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To study the genes which may play a role in the development of the vertebrate central nervous system (CNS) using a subtraction cloning approach, we previously identified a set of novel genes which are predominantly expressed in the mouse embryonic CNS and down-regulated during development. One of these genes, drg, encodes a novel 41 kilodalton GTP-binding protein (DRG), which is highly expressed in the embryonic CNS and shows remarkable evolutionary conservation. To study the biological role of this protein during Xenopus embryonic development, we cloned the Xenopus drg cDNA (Xdrg). The predicted Xenopus DRG protein (XDRG) is more than 95% identical to the mouse DRG. Analysis of Xdrg expression by Northern blots, whole-mount in situ hybridization and RNA-PCR revealed the presence of varying levels of transcript for this gene in embryos and adult tissues. Among the three mRNA species detected by Northern hybridization, two smaller ones show temporally regulated expression patterns during embryonic development.
Fig. 3. Expression of Xdrg mRNA in Xenopus embryos_ (A) Northern blot analysis. Each lane contained 2-5 pg of poly A+ selected RNA Isolated from the total embryos ar the indicated stage of development. The same blot was sequentially hybridized to Xdrg. c-src and EF-1a probes. The positions and sizes of the three transcripts hybridizing to theXdrg probe are indicated by arrowheads. The signals shown here were obtained after approximately 48 h (Xdrg). 24 h (c-src) and 3 h (EF-la) exposures to the Xray films. Longer exposures (not shown here) reveal the presence of Xdrg and c-src hybridizing bands in stage 1and EF-7a band in stage 8 fanes also.
(BI Quantitative analysis of Xdrg expression in embryos. Hybridized filters were exposed to Bas 2000 image analyzer (Fuji) plates for 6-10 hand signals quantitated using software supplied by the manufacturer. The bars represent Xdrg 1.3 kb and 1,8 kb mRNA expression normalized against c-src expression.
Fig. 4. Whole-mount in situ hybridization analysis of XdrgmRNA distribution in Xenopus embryos. Embryos in A through E were hybridized with
an Xdrg antisense probe. No hybridization to a control sense probe was detected under similar conditions (data not shown). (A! Dorsal and(B! lateral views
of the stage 17 (neurula) embryos showing expression in neural folds (nf). IC) A stage 22 embryo showing expression in developing brain (br), otic vesicle
(ov) and the neural tube (ntJ. Note the strong signals In the anterior and posterior regions of the CNS. (D,E) A stage 32 embryo showing expression In
various head structures (brain. br: eye, ey; otic vesicle, ov), branchial arches-like structures (ba) and spinal cord. As a positive control, in (F), a stage 22
embryo was hybridized fa an antisense probe derrved from the Xenopus N-CAM cDNA (Kintner and Melton. 1987). under conditions similar to those used
for the Xdrg probe.
Fig. 5. RNA PCR analysis of dissected embryos.
Stage 22 and 32 embryos were dissected
in various sections as shown in (A), One .ug of
total RNA isolated from these dissected embryos
was reverse transcnbed and subjected to PCR for
35 cycles (B), or 15cycles (C). using a set of genespecific
primersas shown underneath each panel.
(B) shows ethidlum bromide stamedgels. while m
(C). after electrophoresis. samples were transferred
to nylon membranes and hybridized to the
respective p2Pl-labeled probes as indicated. In
(B) and (C), the lane numbers 1-7 correspond to
the different stages and regions of the embryos
as shown in (A). CI andC2are reverse transcriptase
minus and RNA minus controls respectively_ In
(C). the exposure times for Xdrg and EF-1 a were
approximately 6 hand 2 h. respectively. Longer
exposures of Xdrg hybridized membrane in IC)
showed the presence of positive signal in lane 4
also (not shown here).
Fig. 6. Expression of Xdrg in adult tissues. Approximately 5,ug of poly
A+ RNA isolated from various adult tissues was electrophoresed on
agarose/formaldehyde gel. transferred to nylon membrane and sequentially
hybndlzed to Xdrg, c-src and EF-7a probes respectively. The exposure
times for Xdrg, c-src, and EF-l a were approximately 50 h, 24 hand 4 h,
respectively. In longer exposures of Xdrg hybridized membrane, a faint 1.3
kb signal in lane 2, 1.8 kb signals in all except lane 2, and a 2.8 kb signa/In
lane 7, were visible (not shown here). The positions of 2.8, 1.8and 1.3 kb
transcripts (from top to bottom) are indicated by arrowheads. Lanes 1-8
contain RNA from brain, heart, stomach, liver, kidney, testis, ovary and
skeletal muscle respectively.
drg1 (developmentally regulated GTP binding protein 1) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Lateral view: Dorsal up, anterior left
