,
with the addition of 10 µM ZnSO4. The
efficiency of in vitro translation was assayed by running parallel translation reactions performed in the presence of
[35S]methionine (Amersham). The double-stranded synthetic
DNA fragment carrying the binding site for the Ebf/Olf1 protein
(5 Cloning of Mmot1 by PCR-based Differential Screening
We applied a modified RNA fingerprinting protocol
(11)1 to the analysis of differential gene expression in
the embryonic and postnatal mouse brain. By RNA fingerprinting, we
compared the following stages and districts: E12.5, mesencephalon + cerebellar primordium; E12.5, prosencephalon; P4, brainstem + cerebellum; P4, forebrain. As primers we employed a panel of arbitrary
12-mers, some of which were carrying a partially degenerate position at their 3
Genetic Linkage Analysis Defines Mmot1 as a New Member of the
Ebf/Olf1-like Gene Family
Genetic evidence obtained by other authors (32) had assigned
Ebf to proximal mouse chromosome 11. To strengthen our
evidence defining Mmot1 and Ebf as distinct
genes, we set out to localize Mmot1 in the mouse genome by
linkage analysis in the BSS backcross generated and maintained at The
Jackson Laboratory (24). Using a primer pair (p1 and p2) from a region
of low degree homology with Ebf, we amplified a 2.7-kilobase
genomic fragment spanning an intronic sequence in the coding portion of
the gene. The experiment was conducted on the parental strain DNAs of
the BSS backcross (C57BL/6JEi, B6, and SPRET/Ei spretus).
Automated sequencing of the product ends confirmed them as part of the
Mmot1 gene. The PCR product was digested with frequent
cutters (RsaI, Sau3AI, TaqI, and
MspI). An MspI polymorphism was identified
consisting of 2,450- and 250-base pair fragments in B6 DNA and a
2,700-base pair fragment in spretus DNA. This polymorphism
was employed to type the 94 individual N2 progeny of the
BSS backcross by PCR and restriction fragment length polymorphism
analysis. 93 out of 94 progeny were typed successfully. Linkage
analysis performed with MapManager 2.6 unequivocally localized
Mmot1 to mouse chromosome 14, 1.1 centimorgan distal to
Raftk (33) (lod score 25.6) and 2.3 centimorgan proximal to
Nfl3 (34) (lod score 22.6). The
data are summarized in Fig. 2. The human homolog of
Nfl has been mapped to chromosome 8p21 (35, 36).
Cloning of the Full Coding Sequence
Based on the above evidence, we set out to isolate clones
spanning the entire coding sequence of Mmot1. To this end,
we plated out 6 × 105 plaque-forming units from an
embryonic day 11.5 whole-embryo cDNA library
(CLONTECH, ML1027). Again, as a probe, we utilized a region of Mmot1 displaying low degree similarity to
Ebf. After high stringency hybridization and washes, we
isolated five positives, one of which spanned the full-length
transcript (5.4 kilobases, corresponding to the band detected by
Northern analysis and not shown). By adopting a strategy involving both
shotgun cloning and primer walking, we obtained the double strand
sequence of the cDNA. The sequence contains a 1659-base pair open
reading frame preceded by an in-frame stop codon (TAA, Protein Sequence Analysis
The deduced peptide sequence (553 residues) was analyzed with a
variety of local and on-line programs. The primary sequence is 80.6%
identical to the Ebf protein, with more conserved regions clustered
around a putative zinc finger domain (residues 134-147, sequence
HEVMCSRCCEKKSC) and a helix-loop-helix domain (residues 344-387,
sequence KEMLL ... VPRNP). Also perfectly conserved is a putative
nuclear targeting domain (residues 219-223, sequence RRARR). A
Drosophila melanogaster gene named collier
(accession number X97803) was also found to encode a highly similar
protein (20). The deduced peptide sequence of Mmot1 is
illustrated in Fig. 3 and compared with the two other
known members of the subfamily (Ebf/Olf1 and Collier).
Mmot1 Belongs to an Expanding, Phylogenetically Conserved Gene
Family
Conservation of the Mmot1 gene was assessed
experimentally by zoo blot analysis. A Southern blot containing DNAs of
six mammalians, a frog, and chicken was hybridized with a fragment of
the Mmot1 coding sequence (positions 1340-2340) that shows
the highest degree of divergence from Ebf. Both
hybridization and washes were carried out at high stringency conditions
(see "Experimental Procedures"). The experiment suggested the
existence of strongly conserved homologs of Mmot1 in all
organisms tested, including chicken and Xenopus laevis (Fig.
4a).
Because the isolation of Mmot1 in mouse defines a new family
of closely related mammalian HLH proteins, we looked to identify possible new homologs of our gene and Ebf/Olf1 in the
Expressed Sequence Tag (EST) data base (37). The search was conducted with the BLAST programs using the Mmot1 protein as a query sequence and
revealed the existence of at least one other member of the same family,
represented in the murine (W14732) (Fig. 4b) and human
(W21838) EST collections. Murine EST W14732 and human EST W21838 are
probably orthologous to each other and distinct from
Ebf/Olf1 and Mmot1. At the nucleotide level,
Mmot1 and W14732 are 77.1% identical. As mentioned, a
search of GenBankTM revealed a homolog of
Ebf/Olf1 and Mmot1 in Drosophila
melanogaster (collier, accession number X97803). At the
nucleotide level, Mmot1 and collier are 70.9%
identical. One additional member of the same gene family was identified
in Caenorhabditis elegans (accession number C13312).
Finally, a dedicated analysis of the complete yeast genome through the
TBLASTN program identified no high score homologs in that unicellular
eukaryote.
Expression of Mmot1 in the Midgestation Embryo and Adult
To confirm the expression data obtained by RNA fingerprinting
(Fig. 1) and quantitative RT-PCR (not shown) and to finely characterize the distribution of our transcript in the embryo, we performed in
situ hybridization of mouse tissue sections at embryonic days 10.5-14.5 (Fig. 5). As a riboprobe, we employed a
377-nt fragment (position 1340-1717) displaying 66.3% identity to
Ebf.
mRNA in situ hybridization of
embryonic tissue sections at 10.5 (a and b), 12.5 (c-g), 13.5 (h-n and s), and 14.5 (o-q) days postcoitus. b, f,
l, m, n, q, and s are
coronal sections; all others are sagittal sections. g,
k, and n are negative controls, hybridized with a
sense riboprobe. r, schematic summary of transcript distribution at E12.5-13.5.
s, nonradioactive in situ hybridization of an
E13.5 mesencephalic coronal section. t, histochemical
analysis (anti-PCNA monoclonal antibody) of an adjacent section.
Cb, cerebellum; drg, dorsal root ganglia;
fp, floor plate; DT, dorsal thalamus; GE, ganglionic eminence; I, isthmus;
M, mesencephalon; ne, nasal epithelium;
p1-p4, prosomeres; r1-r4, rhombomeres;
rp, roof plate; sc, spinal cord; T,
telencephalon; V, trigeminal ganglion; or, optic
recess.
Mmot1 expression (Fig. 5, a and
b) is localized to the whole rhombencephalon
(r1-r4) and spinal cord caudally and to the mesencephalon (M) and pretectal anlage (prosomere 1) rostrally. Both alar
and basal plate cells are positive, unlike the floor plate
(fp) and roof plate (rp). In the peripheral
nervous system, the gene is expressed in the trigeminal ganglion
(maxillary mesenchyme) and in the dorsal root ganglia. Mmot1
expression was also detected in the dorsal maxillary epithelium.
At this stage (Fig. 5, c-f, negative
control in g), the expression of Mmot1 includes a
rostral domain (metencephalic-mesencephalic territory, excluding the
basal portion of the fossa isthmica), a caudal domain (spinal cord),
and the anlage of the nasal epithelium. Rostrally, expression stops
between mesencephalon and p1, to resume weakly in prosomere
1 and, more strongly, between p1 and the dorsal thalamus
(p2), caudal to the retroflex or habenula-interpeduncular tract; the epithalamus is clearly negative. Moreover, low level signal
is observed in the preoptic area and mamillary region. In the r1-r4
interval, expression spans the cerebellar plate and pontine nuclei.
Caudally, the expression domain stops abruptly at the r4-r5
interrhombomeric boundary, to resume from r7 all the way to the tail,
and is restricted to the alar plate neuroepithelium. The fossa isthmus
does not present labeling in its ventral midline, whereas the
trigeminal and dorsal root ganglia are still positive.
At these stages (E13.5: Fig. 5, h-n,
negative controls in k and n; E14.5: Fig. 5,
o-q) central nervous system areas expressing the gene at
high levels are virtually restricted to the mesencephalon and
metencephalon, whereas the cerebellar plate appears less intense in its
ventricular zone. Very weak labeling is also observed in the alar plate
of the rostral diencephalon and telencephalon. While the alar spinal
cord expresses Mmot1 at E13.5, this expression disappears at
E14.5. The distribution of Mmot1 in the context of the
prosomeric model (3) is schematically summarized in Fig. 5r.
Extraneural labeling for Mmot1 can be detected in the limb
bud. At all stages examined, very strong signal is present in the
olfactory portion of the nasal cavity, with a sharp demarcation at the
boundary with the respiratory epithelium, which does not express the
gene. No other major expression sites could be seen at the embryonic
stages examined.
High resolution, nonradioactive in situ analysis was
conducted on E13.5 sections with an Mmot1 cRNA probe (Fig.
5s). In parallel, immunohistochemical analysis was carried
out on adjacent sections with an anti-PCNA monoclonal antibody (Fig.
5t) used as a marker of proliferating neuroblasts (38). The
comparison reveals that Mmot1 is expressed in a thin
postmitotic stratum of the ventricular wall, apical to the interkinetic
migration range of neuroepithelial cells.
To extend the characterization of Mmot1 expression beyond
embryonic development, we determined the distribution of the transcript in 11 adult mouse tissues by RNase protection assay (Fig.
6). In the adult mouse, Mmot1 is specifically
expressed in the cerebellum, muscle, heart, ovary, and testis. Lower
expression levels are shown in the adult brain. Very low level or
absent signal is observed in the thymus, kidney, liver, spleen, and
intestine.
Mmot1 Binds the Nucleotide Target Site of Olf1 in Vitro
To characterize the DNA binding properties of Mmot1, in a
comparison with its close cognate Ebf/Olf1, we synthesized the protein by in vitro transcription and translation of a full coding
Mmot1 cDNA subclone (ab1). The translation
product (60 kDa) matched the size of our deduced amino acid sequence.
The DNA binding domains of Mmot1 and Ebf/Olf1 are extremely similar,
and we therefore tested whether Mmot1 could recognize the same sequence
bound by Ebf/Olf1. Labeled double-stranded oligonucleotides
corresponding to the wild type and mutant Olf1 recognition sites (29)
were employed in a gel mobility shift assay. A band shift was observed after incubation of the Mmot1 protein produced by in vitro
translation with the wild type double-stranded oligonucleotide but not
with a mutant double-stranded oligonucleotide in which the Olf1 target site was disrupted (Fig. 7).
The present paper reports the successful application of a
PCR-based, internally primed RNA fingerprinting technique (13, 39)1 to the isolation of a gene displaying a restricted
pattern of expression in the midgestation embryonic mouse brain. Among
other potentially significant results,4
this approach has led to the identification of Mmot1, a new
helix-loop-helix-type DNA-binding protein homologous to Ebf/Olf1 (19).
To date, this family included three known members: a rat gene named
Olf1 (19), a virtually identical mouse gene named
Ebf (32), and the Drosophila gene
collier (20).
collier appears to be involved in anterior head patterning
in Drosophila. Its expression is dependent on a gene
expression program involving cytoplasmic polarity genes
(bicoid) and gap rule genes (buttonhead, in
particular) (20). A phenocopy of the collier mutant,
produced by transgenic insertion of an antisense construct, has
displayed down-regulation of the En gene in the intercalary
segment but not in the mandibular one (20). In Drosophila, collier is only expressed in actively proliferating
territories of neuroectodermal origin (20). In a comparison with Mmot1
and Ebf/Olf1, the Drosophila protein appears to lack the
second helix of the HLH domain featured in its mammalian counterparts
(Fig. 3). This suggests that Collier might be unable to assemble into dimers or bind a palindromic sequence and thus might be involved in the
transcriptional regulation of a nonoverlapping developmental pathway
with respect to its mammalian homologs.
Although the mouse gene Ebf and the rat gene Olf1
are reportedly translated from different start sites, they most likely
represent orthologous sequences and encode virtually identical proteins (18, 19), with well characterized regulatory functions in B-cell and
olfactory development. In the present paper, we provide genetic
evidence that Mmot1 represents a separate, phylogenetically conserved member of the same expanding gene family. Moreover, through
the analysis of the EST data base (37, 40), we report the
identification of one high quality homologous sequence (accession number W14732) corresponding to an adult mouse brain cDNA, suggesting the existence of an additional, as yet uncharacterized, Ebf/Olf1-like transcription factor gene in mammalian
genomes.
At all stages tested by in situ hybridization, the
Mmot1 transcript is found in a finely delimited marginal
stratum of the neural tube. The distribution of Mmot1
compared with that of a proliferation marker (PCNA) (38) suggests that
our gene is expressed precociously in a nascent postmitotic,
subventricular layer, possibly involved in the genesis and/or
differentiation of specific neural cell types.
The Mmot1 transcript is expressed from the mesencephalon to
the tail at 10.5 days of embryonic development but becomes restricted to the metencephalon-mesencephalon and nasal neuroepithelium at E14.5,
after which it remains expressed in the adult cerebellum. Between E12.5
and E13.5, the embryonic distribution of the Mmot1 transcript presents the uncommon feature of a sharp anterior expression boundary within the embryonic diencephalon (between prosomere 1 and 2),
with low level expression in prosomeres 3-5 and undetectable levels in
the telencephalon. This property may contribute to the definition of
factors involved in the fine subdivision of the embryonic forebrain,
providing a posterior boundary (between p1 and
p2) in the context of the prosomeric model reviewed in Refs. 3 and 41.
In addition to displaying high expression levels in the cerebellar
primordium, midbrain, and dorsal thalamus, Mmot1 is
transcribed at remarkable levels in the olfactory neurons of the nasal
cavity and vomeronasal organ. Olf1, a closely related gene,
was found by other authors to drive the expression of several
olfactory-specific proteins (19). However, mice homozygous for induced
mutations of Ebf, the mouse ortholog of Olf1,
display no alterations in the morphogenesis of the olfactory area or in
the expression of olfactory proteins (42). Likewise, they present with
no obvious abnormalities in midbrain or hindbrain development. Evidence
of strong similarities in the expression of Ebf/Olf1 (42)
and Mmot1 (present paper) at many sites in the developing
brain and nasal neuroepithelium provides a possible explanation for the
lack of neurodevelopmental and olfactory defects in
Ebf In summary, Mmot1 is a new, developmentally regulated,
restrictedly expressed member of a novel, expanding family of neural transcription factors whose analysis may have considerable impact on
the study of midgestation neural development in general and the
mechanisms of normal and aberrant cerebellar ontogeny in
particular.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U71189. The contribution of Alessandro Bulfone
(Telethon Institute of Genetics and Medicine, Milan) has been truly
essential for completion of this work. Thanks to Antonello Mallamaci
for helpful discussions and to Fabio Grassi for the gift of mouse RNAs.
We thank Lucy Rowe and Mary Barter (The Jackson Laboratory) for sharing
the BSS genetic mapping panel. We are deeply thankful to the late Emilia Viganotti for her generous donation to our study of brain development.
Department of Biological and Technological
Research (DIBIT),
Institute of
Morphological Sciences, University of Murcia School of Medicine,
E-30071 Murcia, Spain
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/
knockout mutants. The
isolation of Mmot1 and of an additional homolog in the
mouse genome defines a novel, phylogenetically conserved mammalian
family of transcription factor genes of potential relevance in studies
of neural development and its aberrations.
helix in basic HLH transcription
factors, which mediates DNA binding. In Ebf, the establishment of
DNA-protein interactions is mediated by an N-terminal domain, inclusive
of a zinc finger element, whereas the HLH domain appears exclusively
involved in dimerization (21).
General Methods
-GACGAGGCTGGA) (final concentration, 4 µM). PCR conditions were 3 min at 94 °C, 2 min at
80 °C in which Taq polymerase was added (hot start),
followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C, 30 s at 72 °C, with a final elongation step of 5 min at 72 °C. 0.1 µl of [
-32P]dCTP was added to each reaction.
Amplified products were separated on a 5% denaturing acrylamide gel
and visualized by autoradiography. Differentially displayed bands were
cut from the gel and electroeluted. The bands were reamplified using
the same 12-mer primers and blunt-end cloned into pBluescript II SK+
(Stratagene) as described (11).
-GGTTGGCCATAGGAACATT and 5
-TCTTTCCAGCTCCCCAGC) as
described under "Results." Its segregation was followed, and linkage analysis was performed with the MapManager 2.6 program (25).
-CGCATCTTCTTGTGCAGTG and
5
-GTTCAGCTCTGGGATGAC). Each reaction was conducted incorporating
0.1 µl of [
-32P]dCTP in the mix. Amplification
products were separated through polyacrylamide gels and quantitated by
densitometry (Image Quant, Molecular Dynamics). At each stage examined,
identical amounts of cDNA from each RT reaction were then used in
two parallel PCR amplifications using Mmot1-specific primers
(p1 and p2, see above). A touchdown PCR (26) was conducted using 3 min
at 94 °C, 2 min at 80 °C in which Taq polymerase
(Perkin-Elmer) was added; 2 cycles of 1 min at 94 °C, 1 min at
62 °C, 1 min at 72 °C; 2 cycles of 1 min at 94 °C, 1 min at
60 °C, 1 min at 72 °C; 2 cycles of 1 min at 94 °C, 1 min at
58 °C, 1 min at 72 °C; 21 cycles of 1 min at 94 °C, 1 min at
56 °C, 1 min at 72 °C; and a final elongation of 5 min at
72 °C.
-32P]UTP (800 Ci/mmol) (Amersham
Life Science, Inc.). 50 µg of RNA from each of 11 different adult
mouse tissues was hybridized, treated, and polyacrylamide gel
electrophoresis-separated as described (28). Normalization was achieved
through a mouse
-actin riboprobe.
-ACCCATGCTCTGGTCCCCAAGGAGCCTGTC) (29) and a control DNA
fragment where the binding site had been mutated
(5
-ACCCATGCTCTGGTCAGCAAGGAGCCTGTC) were end-labeled
with [
-32P]ATP (Amersham) using 20 units of T4
polynucleotide kinase (Ambion Inc.) according to established protocols
(28). In vitro DNA binding and electrophoretic mobility
shift assays were performed as described (30), except that 2 µg of
poly(dI-dC) were used as a nonspecific competitor. In each binding
reaction (20 µl) we employed 0.15 pmol of labeled double-stranded DNA
(about 40,000 cpm); 10 µl were then applied to a nondenaturing 6%
polyacrylamide gel.
ends obtained through computer simulations of PCR experiments run on a nonredundant mouse nucleotide data base.1 At
embryonic day 12.5, band 203 (778 nt, nucleotides 939-1717 of the
full-length transcript), obtained with primer DR34, amplified almost
exclusively in the posterior region (metencephalon-mesencephalon), whereas it failed to show an obvious band at postnatal day 4 (P4) either in the anterior or posterior head (Fig. 1). The
band was gel-excised, reamplified, and cloned into pBluescript II SK
(Stratagene). Clones were screened as described (11), and plasmid
203.14 was manually sequenced. A data base search was run with BLASTN
and BLASTX using the Genetics Computer Group interface (31). The search
revealed 72% identity at the nt level with a mouse gene named
Ebf for early
B-cell factor (18)
encoding a helix-loop-helix transcription factor. Ebf is
virtually identical to an independently cloned rat gene named
Olf1 (olfactory transcription factor 1) (19). To confirm that Mmot1 and
Ebf are indeed different genes, we analyzed the
Mmot1 cDNA by restriction mapping, identifying SacII and HinfI restriction sites absent from
Ebf (or Olf1) (positions 1529 and 1472 of the
Mmot1 transcript, respectively) as predicted from sequence
analysis.
Fig. 1.
RNA fingerprinting experiment comparing
cDNAs from anterior and posterior head territories on the 13th day
of prenatal development (E12.5) and on the 5th day of
postnatal development (P4). RNA fingerprinting RT-PCR
reactions were conducted in duplicate at each stage examined.
Arrowhead, band 203 corresponding to the Mmot1
transcript.
[View Larger Version of this Image (53K GIF file)]
Fig. 2.
Mapping of Mmot1 in the mouse
genome. Above, haplotype and linkage analysis of
Mmot1 and flanking loci on mouse chromosome 14 through the
analysis of the BSS backcross (The Jackson Laboratory). Empty
squares indicate the Mus spretus allele; solid
squares indicate the C57BL/6J allele. Shaded squares,
genotype not determined. Numbers to the right
indicate recombination fractions ± S.E. and lod scores.
Columns represent different haplotypes observed on chromosome 14. Numbers below columns define the number of
individuals sharing each haplotype. Below, position of
Mmot1 on chromosome 14 with respect to nearby markers
independently mapped by others on the BSS backcross. Numbers
on the left represent approximate genetic distances from the
most centromeric chromosome 14 marker in this cross.
[View Larger Version of this Image (36K GIF file)]
66).
Fig. 3.
Sequence alignment of Ebf/Olf1, Mmot1, and
the Drosophila melanogaster protein Collier. Boxed
residues are identical in different proteins, from N terminus to C
terminus. Shaded residues represent the zinc finger element,
nuclear targeting domain, and helix-loop-helix domain, respectively.
The second helix of the conserved HLH domain is not present in the
Collier protein.
[View Larger Version of this Image (92K GIF file)]
Fig. 4.
Phylogenetic conservation. a, high
stringency Southern (zoo blot) analysis of 10 µg of DNA from each of
eight mammalian and nonmammalian species. The filter was hybridized
with an Mmot1-specific probe displaying >40% divergence
from the corresponding region of the Ebf transcript. For
details on hybridization and washing conditions, see "Experimental
Procedures." b, the analysis of the EST data base revealed
the existence of several possible new members of the Ebf family of HLH
factors in the human and mouse genomes. W14732 is an adult mouse brain
EST, clearly distinct from Ebf and Mmot1.
Boxed nucleotides are identical in at least two sequences.
The region shown corresponds to nt 956-1305 of the Ebf gene
and 1038-1387 of the Mmot1 gene, spanning the segment encoding the HLH domain.
[View Larger Version of this Image (64K GIF file)]
Fig. 5.
[View Larger Version of this Image (86K GIF file)]
Fig. 6.
Expression of Mmot1 in 11 adult
mouse tissues tested by RNase protection assay with a 379-nt
Mmot1-specific antisense riboprobe featuring 36%
divergence from the corresponding region of the Ebf
cDNA. Persistent expression in the adult cerebellum and, at
very low levels, in the adult brain is revealed by this assay.
[View Larger Version of this Image (88K GIF file)]
Fig. 7.
Gel mobility shift assay revealing specific
affinity of the Mmot1 protein for the Olf1 nucleotide binding site
described in Ref. 29. Lanes contain the following: no
lysate, -32P end-labeled double-stranded
oligonucleotide; RRL, same as no lysate but with
the addition of rabbit reticulocyte lysate; 4 µl and
1 µl, in vitro synthesized, unlabeled protein
after incubation with 4 and 1 µl
-32P end-labeled
double-stranded oligonucleotide, respectively. A band shift
(doublet) is observed after incubation of Mmot1 with the
wild type (wt) nucleotide site but not with a mutant
(mut) nucleotide site featuring two substitutions that
disrupt the palindrome (see "Experimental Procedures" for sequences
of wild type and mutant sites).
[View Larger Version of this Image (70K GIF file)]
/
knockout mutant mice. The notion of
genetic redundance in the pathway involving Ebf/Olf1 and
Mmot1 is strengthened by in vitro functional
evidence presented in this paper, which proves that Ebf/Olf1 and Mmot1
share DNA binding affinity and specificity (29) as expected based on
the marked similarities in their dimerization and DNA binding domains.
Moreover, similarities in the HLH domains of the two proteins suggest
the possibility that they might assemble as heterodimers in those
territories where the corresponding genes are coexpressed. In this
scenario, the generation of Mmot1 lack- or gain-of-function
mutants and the analysis of double knockout mutants for
Mmot1 and Ebf will assist in the genetic
dissection of functional pathways involving the two genes while
clarifying their role in midbrain/hindbrain subdivision and olfactory
development.
*
This work was supported by Italian Telethon Grant B14 and
European Union Grant BMH4-CT96-0777.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These two authors contributed equally to this work.
**
To whom correspondence should be addressed: DIBIT-HSR, via
Olgettina 58, I-20132 Milano, Italy. Fax: 39-2-26434855; E-mail: consaleg{at}dibit.hsr.it.
1
G. G. Consalez, A. Cabibbo, A. Corradi, C. Alli,
M. Sardella, R. Sitia, and R. Fesce, submitted for publication.
2
The abbreviations used are: PCR, polymerase
chain reaction; HLH, helix-loop-helix; RT, reverse transcription; PCNA,
proliferating cell nuclear antigen; contig, group of overlapping
clones; BSS, (C57BL/6j × SPRET/Ei)F1 × SPRET/Ei; nt,
nucleotide(s); EST, expressed sequence tag.
3
R. Turner and J. Nadeau, unpublished data.
4
A. Corradi and G. G. Consalez, unpublished
data.
A Practical Approach, IRL Press at Oxford University Press, Oxford
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.