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cells did not die. Microarray was used to identify candidate genes responsible
for this TRDN-induced, rhabdomyolysis-like phenotype. They compared
nontransgenic prometamorphic (NF55) limbs and frog limbs after 2 and
6 weeks of transgene induction from NF55. Because hind limb is very sen-
sitive to TH, NF55 limbs have experienced significant TH-induced devel-
opmental changes. Candidate genes were those induced or repressed in
TRDN-expressing transgenic animals relative to NF55 controls and oppo-
site to expression changes in the nontransgenic frog limb. They found 24
candidate genes with altered regulation after 2 weeks of induction, most
of which had known muscle expression. Because the muscle lysis phenotype
became visible within 2 days, highly regulated genes were analyzed by
in situ
hybridization, and calsequestrin 1 was found to have downregulation within
1 day after induction of the TRDN transgene. Roles for the five other can-
didate genes tested, which were downregulated by 4 days, are also possible.
The other 18 candidate genes await similar analysis. Another pattern ob-
served from this analysis was that a number of genes downregulated in tail
just before tail resorption were repressed in limb muscle expressing the
TRDN transgene. Thus, tail degeneration requires T3 induction, and limb
degeneration requires the blockade of T3 induction.
2.4. Intestine
The intestine remodels extensively during metamorphosis going from a
long, coiled herbivorous gut with a single infolding to a short, carnivorous
gut with a villus-trough structure in cross section (
Shi & Ishizuya-Oka,
2001
). The three main tissue types, epithelium, connective tissue (mostly
fibroblasts), and muscle, have distinct responses to TH. During transforma-
tion, most cells of the larval epithelium die by apoptosis, and later adult
epithelial cells appear and proliferate to form the adult absorptive surface.
Fibroblasts and muscle cells also proliferate and their layers become thicker.
The same remodeling events are believed to occur whether induced by TH
or allowed to progress naturally.
To understand gene programs involved in remodeling the intestine, two
microarray analyses were carried out, one on natural metamorphosis and one
on TH-induced metamorphosis (
Buchholz, Heimeier, Das, Washington, &
Shi, 2007; Heimeier, Das, Buchholz, Fiorentino, & Shi, 2010
). For natural
metamorphosis, intestines were collected at different stages, namely
premetamorphosis (NF53, prior to endogenous TH in circulation),
prometamorphosis (NF58, beginning of larval epithelial apoptosis), climax
