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The Xenopus laevis gene tumorhead (TH) is a regulator of cell proliferation of the ectodermal germ layer during embryonic development. TH overexpression results in increased cell proliferation within the developing ectoderm, causing an expansion of the neural plate. Conversely, loss of TH function results in inhibition of proliferation of ectodermal cells. Embryos with altered levels of TH protein are unable to express neural differentiation markers, indicating that the effect of TH in proliferation is linked with differentiation in the nervous system. To date, the molecular mechanism by which TH affects cell proliferation during embryogenesis is unknown. We have utilized the yeast two-hybrid system to identify protein partners of TH that could lead us to define the mechanism or pathway through which TH functions. Using this assay we have identified a new variant of TH designated TH-B, as a potential protein partner of the original TH, now referred to as TH-A. The sequence for TH-B was found to be 85% identical at the amino acid level to the TH-A sequence. Further characterization of the TH-B variant using RT-PCR indicates that it is expressed ubiquitously throughout development from early cleavage stages until at least the tadpole stage. TH-B association with TH-A was confirmed in co-immnoprecipitation studies in Xenopus, indicating that the two variants may function as an oligomer in vivo. These studies reveal the presence of an isoform of TH that may possess novel functional capabilities.
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16525938
???displayArticle.link???Int J Dev Biol ???displayArticle.grants???[+]
Fig. 3 (Left column). Temporal analysis of TH variants expression during embryogenesis. Embryos at the indicated developmental stages were
collected and total RNA preparations were made from cell extracts from these embryos. Expression of the TH-A and TH-B variants was detected by
RT-PCR using the different RNA preparations as templates and specific primer sets for the different isoforms. 1.2 μg of total RNA was used as template
for each reaction. PCR with primers for the amplification of the eF1α message was performed as an internal experimental control. Reactions were
performed in the presence (+) or absence (-) of reverse transcriptase to assess the presence of contaminant DNA in our preparations. Amplified PCR
products were analyzed in a 2% agarose gel.
Fig. 4 (Right column). Spatial analysis of expression of TH variants during embryogenesis. Embryos at the indicated developmental stages were
collected and dissected into their animal (A) and vegetal (Vg) hemispheres, or dorsal (D) and ventral (Vt) regions, according to the developmental stage.
Total RNA preparations were made from cell extracts from whole (W) embryos or the embryos that were dissected. Expression of the TH-A and THB
variants was detected by RT-PCR using the RNA preparations and specific primer sets for the different isoforms. 800 ng of total RNA was used
as template for each reaction. PCR reactions for the amplification of TH-B were also performed in the absence of reverse transcriptase (-RT) to
determine if contaminant DNA was present in our preparations. Reactions containing primers for the amplification of the eF1α message were included
as an internal control. Amplified PCR products were analyzed in a 2% agarose gel.
Fig. 5. TH-B interacts with TH-A in Xenopus embryos. Embryos at the
two-cell stage were injected with myc-TH-A mRNA, HA-TH-B mRNA, or
a combination of both transcripts. Embryos were grown to the neurula
stage (stage 17) and cell extracts were made. Fractions of the lysates
were removed to analyze the expression of the myc-TH-A and HA-TH-B
proteins. The lysates were then incubated with anti-HA antibody (A), or
anti-myc antibody (B), followed by the addition of protein-A and protein-
G sepharose beads to trap the immune complexes. After washing the
beads, these were boiled in SDS-PAGE loading buffer. The lysate (L) and
the immunoprecipitate (IP) samples were run in 10% SDS polyacrylamide
gels, followed by anti-HA or anti-myc Western blots. Note the presence
of myc-TH-A only in the HA-IP containing HA-TH-B (A) and the presence
of HA-TH-B only in the myc-IP containing myc-TH-A (B).
