POU-V factors antagonize maternal VegT activity and beta-Catenin signaling in Xenopus embryos.

VegT and beta-Catenin are key players in the hierarchy of factors that are required for induction and patterning of mesendoderm in Xenopus embryogenesis. By descending the genetic cascades, cells lose their pluripotent status and are determined to differentiate into distinct tissues. Mammalian Oct-3/4, a POU factor of subclass V (POU-V), is required for the maintenance of pluripotency of embryonic stem cells. However, its molecular function within the early embryo is yet poorly understood. We here show that the two maternal Xenopus POU-V factors, Oct-60 and Oct-25, inhibit transcription of genes activated by VegT and beta-Catenin. Maternal POU-V factors and maternal VegT show an opposite distribution along the animal/vegetal axis. Oct-25, VegT and Tcf3 interact with each other and form repression complexes on promoters of VegT and beta-Catenin target genes. We suggest that POU-V factors antagonize primary inducers to allow germ layer specification in a temporally and spatially coordinated manner.

XBP1 forms a regulatory loop with BMP-4 and suppresses mesodermal and neural differentiation in Xenopus embryos.

The active form of the Xenopus X-box binding protein 1 (xXBP1) partially synergizes and partially antagonizes with BMP-4 signaling. xXBP1 overexpression inhibits mesoderm differentiation and formation of neural tissues. A functional knockdown promotes differentiation of lateral and dorsal mesoderm but not of ventral mesoderm and of neuroectoderm. We show that the active form of xXBP1 in gastrula and early neurula stage embryos is generated by removal of exon 4 and not by an endoribonuclease activity in the endoplasmic reticulum. The N-terminal region of xXBP1 which contains the basic leucine-zipper also contains a nuclear localization signal and both, the N-terminal as well as the C-terminal regions are required for xXBP1 function. The effects of xXBP1 are in part correlated to a regulatory loop between xXBP1 and BMP-4. xXBP1 and BMP-4 stimulate mutually the transcription of each other, but xXBP1 inhibits the BMP-4 target gene, Xvent-2. Both, in vitro and in vivo assays demonstrate that xXBP1 interacts with BMP-4 and Xvent-2B promoters. GST-pulldown assays reveal that xXBP1 can interact with c-Jun, the transcriptional co-activator p300 and with the BMP-4 responsive Smad1. On the other hand, xXBP1 also binds to the inhibitory Smads, Smad6 and Smad7, that can act as transcriptional co-repressors. Based on these data, we conclude that xXBP1 might function as an inhibitor of mesodermal and neural tissue formation by acting either as transcriptional activator or as repressor. This dual activity depends upon binding of co-factors being involved in the formation of distinct transcription complexes.

The POU factor Oct-25 regulates the Xvent-2B gene and counteracts terminal differentiation in Xenopus embryos.

The Xvent-2B promoter is regulated by a BMP-2/4-induced transcription complex comprising Smad signal transducers and specific transcription factors. Using a yeast one-hybrid screen we have found that Oct-25, a Xenopus POU domain protein related to mammalian Oct-3/4, binds as an additional factor to the Xvent-2B promoter. This interaction was further confirmed by both in vitro and in vivo analyses. The Oct-25 gene is mainly transcribed during blastula and gastrula stages in the newly forming ectodermal and mesodermal germ layers. Luciferase reporter gene assay demonstrated that Oct-25 stimulates transcription of the Xvent-2B gene. This stimulation depends on the Oct-25 binding site and the bone morphogenetic protein-responsive element. Furthermore, Oct-25 interacts in vitro with components of the Xvent-2B transcription complex, like Smad1/4 and Xvent-2. Overexpression of Oct-25 results in anterior/posterior truncations and lack of differentiation for neuroectoderm- and mesoderm-derived tissues including blood cells. This effect is consistent with an evolutionarily conserved role of class V POU factors in the maintenance of an undifferentiated cell state. In Xenopus, the molecular mechanism underlying this process might be coupled to the expression of Xvent proteins.

Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis.

We report on the temporal and spatial expression pattern of two novel genes of the Xenopus fork head/winged helix family, xFoxB2 and xFoxI1c. xFoxB2 is activated at the late blastula stage and first expressed within the dorsolateral ectoderm except for the organiser territory. During gastrulation, xFoxB2 is found in two ectodermal stripes adjacent to the dorsal midline. Expression is completely down-regulated during neurulation. However, two distinct sets of cells expressing xFoxB2 re-appear in the rhombencephalon of swimming tadpoles. xFoxI1c is initially expressed at the early neurula stage in an epidermal ring around the neural field. Subsequent expression is found to be increased, and is exclusively localised to placodal precursor cells. The placodal expression remains until stage 40, when it is restricted to a distinct region in the lateral body wall behind the gills.