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The deleted in colorectal cancer (DCC) gene has been identified as a candidate tumor suppressor gene on the basis of frequent allelic loss and decreased or absent gene expression in several human cancer types, as well as somatic mutations in the gene in colorectal tumors. We have identified a Xenopus DCC homologue (XDCC alpha) predicted to encode a protein of 1427 amino acids and have characterized XDCC expression in developing embryos and adult tissues. The predicted amino acid sequences of XDCC alpha and human DCC are greater than 80% identical; each has four immunoglobulin-like domains, six fibronectin type III domains, and a cytoplasmic domain of about 325 amino acids. While RNase protection assays and immunoblotting studies failed to detect XDCC alpha expression in embryos prior to developmental stage 15, XDCC alpha expression was present in embryos from stages 19 to 46. Whole mount in situ hybridization studies localized XDCC alpha expression to developing forebrain, midbrain, and hindbrain regions. DCC expression was inhibited by treatments that altered the development of mature neural structures; specifically, uv-ventralized embryos and exogastrulae had reduced DCC expression. These results indicate that XDCC alpha is developmentally regulated and expressed as a consequence of neural induction. Moreover, unlike some well-characterized tumor suppressor genes, such as the p53 and retinoblastoma genes, that are not differentially expressed in developing Xenopus embryos, the DCC gene may have a specific role in the morphogenesis of the brain and perhaps other tissues and organs.
FIG. 1. Comparison of the predicted open reading frames of the human and Xenopus DCC eDNA sequences. (A) The amino acids (in single
letter code) comprising the complete open reading frame of the human (Hum) sequence and one of two Xenopus DCC (XLa) homologues are
shown. A portion of the sequf'll('f'!': for a second Xenn]m.<: nee (XLjl) homologue have heen charact.P.rizerl Only differenCf'R hPtWef'n thf' sequencf!S
are noted. The majority of the sequences for XL/3 from amino acids 1 to 400 have been obtained; those that have not yet been obtained are
indicated by asterisks. Gaps introduced in the XLa and Hum DCC sequences for improved alignment are indicated by dashes. Both the Hum
and XLa sequences terminate at the phenylalanine (F) noted. (B) The human and XLa homologues are each predicted to encode a protein with
a signal sequence, four immunoglobulin (lg)-like domains, six fibronectin type III domains, a membrane spanning region (TM), and a cytoplasmic
domain. The percentage identity at the amino acid level for the various domains is indicated. The two regions known to be affected by
a\ternative splicing in human DCC are noted by arrows.
FIG. 2. Analysis of XDCCa gene expression at selected developmental
stages. A ribonuclease protection assay was carried out with a 630-
bp XDCCrr. anti-sense riboprobe containing sequences from the sixth
fibronectin type III domain, the transmembrane region, and the proximal
region of the cytoplasmic domain of XDCCa. For each sample 20
stg of total RNA was incubated with the 82P-Iabeled antisense riboprobe.
Approximately 1.0 X lOS cpm of undigested probe was loaded in
the lane at the far left (probe). Yeast torula RNA (tRNA) was used
as a negative control. The expected protected fragment of 582 bp was
detected in stages 19-40 and is indicated. In a ll samples, a fraction
of the XDCCa riboprobe was not fully digested by the ribonuclease
treatment (indicated as probe).
FIG. 3. Studies of XDCC protein expression in selected Xenopus developmental stages. (A} Protein extracts from Xenopus embryos of selected
developmental stages were analyzed by SDS-PAGE on 7.5% polyacrylamide gels. After transfer to Immobilon membranes, the expression of
XDCC was studied by enhanced chemiluminescence (ECL) immunoblotting using an affinity-purified polyclonal rabbit antiserum that was
raised against a bacterial fusion protein containing XDCCa cytoplasmic sequences (antiserum 723). The M, of protein markers (X10-3) is
indicated. XDCC proteins with apparent M, of about 175-190 X 103 were detected in stages 18/19-40. (B) Combined immunoprecipitation and
immunoblotting analysis of XDCC protein expression. Extracts from selected stages were immunoprecipitated with rabbit polyclonal antisera
raised against XDCCa cytoplasmic sequences (723 and 724) or control rabbit immunoglobulin (lgG ). The immunoprecipitates were electrophoresed
and analyzed by ECL immunoblotting as described in A, except that a mouse polyclonal antiserum (6) raised against a bacterial fusion
protein containing Xenopus sequences was used.
FIG. 4. Whole mount in situ hybridization with digoxigenin-labeled XDCCa riboprobes. Stage 37/38 Xenopus embryos were studied by whole
mount in situ hybridization using a 630-bp anti-sense (A) or sense XDCCa (B) riboprobe. The probes were digoxigenin-labeled and alkaline
phosphatase-conjugated, anti-digoxigenin antibodies were used to detect hybridization signals. Specific hybridization to developing forebrain,
midbrain, and hindbrain regions was seen with the anti-sense XDCCa probe (A, arrowheads). Nonspecific staining of some areas was seen with both the sense and anti-sense XDCCa probes.
FIG. 5. Localization of XDCCa transcripts in developing brain regions of stage 37/38 embryos. As described under Materials and Methods and
the legend to Fig. 4, in situ hybridization was carried out on whole mount embryos with digoxigenin-labeled anti-sense or sense XDCCo: riboprobes
and alkaline phosphatase-conjugated anti-digoxigenin antibodies were used for detection. The embryos were embedded in paraplast and
sectioned. XDCCa transcripts were localized with the anti-sense riboprobe in forebrain (FB) and midbrain (MB) (A), midbrain (C), and hindbrain
(HB) (E) regions. Comparable sections from an embryo hybridized to the sense (control) XDCCa probe are shown in B, D, and F. While
signals in the eye (E) with tile XDCC« anti-sense I~robe were greater than those seen with the XDCCa sense I~robe, no specific signal with the
anti-senseXDCCa probe was seen in the headmesenchyme (HM), notochord (NC), or Floorplate (FP). The otic vesicle (OV) also failed to react
specifically with the XDCCa anti-sense probe.
FIG. 6. ECL-immunoblot studies of XDCC protein expression in uv-ventralized and LiCl-dorsalized embryos and exogastrulae. (A) Studies of
uv-ventralized stage 40 embryos. uv-treated embryos were allowed to develop until the equivalent of stage 40 and were then separated on the
basis of their dorsoanterior index (DAI). Extracts from pools of three embryos of equivalent DAl were prepared for each DAI value. The
schematic representation of each OAT is shown above the corresponding lane. ECL-immunoblot analysis was performed using anti-DCC antiserum
723 as described in the legend to Fig. 3. DCC protein and mobility of molecular weight markers are indicated. (B) Studies of LiCldorsalized
embryos. The analysis of XDCC expression was performed essentially as described above for the uv-ventralized embryos. (C) Studies
of stage 23 embryos and exogastrulae. Extracts from stage 23 exogastrulae (Exo) and untreated stage 23 and 40 embryos were studied by ECLimmunoblot
analysis as described above.
