Sullivan SA et al. (2001), foxD5a, a Xenopus winged helix gene, maintains ...

XB-ART-8913

Dev Biol 2001 Apr 15;2322:439-57. doi: 10.1006/dbio.2001.0191.

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foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent on the C-terminal domain.

Sullivan SA , Akers L , Moody SA .

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Xenopus foxD5a, the full-length fork head gene previously described as a PCR fragment (XFLIP), is first detectable at stage II of oogenesis. Low-abundance maternal transcripts are localized to the animal hemisphere of the cleavage embryo, and protein can be translocated to the nucleus prior to the onset of zygotic transcription. Zygotic expression is strongest in the presumptive neural ectoderm at gastrula and neural plate stages, but there is minor paraxial mesodermal expression during primary gastrulation that becomes significant in the tail bud during secondary gastrulation. Expression of foxD5a in animal cap explants induces elongation and expression of mesodermal, neural-inducing, and early neural-specifying genes, indicating a role in dorsal axis formation. Zygotic foxD5a expression is induced strongly by siamois, moderately by cerberus, weakly by Wnt8 and noggin, and not by chordin in animal cap explants. Expression of foxD5a in whole embryos has differential dorsal and ventral effects. Ventral mRNA injection induces partial secondary axes composed of expanded mesodermal and epidermal tissues, but does not induce ectopic neural tissues. Dorsal mRNA injection causes hypertrophy of the neural plate and expansion of early neural genes (sox3 and otx2), but this is not the result of increased proliferation or expanded neural-inducing mesoderm. The neural plate appears to be maintained in an immature state because otx2 expression is expanded and expression of en2, Krox20, proneural genes (Xnrgn1, neuroD) and a neural differentiation gene (n-tubulin) is repressed in foxD5a-expressing cells. These results indicate that foxD5a maintains an undifferentiated neural ectoderm after neural induction. Expression of foxD5a constructs fused with the engrailed repressor domain or with the VP16 activation domain demonstrates that FoxD5a acts as a transcriptional repressor in axis formation and neural plate expansion. Deletion constructs indicate that this activity requires the C-terminal domain of the protein.

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Species referenced: Xenopus
Genes referenced: actl6a cer1 chrd dnai1 eef1a2 egr2 elavl1 en2 foxa2 foxd4l1.1 foxd4l1.2 gdf1 gmnn h4c4 myc neurod1 neurog2 nodal3.1 nodal3.2 nog otx2 sia1 sox2 sox3 tbx2 tubb2b wnt8a wnt8b

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	FIG. 1. Sequence analysis of Xenopus foxD5a. (A) Schematic representations of foxD5a mRNA and expression constructs derived from it. For some experiments, six copies of a myc epitope (not shown) were added to the N-terminus. (B) Alignment of Xenopus FoxD5a to some high-scoring BLAST hits. Vertebrate representatives of all Class D subtypes are shown; rat HNF3b, a low-scoring match from Class A, is included to illustrate typical similarities and differences between known gene-repressing WHD proteins (2) and transactivating WHD proteins (1). Identity to FoxD5a is indicated by a dash. WHD boundaries and nuclear localization signals (5 5 5) are as in Kaufman and Kno¨ chel (1996). Region II boundaries are reduced compared to those reported for HNF3 proteins by Pani et al. (1992), reflecting more limited conservation. Two regions of conserved amino acid composition (rather than sequence) are highlighted: acidic residues are shaded in the boxed N-terminal “blob,” whereas proline/alanine/glutamine (P/A/Q) residues are shaded in the C-terminal region between the WHD and region II.
	FIG. 2. Expression of foxD5a is both maternal and zygotic. (A) In situ hybridization of a piece of ovary using full-length antisense foxD5a probe demonstrates that this gene is transcribed in early oogenesis, starting around stage II. Signal is found throughout the cytoplasm and in perinuclear clumps (arrows). Asterisks denote unstained stage I oocytes. (B) Animal (An) and vegetal (Veg) hemispheres of 16-cell embryos were dissected, their RNA extracted and analyzed by PCR. foxD5a mRNA, like that of Xwnt8b (Cui et al., 1995), is present in the animal hemisphere but not the vegetal hemisphere. Vg1 is a vegetally localized control (Kessler, 1999). The histone H4 was used as a loading control. (C) Exogenous FoxD5a-myc protein, whose mRNA was injected into the one-cell embryo, can be detected in the nucleus (arrows) as early as the 64-cell stage. Protein was detected by PAP immunohistochemistry. (D) Stage 8 embryo, prepared as in C, showing that exogenous FoxD5a-myc protein appears nuclear (arrows). (E) FoxD5a-myc protein (arrows), detected by a Texas Red secondary antibody, in some cells of a stage 7.5 embryo. (F) Same microscopic field as in E. Hoescht DNA staining (arrows) colocalizes with myc-staining, demonstrating that FoxD5a-myc protein is nuclear. (G) Dorsal view at stage 10. Zygotic foxD5a is expressed in a broad band on the dorsal side at a distance from the site of involution at the blastopore (arrow). The unlabeled tissue between the foxD5a band and the blastopore is the suprablastoporal endoderm and noninvoluted deep mesoderm (Keller, 1991). (H). Midsagittal section at stage 11 demonstrates foxD5a expression in only the superficial cells of the dorsal lip (dark purple between arrows), whereas chordin (light blue) is expressed in the underlying mesoderm. (I) Vegetal view at stage 11; foxD5a is expressed in the presumptive neural ectoderm of the dorsal lip and weakly in paraxial mesoderm (arrows). (J) Vegetal view at stage 12; foxD5a is expressed in the elongating neural ectoderm. (K) Midsagittal section of stage 13 embryo. foxD5a expression extends throughout the anterior–posterior extent of the neural plate (between arrows). bc, blastocoele; yp, yolk plug. (L) Transverse section through a stage 13 embryo. foxD5a expression extends from the midline (vertical arrow) nearly to the lateral boundary of the neural plate (oblique arrow). Weak staining in the paraxial mesoderm can be discerned (small horizontal arrow). (M) Transverse section through a stage 13 embryo at level comparable to that in L. Sox3 expression is absent at the midline (vertical arrow) and the lateral limit of its expression (oblique arrow) marks the lateral boundary of the neural plate. Small horizontal arrow denotes lateral mesodermal expression. (N) Stage 33/34 embryo demonstrating foxD5a expression in the tail bud. Lines denote level of sections shown in O and P. (O) Transverse section through rostral tail bud demonstrating foxD5a expression throughout the newly formed neural tube (nt) and the paraxial mesoderm (so). Note lack of staining in the notochord (asterisk) and postanal gut tube (g). (P). Transverse section caudal tail bud demonstrating foxD5a expression throughout the tissue surrounding the neurenteric canal (nec), which comprises both of presumptive neural and mesodermal components.
	FIG. 3. Expression of foxD5a in animal cap explants causes dorsal axis differentiation. (A) Animal cap explants injected with foxD5a mRNA at the two-cell stage elongate after 2 days of culture. (B) Tissue sections of control, uninjected explants cultured for 2 days and stained for the 12/101 antigen. They do not express this somitic muscle marker. (C) Tissue sections of elongated foxD5a-injected explants cultured for 2 days express the 12/101 antigen (brown PAP reaction product). (D) foxD5a-injected explants cultured for 1 day have not yet elongated, but express the early neural plate marker sox3 (blue). (E) Tissue section of a foxD5a-injected explant cultured for 1 day expressing sox2 (blue). Arrow indicates a cluster of sox-2-positive cells. (F) foxD5a-injected animal cap explants express some mesoderm markers, one neural inducer, and some neural markers. Explants analyzed for Xbra1, Xnr3, noggin, and geminin were collected at stage 12; those for Xngnr1 were collected at stage 14; those for m-actin and n-tubulin at stages 18–20. Un, uninjected, stage-matched control explants; WE, stage-matched whole embryo with (1) or without (2) reverse transcription. EF1a served as a positive control.
	FIG. 4. siamois and select neural inducers upregulate foxD5a zygotic expression. (A) Ectopic circumblastoporal expression of foxD5a (purple) after bilateral injection of Wnt8 mRNA. Asterisk denotes the normal dorsal expression domain of foxD5a. Compare to uninjected controls (Figs. 2I and 2J). Vegetal view; b, blastoporal region. (B) Ectopic ventral expression of foxD5a (arrows) after bilateral injection of siamois mRNA. Asterisk denotes the normal dorsal expression of foxD5a in the dorsal lip at stage 12. Ventral, vegetal view. (C) Ectopic ventral expression of foxD5a (arrow) after bilateral injection of noggin mRNA. Asterisk denotes the normal dorsal expression domain of foxD5a. Ventral view. (D) foxD5a expression in animal caps is upregulated robustly by siamois (S) and cerberus (Ce), moderately by Wnt8 (W) and Noggin (N), and not by chordin (Ch), under conditions that induce neural genes (Xngnr1) and not muscle genes (m-actin). WE, whole embryo with (1) or without (2) reverse transcription. Un, uninjected control caps. EF1a served as a positive control. All explants and whole embryos were harvested between stages 12/13, when foxD5a expression in the whole embryo is maximum.
	FIG. 5. Ectopic expression of foxD5a causes expansion of axial tissues in embryos. (A) Embryo in which foxD5a mRNA was injected into one ventral vegetal blastomere. A partial secondary axis is apparent (arrows). (B) Tissue section from a similarly injected embryo demonstrating expansion of the epidermis, marked by keratin immunostaining (dark blue), in the partial axis (arrow). Double-labeled cells expressing foxD5a, which were marked by GFP, appear light blue within the ectopic tissue. (C) Tissue section from a similarly injected embryo demonstrating expansion of the somitic mesoderm, marked by 12/101 immunostaining (red), in the partial axis (arrow). Cells expressing foxD5a, marked by GFP, are double-labeled (yellow) within the expanded somite. (D) Embryo in which foxD5a mRNA was injected into one dorsal midline, animal blastomere. A tissue bulge emanating from the neural tube (arrow) obscures the eye. (E) DIC image of transverse tissue section of hindbrain from a similar embryo. Neural tube is thicker on the injected side (right) compared to that on the control side (left). Dashed line demarcates the basal lamina of the neural tube. n, notochord. (F) DIC image of transverse tissue section of hindbrain from an embryo injected bilaterally with EnRfoxD5a mRNA. Neural tube is expanded on both sides to appear duplicated. Dashed line denotes basal lamina of the neural tube. There is only one notochord (n). (G) Anterior view of whole-mount in situ hybridization preparation of control embryo (stage 15) stained for sox3 expression, which is absent at the midline. (H) Anterior view of a foxD5a mRNA-injected embryo (stage 15) stained for sox3 expression. The neural plate on the injected side (right) is about 25% broader than that on the uninjected side (left), as measured from the unstained midline. The clearing of sox3 expression in the anterior neural plate (asterisk) may indicate a secondary floor plate. (I) Dorsal view of a foxD5a mRNA-injected embryo (stage 14) stained for sox3 expression. The neural plate on the injected side (right) is nearly twice as broad as that on the uninjected side (left). (J) Dorsal view of stage 14 foxD5a-injected embryo treated with HUA at stage 102. The neural plate is demarcated by sox3 expression (blue) and the foxD5a-expressing cells are red (bgal). The injected side of the neural plate (right) is nearly 50% broader than the control side (left). (K) Dorsal view of control embryo (stage 15) stained for chordin expression (blue). Posterior is to the top. (L) Dorsal view of foxD5a-injected embryo (stage 15). Chordin expression (blue) does not expand laterally, concomitant with foxD5a expression (red). (M) Anterior view of stage 14 foxD5a-injected embryo. Xbra1 expression (blue) is confined to the periblastoporal region (not seen from this view), and does not expand into the anterior domain of the foxD5a-expressing cells (red). (N) Dorsal view of stage 11 foxD5a-injected embryo. Xbra1 expression (blue) encircles the blastopore (arrow) and does not extend anteriorly into the domain of the foxD5a-expressing cells (red).
	FIG. 6. Expression of exogenous foxD5a in the neural plate expands anterior markers and represses differentiation markers. (A) Anterior view of a control embryo (stage 14) stained for otx2 expression (blue). Staining encircles the anterior neural plate and does not extend into the hindbrain/spinal cord domain (arrow). (B) Dorsal view of a foxD5a mRNA-injected embryo (stage 14) stained for otx2 expression. Arrow denotes comparable level of arrow in A. Otx2 expression expands posteriorly (toward the bottom). (C) Transverse section of embryo similar to that in B, demonstrating ectopic otx2 expression (blue) in the neural ectoderm at the level of the blastopore (bp). (D) Anterior view of stage 16 embryo stained for en2 expression (blue). On the side of foxD5a injection (red) the en2 stripe (arrow) is smaller and slightly more posterior than the control stripe (right side). (E) Anterior view of stage 18 embryo. en2 expression (blue) is repressed (arrow) in the cells expressing injected foxD5a mRNA (red). (F) Anterior view of stage 18 embryo. On the side of foxD5a injection (red) the Krox20 stripes (blue) are more posterior (arrows) than the control stripes (right side). The rhombomere 3 stripe also is significantly smaller. (G) Dorsal view of stage 20 embryo. Krox 20 expression is completely repressed on the side of foxD5a injection (red). Posterior is to the top of the figure. (H) Anterior view of stage 15 embryo. Xngnr1 expression (blue) on the control side (left) is expressed in five patches (arrows). These patches are repressed in cells expressing injected foxD5a mRNA (red). (I) Dorsal view of stage 15 embryo. One horizontal stripe (large arrow) of Xngnr1-expressing cells (blue) is missing and one longitudinal stripe (small arrow) is smaller on the side of injected foxD5a-expressing cells (red). Posterior is to the top of the figure. (J) Anteriodorsal view of stage 18 embryo stained for n-tubulin expression (blue). The anterior patch (arrow) of the trigeminal ganglion is repressed on the side of injected foxD5a-expressing cells (red). (K) Transverse section from the spinomedullary level of stage 16/17 embryo demonstrating an area (arrow) in which n-tubulin expression (blue) is repressed on the side of injected foxD5a expression (red). Asterisk denotes a cluster of n-tubulin-expressing cells on the control side. (L) Transverse section from the caudal spinal level of stage 19/20 embryo demonstrating expansion (arrows) of clusters of n-tubulin-expressing cells (blue) on the side of injected foxD5a (red). Asterisk denotes a cluster of n-tubulin-expressing cells on the control side.