Paracentrotus lividus Genes in Development: Lithium - Sensitive Kinase

GSK3B/shaggy

Function

GSK3B/shaggy is a litium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus.
In the sea urchin embryos the level of GSK3B activity controls the position of the boundary between the presumptive ectoderm ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos.
Southern blot analysis demonstrated that a GSK3B is a single copy gene which produces 3 different transcripts (SU-G3, SU-G8, SU-G9). SU-G3 and SU-G9 are about 6.5 kb long and SU-G8 is about 2.2 kb long (Emily-Fenouil et al., 1998).

Protein

The three GSK3B transcripts produce 3 different proteins by alternative splicing.
These three transcripts encode different proteins which are identical for about 90% of their length and sequence of their short N-terminal region. The N-terminal sequence of the SU-G3 protein is most similar to GSK3B from other organisms.
The SU-G9 protein is longer by 18 amino acids and SU-G8 is shorter by 15 residues. The protein coded for by SU-G3 (Su-GSK3B) is predicted to be 414 amino acids long giving a Mr of 46300 (Emily-Fenouil et al., 1998).
GenBank: 2959981 (Su-GSK3B)

Subcellular location

Expression Pattern

Nothern blot analysis detected the transcripts of about 6.5 kb. This indicates that clones SU-G3 and SU-G9 were nearly full- length cDNAs, while the SU-G8 cDNA was truncated.
Su-GSK3B transcripts are present in the unfertilized egg, and the maternal level is mantained during the early cleavage stages. The transcript level then declines rapidly at the blastula stage and remains low at all stages from hatching blastula to pluteus.
SU-G3 (which is largely prevalent) and SU-G9 both showed this expression profile, while SU-G8 transcripts were found to be expressed at a very low but almost constant level throughout development.
Whole-mount in situ hybridization using the probe which detects all types of the transcripts, gave strong labeling in unfertilized eggs and early stages. The signal decreased at late blastula and was faint at the gastrula stage, in agreement with nother blot data. Transcripts did not display any obvious localization in unfertilized eggs and did not become restricted to any particular lineage or area in early embryos (Emily-Fenouil et al., 1998).

mRNA level

Temporal accumulation

Method: Nothern blot analysis
Reference: Emily-Fenouil et al., 1998

Stage
Unfertilized egg
16-cell stage
60-cell stage
6h blastula
7h blastula
8h blastula
11h blastula
Mesenchyme blastula blastula
Gastrula
Prism
Pluteus
SU-G3 level
+
+
+
+
+
+
+ -
+ -
+ -
+ -
+ -
SU-G9 level
+
+
+
+
+
+ -
+ -
+ -
+ -
+ -
+ -
SU-G8 level
+ -
+ -
+ -
+ -
+ -
+ -
+ -
+ -
+ -
+ -
+ -

Ectopic expression

Effect of injection of kinase-dead GSK3B mRNA into unfertilized eggs

mRNA coding for kinase-dead GSK3B were injected into unfertilized eggs at dozes varying from about 0.4 pg to 4 pg per egg (4* 105 to 4* 105).
Doses below 0.4 pg had no visible effect on embryo morphology. Around the threshold value of 0.4 pg, most embryos (>95%) were unaffected, but a few had an abnormal pluteus-like shape.
At about 0.8 pg injected embryos displayed a range of phenotypes. The digestive tract was greatly enlarged and was formed by exogastrulation, reflecting overdevelopment of the endoderm at the expense of the ectoderm. Spicules had abnormal shapes and were strongly reduced in most cases. There are classic features of the vegetalized morphology obtained by treatment with vegetalizing agents such as lithium.
At 2 pg up to 4 pg, nearly all embryos ( >95%) consisted of a hollow sphere of thick epithelium, with a small invagination, one or two clusters of pigmented cells and a few other cells within the central cavity. Spicules were totally absent.
Such extreme phenotypes have never been observed following lithium treatment, probably because high concentrations of lithium are toxic. Except for this extreme form, the series of increasingly abnormal phenotypes closely resembled that obtained with increasing concentrations of lithium.
These results suggest that GSK3B is the target for lithium in the sea urchin embryo, with the kinase-dead GSK3B having a dominant- negative effect that mimics lithium inhibition.
Injection of kd-GSK3B also reduce the HE expression domain. The results resemble those observed while treatment with lithium (Ghihlione et al., 1993) but the effect is stronger since HE can be repressed to an undetectable level (Emily-Fenouil et al., 1998).

Dose
<0.4 pg
~0.4 pg
~0.8 pg
2-4 pg
Effect (48 hour embryos) No visible effect Most embryos (>95%) were unaffected, but a few had an abnormal pluteus-like shape Embryos displayed a range of phenotypes. The digestive tract was greatly enlarged and was formed by exogastrulation. Spicules had abnormal shapes and were strongly reduced in most cases Nearly all embryos ( >95%) consisted of a hollow sphere of thick epithelium, with a small invagination, one or two clusters of pigmented cells and a few other cells within the central cavity. Spicules were totally absent

Animalization by misexpression of wild-type GSK3B

Microinjection of about 1 pg per egg of either sea urchin or Xenopus GSK3B produced embryos with a narrow range of altered phenotypes.
Embryos overexpressing GSK3B did not gastrulate. In a very few cases ( <1%), embryos had the overall shape of a gastrula with apparently normal spicules but no archenteron. In most cases the morphology was simpler: embryos were spherical, sometimes slightly flattened, and frequently had very long cilia. The epithelium was thickened on one side, indicating morphological polarity. One single, small abnormal spicules or no spicules were seen. Very few or no cells were present inside the blastocoel. These features characterize the animalized phenotype, which can be obtained by treatment with a variety of chemical agent, including zinc.
Misexpression of the wild-type GSK enlarges the HE spatial domain. The enlargement in the HE expression territory parallels the change in morphology provoked by overexpression of wt-GSK3B, and suggests that wt-GSK3B produce a true animalization, resulting from an extension of the presumptive ectoderm territory (Emily-Fenouil et al., 1998).

Dose
~1 pg per egg
Effect (48 hour embryos) <1% of embryos had overall shape of gastrula with apparently normal spicules but no archenteron.
In most cases embryos were spherical, sometimes slightly flattened, and frequently had very long cilia. The epithelium was thickened on one side, one single, small abnormal spicules or no spicules were seen. Very few or no cells were present inside the blastocoel

Sequences

GenBank:

Regulatory Regions

Regulatory Connections

Upstream Genes

GSK3B/shaggy

Downstream Genes

Evolutionary Homologues

Links

Bibliography
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Comments are welcome to Sveta Surkova
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