Pou5f1 contributes to dorsoventral patterning by positive regulation of vox and modulation of fgf8a expression.

Pou5f1/Oct-4 in mice is required for maintenance of embryonic pluripotent cell populations. Zebrafish pou5f1 maternal-zygotic mutant embryos (spiel ohne grenzen; MZspg) lack endoderm and have gastrulation and dorsoventral patterning defects. A contribution of Pou5f1 to the control of bmp2b, bmp4 and vox expression has been suggested, however the mechanisms remained unclear and are investigated in detail here. Low-level overexpression of a Pou5f1-VP16 activator fusion protein can rescue dorsalization in MZspg mutants, indicating that Pou5f1 acts as a transcriptional activator during dorsoventral patterning. Overexpression of larger quantities of Pou5f1-VP16 can ventralize wild-type embryos, while overexpression of a Pou5f1-En repressor fusion protein can dorsalize embryos. Lack of Pou5f1 causes a transient upregulation of fgf8a expression after mid-blastula transition, providing a mechanism for delayed activation of bmp2b in MZspg embryos. Overexpression of the Pou5f1-En repressor induces fgf8, suggesting an indirect mechanism of Pou5f1 control of fgf8a expression. Transcription of vox is strongly activated by Pou5f1-VP16 even when translation of zygotically expressed transcripts is experimentally inhibited by cycloheximide. In contrast, bmp2b and bmp4 are not activated under these conditions. We show that Pou5f1 binds to phylogenetically conserved Oct/Pou5f1 sites in the vox promoter, both in vivo (ChIP) and in vitro. Our data reveals a set of direct and indirect interactions of Pou5f1 with the BMP dorsoventral patterning network that serve to fine-tune dorsoventral patterning mechanisms and coordinate patterning with developmental timing.

Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade.

pou5f1, also known as Oct4, is required to establish the pluripotent cell population necessary for embryogenesis in mouse. Additional roles during development, including endoderm formation, have been proposed. In zebrafish, the zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate patterning defects and reduced endoderm at the tailbud stage. To investigate the function of maternal and early zygotic pou5f1 expression, we rescued zygotic spg(m793) mutants by injecting pou5f1 mRNA at the one-cell stage and raised them into fertile homozygous spg(m793) adults that mate to produce maternal-zygotic spg (MZspg) mutant embryos. Although neurectoderm, mesoderm, and germ cells develop in MZspg mutants, gastrulation is delayed and proceeds abnormally. Further, MZspg mutants do not maintain expression of sox32/casanova, express little or no sox17, and fail to develop endodermal tissue. Constitutively active Nodal receptor TARAM-A or sox32 overexpression induces ubiquitous sox17 expression in wild-type embryos, but not in MZspg mutants. Overexpression of a Pou5f1-VP16 activator fusion protein can rescue gastrulation and endodermal tissues in MZspg mutants. We propose that pou5f1 plays an activating role in zebrafish endodermal development, where it maintains sox32 expression during gastrulation and acts with sox32 to induce sox17 expression in endodermal precursor cells.

Activation of the knirps locus links patterning to morphogenesis of the second wing vein in Drosophila.

The adjacent knirps (kni) and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. Here, we identify a 1.4 kb cis-acting enhancer element from the kni locus that faithfully directs gene expression in the L2 primordium. We find that three independent ri alleles have alterations mapping within the L2-enhancer element and show that two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri(53j) mutant. We also find that misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium.

spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.