The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova

The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain.

DNA binding by the Oct-1 protein is directed by its POU domain, a bipartite DNA-binding domain made up of a POU-specific (POUS) domain and a POU-homeo (POUH) domain, two helix-turn-helix-containing DNA-binding modules that cooperate in DNA recognition. Although the best-characterized DNA target for Oct-1 binding is the octamer sequence ATGCAAAT, Oct-1 also binds a number of different DNA sequence elements. For example, Oct-1 recognizes a form of the herpes simplex virus VP16-responsive TAATGARAT element, called the (OCTA-)TAATGARAT site, that lacks octamer site similarity. Our studies suggest two mechanisms by which Oct-1 achieves flexible DNA sequence recognition. First, an important arginine found in the Oct-1 POUS domain tolerates substitutions of its base contacts within the octamer site. Second, on the (OCTA-)TAATGARAT site, the POUS domain is located on the side of the POUH domain opposite from where it is located on an octamer site. This flexibility of the Oct-1 POU domain in DNA binding also has an impact on its participation in a multiprotein-DNA complex with VP16. We show that Oct-1 POUS domain residues that contact DNA have different effects on VP16-induced complex formation depending on whether the VP16-responsive element involved has overlapping octamer similarity or not.     

Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice

Purpose

To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).

Methods

Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1^loxP and Pou4f2^loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.

Results

Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.

Conclusion

Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.     

Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.