XB-ART-22866Dev Biol 1993 Feb 01;1552:361-70. doi: 10.1006/dbio.1993.1035.
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XLPOU-60, a Xenopus POU-domain mRNA, is oocyte-specific from very early stages of oogenesis, and localised to presumptive mesoderm and ectoderm in the blastula.
POU-domain proteins are a large family of transcriptional regulatory proteins, related to the homeodomain proteins, many of which are implicated in the control of gene expression during early development. We describe here the isolation of a cDNA encoding a Xenopus POU-domain protein, XLPOU-60. The predicted protein sequence of this cDNA is most closely related to the mouse germ line-specific transcription factor Oct-3/4. The XLPOU-60 gene is specifically expressed in oocytes of newly metamorphosed frogs, from the earliest stages at which transcription is known to occur. The mRNA is concentrated in the animal half of fully grown oocytes and is inherited maternally by the embryo, where it remains localised to animal cap and marginal zone cells of the blastula. Transcripts decline abruptly to a low level during gastrulation, but remain detectable throughout larval stages. However, unlike Oct-3/4, the transcript is not detectable in primordial germ cells, and XLPOU-60 is therefore probably not the functional homologue of the murine gene. We suggest that XLPOU-60 is one of the earliest genes to be transcribed in oocyte development, and that the XLPOU-60 protein may therefore be involved in initiating oocyte-specific patterns of transcription. Localisation of the transcript in the embryo may indicate that XLPOU-60 is also required for the initiation of mesoderm- and ectoderm-specific patterns of transcription in the embryo.
PubMed ID: 8432392
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: krt12.4 pou5f3.3
Article Images: [+] show captions
|FIG. 1. The sequence shown here was derived from two separate cDNAs, one of which extends from nucleotides 178 to 2629, and a second which extends from nucleotides 1 to 763 (see Materials and Methods). The POUS and POUHD domains are boxed, and two consensus polyadenylation sequences are underlined. The putative methionine start codon is in bold.|
|FIG. 2. A comparison of the amino acid sequences of the POU domains of XLPOU-60 and Oct-3/4. Identical residues are indicated with two dots, while consel'vative changes are shown with one dot. The XLPOU-60 Se<!UCllCe differs at seven positions within the POU domain that are otherwise fully conserved between class 1-V POU-domain proteins. These seven amino acids are indicated in bold. The methionine residue in the basic region at the N-terminus of the PO Uno is underlined.|
|FIG. 3. (a) Northern blot showing expression of XLPOU-60 mRNA in stage 1-VI oocytes. Stages were classified according to Dumont (1972). CK, cytokeratin transcript (loading control). (h) Localisation of the XLPOU-60 transcript in the stage VI oocyte. Lanes are as labelled.|
|FIG. 4. RNase protection assay of RNA derived from tissues of newly metamorphosed frogs. Tissues are shown above the lanes. tRN A, control Jane.|
|Ftc. 5. (a) Whole mount i·nsit·u hybridisation to dissected regions of newly metamorphosed fr·ogs. The dissected region includes the kidney (K), fat body (FB), and developing gonads (0, ovary; T, testis). (i) Female, hybridised to a sense (control) probe. (ii) Female, hybridised to an antisense probe. The dark blue/black colour indicates positive staining. (iii) Male. hybridised to an antisense probe. Scale bar, 500 ,.m. (b) Higher power view of (ii), showing expression in individual oocytes 20-30 ~<Ill in diameter. Scale bar, 50 I'm. (c) Section through the ovary in (b), viewed using Nomarski optics. The blue colou1· is found in the oocyte cytoplasm surrounding the large nuclei. No expression is detected in follicle cells surrounding the oocytes. Scale bar, 50 pm. (d) Higher power bright-field view of the section in (c). (e) The same as in (d), stained with DAPl to show nuclei. The larger oocytes are surrounded by follicle cells. Scale bar, 50 um.|
|!''IG. 6. Northern blot showing expression of XLPOU-60 in embryos. The embryonic stage is shown above each lane. Stage VI oocytes and testis are also included. The lower panel shows the same blot reprobed for the ubiquitously expressed gene EF-ln, which shows a complementary pattern of expression to XLPOU-60 (low maternal levels; increased expression following the MBT) and acts as a loading control.|
|FIG. 7. RNase protection showing XLPOU-60 expression in later larval stages. 5S, same RNA samples probed with a probe specific to the 5S rRNA gene as a loading control.|
|Fie. 8. Whole mount in situ hybridisation to embryos and larvae using an antisense XLPOU-60 probe. (a) Transverse section of a blastula (stage 8) embryo. A blue colour indicates positive staining. The transcript is perinuclear and is localised to the animal cap and marginal zone cells of the embryo. No staining is seen surrounding nuclei in the vegetal region (black arrowheads). Some superficial cells of the animal cap and marginal zone appear to lack the transcr ipt (white arrows). (b) Dissected region of a stage 44 larvae. No blue stain is seen on the surface of the dorsal body wall, where the primordial germ cells are located at this stage (arrowhead). Some blue stain is seen in the tail fin, but this is nonspecific; it was also present in larvae hybridised to a sense probe. (c) Drawing of a stage 44 tadpole to explain the dissection in (b). The dashed region was removed, leaving the tail. and the exposed dorsal body wall, with attached primordial germ cells (solid line). The dotted box indi~ates the region enlarged in (d) and (c). (d) Stage 44 tadpole as in (b), showing the primordial germ cells (arrowheads). No blue colour is detectable in these cells. (e) Drawing of (d) shows the position of the primordial germ cells on the dorsal body wall (arrowheads).|
|FIG. 9. RNase protection assay of RNA from dissected stage 8 embryos, showing localisation of the XLPOU-60 transcript in the animal cap. The marginal zone cells were trimmed away in this dissection.|