Annual Report 2006

Expression patterns of two novel
genes isolated from regenerating
individuals of an oligochaete
annelid,
The spatiotemporal expression profiles of
two novel genes, and which
were isolated from regenerating
were examined by RT-PCR and
whole mount hybridization (WISH). For
both genes, the expression was scarcely
detectable in intact worms, but was dramatically
activated following amputation. The expression
levels were highest at the blastema formation
stage (around 12-24 hours after amputation).
The results suggest that these genes play
important roles in annelid regeneration (Fig. 3).
Ppet genes have a function on
differentiation / proliferation of
ES cells and also early
development in the mouse
We have previously reported that mouse
ES cells were divided into three subpopulations
according to the expression levels of platelet
endothelial cell adhesion molecule 1 (PECAM-1)
and stage-specific embryonic antigen (SSEA)-1.
Quantitative RT-PCR and chimera formation
revealed that the expression level of PECAM-1
and SSEA-1 were positively correlated with
pluripotency of ES cell subpopulations. In order
to identify novel regulatory factors in ES cell
differentiation, we have performed comparison
of gene expression profiles by oligo-DNA array
analysis between three subpopulations. In this
fiscal year, we focused on uncharacterized 23
genes (Ppet: PECAM-1 Positive ES cell-derived
Transcripts) and attempt to elucidate the
functions of them in ES cells and early embryos
by small interfering RNA (siRNA) mediated
gene knockdown.
When fluorescein-labeled Ppet siRNAs
were transfected into ES cells, plating efficiency
of these ES cells showed no significant
difference between fluorescein negative and
positive cells in all groups. Akaline phosphatase
(AL-P, a marker for undifferentiated ES cells)
positive colonies were decreased by
knockdown of Ppet 002, 005, 010, 015, 021 and
023 gene as well as Oct3/4 (Fig. 4). In contrast,
AL-P positive colonies were increased by
knockdown of Ppet 001, 003, 004 and 019 genes.
Next, we performed knockdown of Ppet gene in
early mouse embryos. The embryos that
injected Ppet015, 019 and 021 siRNA exhibited
developmental retardation and degradation. In
cases of Ppet 019 and 021 gene knockdown,
total cell numbers of blastocysts were reduced
without morphological abnormality. Conversely,
Ppet 010, 022 and 023 gene knockdown caused
developmental facilitation. In cases of Ppet 022
Fig. 3
Expression patterns of and
RT-PCR was performed using the total RNAs from intact and regenerating at 3-96 hours after
amputation (A). The number of PCR cycles used are indicated at the right of each panel. WISH analysis was
performed using DIG-labeled antisense riboprobes against regenerating fragments at 24 (B-D) or 36 hours after
amputation (E). Lateral views, the anterior is to the left. Arrowheads indicate the base of the blastema. e,
esophagus; g, gut.
Scale bar = 200 µminB.