Dev Growth Differ
September 1, 2012;
Pou-V factor Oct25 regulates early morphogenesis in Xenopus laevis.
POU-V class proteins like Oct4
are crucial for keeping cells in an undifferentiated state. An Oct4
homologue in Xenopus laevis, Oct25
, peaks in expression during early gastrulation, when many cells are still uncommitted. Nevertheless, extensive morphogenesis is taking place in all germ layers at that time. Phenotypical analysis of embryos with Oct25
overexpression revealed morphogenesis defects, beginning during early gastrulation and resulting in spina-bifida-like axial defects. Analysis of marker genes and different morphogenesis assays show inhibitory effects on convergence and extension and on mesoderm
internalization. On a cellular level, cell-cell adhesion is reduced. On a molecular level, Oct25
overexpression activates expression of PAPC
, a functional inhibitor of the cell adhesion molecule EP/C-cadherin. Intriguingly, Oct25
effects on cell-cell adhesion can be restored by overexpression of EP/C-cadherin or by inhibition of the PAPC
function. Thus, Oct25
affects morphogenesis via activation of PAPC
expression and subsequent functional inhibition of EP/C-cadherin.
Dev Growth Differ
[+] show captions
Fig. 1. Morphogenesis phenotypes of Oct25 mRNA injected embryos. (A-D) Vegetal views of a uninjected blastula stage 9 (A), gastrula stages 10 (B) and 11 (C), and a dorsal view at early neurula stage 14 (D). (E-H) The corresponding stages of embryos injected in the ani- mal pole with Oct25 mRNA. (I) Dorsal view of an uninjected embryo at the neural tube stage 19. (J) Dorsal view of an uninjected embryo at tadpole stage 28. (K-L) Corresponding stages of Oct25 mRNA injected embryos showing the weaker phenotype with condensed axial and head structures. (M-N) Corresponding stages of Oct25 mRNA injected embryos showing the stronger phenotype with split axial structures
Fig. 2. Expression pattern of germ layer specific markers in Oct25 mRNA injected embryos. Shown are the mesodermal marker Xbra (vegetal view), mesodermal expression of XSna (vegetal view), the paraxial mesodermal marker PAPC (dorso-vegetal view), the endoder- mal marker Xsox17a (vegetal view), and the early dorsal and neural marker Sox3 (dorsal view). (A-E) Expression of these markers in uninjected embryos at midgastrula stages 11-11.5. (F-J) Corresponding expression in embryos injected with Oct25 mRNA in the animal pole. (K-O) Corresponding expression in embryos after injection of Oct25 mRNA in the marginal zone. Appendant values are listed in Table S2.
Fig. 3. Expression pattern of axial and paraxial markers in Oct25 mRNA injected embryos show axis formation defects. Shown are dor- sal to dorso-vegetal views of the mesodermal marker Xbra, the axial protocadherin AXPC, the axial marker Chd, the paraxial PAPC, and the paraxial expression of the early muscle marker MyoD. (A-E) Expression of these markers in uninjected embryos at the late gastrula stage 12. (F-J) Corresponding expression in embryos injected with Oct25 mRNA in the animal pole. (K-O) Expression of these markers in uninjected embryos at stages that have a similar blastopore size as the Oct25 mRNA injected embryos. Appendant values are listed in Table S3.
Fig. 4. Injection of Oct25 mRNA in the ectodermal animal cap (AC) regulates the paraxial marker mesodermal PAPC independent of mesendoderm induction. Animal views (with dorsal up) of embryos from whole mount in situ hybridization, labeled for PAPC, mesoder- mal marker Xbra, endodermal marker Xsox17a, epidermal ectoderm marker XK81A1 (epidermal keratin), neurectodermal marker Sox2. Corresponding values are listed in Table S4. (A) Animal view of an uninjected embryo labeled for PAPC. (B) Frontal section of an unin- jected embryo labeled for PAPC. The insert shows a magnification from the indicated region. (C, D) Animal views of uninjected embryos labeled for Xbra and Xsox17a. (E, F, G, H) Corresponding embryos and section injected with Oct25 mRNA in the animal pole. The insert in (F) shows the ectopic PAPC expression. (I, J) Animal views of uninjected embryos labeled for XK81A1 and Sox2. (K, L) Corresponding embryos injected with Oct25 mRNA in the animal pole. (M) Reverse transcription-polymerase chain reaction (RT-PCR) on uninjected whole embryos (WE), uninjected ACs (AC), Oct25 mRNA injected ACs (Oct25 AC), water control (H2O), one of the corresponding - RT controls. The mesodermal markers PAPC and Xbra, the endodermal marker Xsox17a, the epidermal keratin (XK81), the neurectodermal Sox2, the classical EP/C-cadherin, and H4 were analyzed. (N) Effects of the Oct25 knock down on expression of paraxial protocadherin (PAPC) and Xbra.
Fig. 5. The Effects of Oct25 on convergence and extension movements analyzed with different assays. (A) Shape changes of explants of the dorsal marginal zone (DMZ) were measured by calculating the quotient of length and width (l/w). Explants were excised with l/w % 1. After cultivation for 24 h, explants were classified as shown. l/w 1.5 is defined as "not elongated", 1.5 < l/w < 2.5 is defined as "weak elongation", l/w > 2.5 is defined as "strong elongation". (B) Changed elongation after injection of Oct25 mRNA in the animal pole (Oct25 in animal cap [AC]), in the marginal zone (Oct25 in MZ), and in the vegetal pole (Oct25 in VG). In addition, the effect on Acti- vin A induced elongation is shown by comparing Activin A induced ACs with (Oct25 in AC+act.) and without Oct25 injection (uninj. AC +act.). Asterisks indicate high significance (v2 test, a 0.01). (C) AC explants (AC) without treatment (left), AC induced with Activin A (middle), AC injected with Oct25 mRNA and treated with Activin A (right). (D) Photographs of embryos labeled with red EosFP in the DMZ. The anterior-posterior axis is diagonal, the blastopore to the lower left corner (dashed line). Left side: Uninjected embryo at begin- ning of gastrulation and at the end. Right side: Embryo injected with Oct25 mRNA at the corresponding stages. Note that the blastopore remains open in the injected embryos. Corresponding values are shown in Table S5.
Fig. 6. Effects of Oct25 on cell adhesion as analyzed by dissociation assays. Animal cap (AC) explants are cultivated in dissociation buffer and photographed at the indicated times (0, 10, 20, 30, 60 min). To ensure exactly identical treatment, control and test samples were placed in one dish. Corresponding values are shown in Table S6. (A) AC explants from uninjected embryos (AC, left side) and from Oct25 mRNA injected embryos (Oct25, right side). (B) Explants from Oct25 mRNA injected embryos (Oct25, left side) and from embryos injected with Oct25 and EP/C-cadherin (Oct25 + EP/C, right side). (C) Explants from Oct25 mRNA injected embryos (Oct25, left side) and from embryos injected with Oct25 and the dominant inhibitory PAPC construct SXPAP (Oct25 + SXP, right side).
Fig. 7. Effects of Oct25 on cell adhesion as analyzed by reaggregation assays. Cells from dissociated animal caps (ACs) were cultivated in Modified Barth's solution (MBS) on a mutator to allow reaggregation. To analyze level of reaggregation, photographs were taken at the indicated times (0, 2, 24 h). Related values are listed in Table S7. (A) AC cells from uninjected embryos (uninj., left column) and from Oct25 mRNA injected embryos (oct25, right column). (B) Comparison of reaggregation levels after injection of Oct25 (left column) and coinjection of Oct25 and EP/C-cadherin (right column). Reaggregation times are indicated. (C) Comparison of reaggregation levels after injection of Oct25 (left column) and coinjection of Oct25 and the dominant inhibitory PAPC construct SXPAP (right column). Reaggrega- tion times are indicated. (D) Model of Oct25 effects on convergence and extension via regulation of cell adhesion molecules. Oct25 induces expression of PAPC. PAPC inhibits the function of EP/C-Cadherin. EP/C-cadherin affects convergence and extension via changes in cell adhesion.