Robertson AJ et al. (2013), Sea urchin akt activity is Runx-dependent and r...

ECB-ART-42919

Biol Open 2013 Mar 25;25:472-8. doi: 10.1242/bio.20133913.

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Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division.

Robertson AJ , Coluccio A , Jensen S , Rydlizky K , Coffman JA .

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In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

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Genes referenced: irak1bp1 LOC100887844 LOC100893907 LOC105441782 LOC115919910 LOC115929578 LOC373206 LOC582192 LOC583082 runx2
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	Fig. 1. Summary of the effects of Runt-1 knockdown in relation to the temporal pattern of cell proliferation and runt-1 expression in the sea urchin embryo.(A) The canonical temporal pattern of cell proliferation during sea urchin embryogenesis (black curve) (adapted from Davidson, 1986), related to the temporal pattern of runt-1 transcript accumulation (dotted blue line) (adapted from Coffman et al., 1996). Runt-1 transcripts accumulate 10-fold (per embryo) from early to late blastula stage, i.e. between 12 and 24 hours post-fertilization (hpf) (Coffman et al., 1996). (B) Effects of Runt-1 knockdown. The first obvious effect in runt-1 morphants consists of impaired cell proliferation between 18–24 hpf (Robertson et al., 2008); subsequently the morphants exhibit widespread apoptosis and differentiation defects (Coffman et al., 2004; Dickey-Sims et al., 2005). The gene expression microarray analysis (NCBI GEO accession number GSE19751) was performed on 18 hr embryos.
	Fig. 2. Sp-akt-1 and Sp-akt-2 gene schematics and mRNA levels over developmental time in normal and runt-1 morphant blastulae.(A) Schematic of the Sp-akt-1 and Sp-akt-2 loci from build 3.1 of the S. purpuratus genome, with exons depicted as boxes (non-coding grey, coding black), and putative Runx target sites matching the consensus TGT/CGGT (or its reverse complement) marked as thin lines below. A 600 bp repeat sequence in intron 4 (*) contains a tandem array of 56 potential Runx target sites (see supplementary material Fig. S2). (B) Relative transcript levels from both genes at eight time points spanning embryogenesis, with maternal akt-2 set to 1. Error bars represent the standard deviations of replicate measurements. (C) Relative akt-1 and akt-2 mRNA levels in runt-1 morphants (black bars) compared to controls (grey bars) in two different batches of experimental embryos, one collected at 17 hpf (average of four replicate measurements ± the standard deviation), the other at 19 hpf (average of three replicate measurements ± standard deviation). Relative levels are with respect to levels measured in normal 12 hr embryos in the experiment depicted in (B). The fact that akt-2 transcript levels are somewhat higher and much less strongly affected at 17 hrs than at 19 hrs most likely reflects biological variability between the batches of embryos (obtained from different outbred crosses). This amount of biological variability in expression levels is not unusual in sea urchins.
	Fig. 3. Effects of perturbing Akt function on development and blastula stage cell proliferation.(A) Morphological effects of MASOs directed against akt-1 and akt-2. Scale bar: 20 µm. (B) Cell numbers in control and akt-2 morphants, and in embryos over-expressing wild type and kinase-dead (K186M) akt-1 mRNA, from 10–15 hpf. (C) Confocal Projections of control and akt-2 morphants labeled with EdU from 14–20 hpf. The EdU label (red) is incorporated into replicating DNA during the period of exposure; the DAPI (blue) indicates the total nuclear DNA. Scale bar: 20 µm. (D) Quantification of EdU labeling from the experiment shown in (B), showing the average number of nuclei per embryo displaying an EdU fluorescence intensity above a specific threshold (>25% of the DAPI signal), ± the SD. (E) Projections of fluorescent confocal images taken of untreated control embryos and embryos treated immediately after fertilization with the Akt inhibitor API-2 (25 µM) then labeled with EdU (red) for 2 hrs, beginning at 18 hrs post-fertilization; the DAPI (blue) indicates the total nuclear DNA. Scale bar: 20 µm. (F) Quantification of the results of an EdU labeling experiment, showing the number of nuclei displaying an EdU fluorescence intensity above a specific threshold (>25% of the DAPI signal), ± the SD. A total of 200 nuclei were counted for each sample, representing 2 control and 4 treated embryos. (G) Effects of API-2 treatment (5 µM) on total cell numbers at late blastula stage. Bars indicate average cell number per embryo from 10–15 embryos, ± the SD. Significance values calculated by t-test.
	Fig. 4. Rescue of development in runt-1 morphants with akt-1 mRNA.(A) Two day runt-1 morphant displaying gastrulation defective phenotype, and runt-1 morphant co-injected with full-length akt mRNA at 0.3 mg/ml. Scale bar: 20 µm. (B) Quantification of gastrulation in runt-1 morphants co-injected with increasing [akt mRNA]. (C) Average cell numbers ± SD in 24 hr uninjected late blastula stage embryos, runt-1 morphants, and runt-1 morphants co-injected of akt-1 and akt-1-K186M mRNA. Six to ten embryos were counted in each group. Significance value calculated by t-test.
	Fig. 5. A hypothetical regulatory circuit through which Runt-1 regulates cell proliferation in the mid-to-late blastula stage embryo.Gene names are italicized below the symbol for the corresponding gene. Protein products are capitalized. Positive cis-regulatory inputs are shown as arrows terminating on genes; negative (inhibitory) protein–protein regulatory interactions are shown as bars terminating on the named proteins; stimulatory effects are shown as arrows. Solid lines represent experimentally verified or well-established interactions/effects; dashed lines represent hypothetical interactions/effects. Interactions/effects not analyzed in this paper are shown in the background. Evidence and supporting references for the other interactions are given in Robertson et al. (Robertson et al., 2008).

References [+] :

Aoki, The akt kinase: molecular determinants of oncogenicity. 1998, Pubmed