Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development.

In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.

Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates.

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.

The Meis3 protein and retinoid signaling interact to pattern the Xenopus hindbrain.

In Xenopus embryos, proper hindbrain formation requires activities of both XMeis3 protein and retinoic acid (RA) signaling. In this study, we show that XMeis3 protein and RA signaling differentially interact to regulate hindbrain patterning. The knockdown of XMeis3 protein prevented RA-caudalizing activity from inducing hindbrain marker expression in both explants and embryos. In contrast, inhibition of RA signaling differentially modulated XMeis3 activity. Target genes that are jointly activated by either RA or XMeis3 activities could not be efficiently induced by XMeis3 when RA signaling was inhibited. However, transcription of an XMeis3 target gene that is not an RA target gene was hyper-induced in the absence of retinoid signaling. Target genes jointly induced by RA or XMeis3 protein were synergistically activated in the presence of both activities, while RA treatment inhibits the ability of XMeis3 to activate transcription of neural genes that are not RA targets. HoxD1, an RA direct-target gene was also identified as an XMeis3 direct-target gene. HoxD1 protein acts downstream of XMeis3 to induce hindbrain marker gene transcription. To pattern the hindbrain, RA requires functional XMeis3 protein activity. XMeis3 protein appears crucial for initial hindbrain induction, whereas RA signaling defines the spatial limits of hindbrain gene expression by modifying XMeis3 protein activity.

Replication and/or separation of some human telomeres is delayed beyond S-phase in pre-senescent cells.

Cultured primary human cells, which lack telomerase, enter a state of replicative senescence after a characteristic number of population doublings. During this process telomeres shorten to a critical length of approximately 5-7 kb. The mechanistic relationship between advanced cell passage, cellular senescence and telomeric function has yet to be fully elucidated. In the study described here, we investigated the relationship between changes in telomeric replication timing and/or sister chromatid separation at telomeric regions and advanced cell passage. Using fluorescence in situ hybridization, we analyzed the appearance of double hybridization signals (doublets), which indicate that the region of interest has replicated and the replicated products have separated sufficiently to be resolved as two distinct signals. The results showed that the replication and separation of several telomeric regions occurs during the second half of S-phase and that a delay in replication and/or separation of sister chromatids at these regions occurs in pre-senescent human fibroblasts. Surprisingly, in a significant percentage of pre-senescent cells, several telomeric regions did not hybridize as doublets even in metaphase chromosomes. This delay was not associated with extensive changes in methylation levels at subtelomeric regions and was circumvented in human fibroblasts expressing ectopic telomerase. We propose that incomplete replication and/or separation of telomeric regions in metaphase may be associated with proliferative arrest of senescent cells. This cell growth arrest may result from the activation of a mitotic checkpoint, or from chromosomal instability consequent to progression in the cell cycle despite failure to replicate and/or separate these regions completely.

High glucose-induced replicative senescence: point of no return and effect of telomerase.

Primary human cells enter senescence after a characteristic number of population doublings (PDs). In the current study, human skin fibroblasts were propagated in culture under 5.5mM glucose (normoglycemia); addition of 16.5mM D-glucose to a concentration of 22 mM (hyperglycemia); and addition of 16.5mM L-glucose (osmotic control). Hyperglycemia induced premature replicative senescence after 44.42+/-1.5 PDs compared to 57.9+/-3.83 PDs under normoglycemia (p<0.0001). L-Glucose had no effect, suggesting that the effect of hyperglycemia was not attributed to hyperosmolarity. Activated caspase-3 measurement showed a significantly higher percentage of apoptotic cells in high glucose medium. Telomerase overexpression circumvented the effects of hyperglycemia on replicative capacity and apoptosis. The "point of no return," beyond which hyperglycemia resulted in irreversible progression to premature replicative senescence, occurred after exposure to hyperglycemia for as few as 20 PDs. These results may provide a biochemical basis for the relationship between hyperglycemia and those complications of diabetes, which are reminiscent of accelerated senescence.