April 1, 1996;
Cloning and expression of Xenopus CCT gamma, a chaperonin subunit developmentally regulated in neural-derived and myogenic lineages.
containing TCP-1 (CCT
) is a eukaryotic cytoplasmic chaperonin
, consisting of multiple distinct subunits in a double-toroid structure. In vitro, the CCT
has been shown to assist in the folding of tubulin and actin into active conformations through an ATP-dependent mechanism. The function and distribution of these proteins in vivo are also not known. In this report, we show that the expression of two CCT
subunits (alpha and gamma) are developmentally regulated in neural-derived and myogenic lineages. While expression in the central nervous system
is consistent with a role in tubulin and actin conformation, we also detect robust expression in the developing cranial neural crest
. Enrichment in the neural crest
may represent the presence of a novel substrate for the CCT
. We have also cloned the complete cDNA for the Xenopus ortholog of CCT
gamma, which has 87% amino acid identity with the mouse protein. This remarkable evolutionary conservation suggests a conserved function for this protein among vertebrates, and possibly among all eukaryotes.
[+] show captions
Fig. 2. Expression of CCTy in whole mount in situ preparations of
stage 30 Xenopus embryos. Hybridization with an antisense RNA probe
(top) identifies transcripts in the neural tube (arrowhead), pharyngeal
arches (thin arrow), and dorsolateral mesoderm (thick arrow). A sense
RNA probe (bottom) shows no specific cellular staining. There is faint
staining in the pharyngeal cavity c); histological analysis confirms that
this is not associated with cells (not shown).
Fig. 3. Expression of CCT-y in neural crest and neural tube in Xenopus
embryos. Whole embryos were stained by whole mount in situ hybridization
(see Experimental Procedures) using an RNA probe synthesized
from the full-length cDNA. Embryos were sectioned in the
horizontal plane. A: Stage 23 embryo shows expression in the neural
ectoderm (n), eye anlage (e), and migrating cranial neural crest (arrows).
B: Expression in the visceral arches of a stage 28 embryo. C: A more
dorsal section of the embryo in B shows hybridization in the neural tube
(n). eye vesicle (e), and neural crest (arrow). D: Expression is also seen
in the visceral arches of the stage 35 embryo. ph, pharynx; cg, cement
Fig. 4. CCTy is expressed in muscle. Embryos were prepared as in
Figure 3. Transcripts are detected in the somites of stage 28 (A) and 35
(8) embryos. Expression is also seen in the heart (h) of a stage 35
embryo (C). In transverse sections of stage 41 embryos (D and E), expression
of CCTy is seen in the somites (s), the facial muscle (m), and the
endoderm at the ventral midline of the oral cavity (arrow). pr, pigmented
Fig. 5. Expression pattern of CCTa during Xenopus embryogenesis
is similar to that of CCTy. In situ hybridization was performed as for
CCTy, using an RNA probe transcribed from a partial clone of Xenopus
CCTa. A: Expression in the branchial arches of stage 35 embryo. Transcripts
are also detected in the somites of a stage 28 embryo (B) and in
the facial muscle and ventral oral endoderm at stage 41 (C).
Fig. 6. Northern analysis of CCTy and CCTa transcripts. Total RNA from staged Xenopus embryos was
hybridized with random-primed DNA probes using a 500 bp PCR product (CCTy) or a partial cDNA clone
(CCTa) as a template. The 28s rRNA band is shown as a loading control.
cct3 ( chaperonin containing TCP1, subunit 3 (gamma)) gene expression in Xenopus laevis embryo, NF Stage 30, as assayed by in situ hybridization. Lateral view: Dorsal up, Anterior left
tcp1 (t-complex 1) gene expression in Xenopus laevis embryo , NF Stage 35, as assayed by in situ hybridization. Horizontal section of the head, anterior up