Yan B et al. (2009), foxD5 plays a critical upstream role in regulat...

XB-ART-39749

Dev Biol May 1, 2009; 329 (1): 80-95.

foxD5 plays a critical upstream role in regulating neural ectodermal fate and the onset of neural differentiation.

Yan B , Neilson KM , Moody SA .

Abstract
foxD5 is expressed in the nascent neural ectoderm concomitant with several other neural-fate specifying transcription factors. We used loss-of-function and gain-of-function approaches to analyze the functional position of foxD5 amongst these other factors. Loss of FoxD5 reduces the expression of sox2, sox11, soxD, zic1, zic3 and Xiro1-3 at the onset of gastrulation, and of geminin, sox3 and zic2, which are maternally expressed, by late gastrulation. At neural plate stages most of these genes remain reduced, but the domains of zic1 and zic3 are expanded. Increased FoxD5 induces geminin and zic2, weakly represses sox11 at early gastrula but later (st12) induces it; weakly represses sox2 and sox3 transiently and strongly represses soxD, zic1, zic3 and Xiro1-3. The foxD5 effects on zic1, zic3 and Xiro1-3 involve transcriptional repression, whereas those on geminin and zic2 involve transcriptional activation. foxD5''s effects on geminin, sox11 and zic2 occur at the onset of gastrulation, whereas the other genes require earlier foxD5 activity. geminin, sox11 and zic2, each of which is up-regulated directly by foxD5, are all required to account for foxD5 phenotypes, indicating that this triad constitutes a transcriptional network rather than linear path that coordinately up-regulates genes that promote an immature neural fate and inhibits genes that promote the onset of neural differentiation. We also show that foxD5 promotes an ectopic neural fate in the epidermis by reducing BMP signaling. Several of the genes that are repressed by foxD5 in turn reduce foxD5 expression, contributing to the medial-lateral patterning of the neural plate.

PubMed ID: 19250931
PMC ID: PMC2672197
Article link: Dev Biol
Grant support: [+]
Genes referenced: acss2.2 foxd4l1.1 gal.2 gem gmnn irx1 irx2 irx3 klf6 krt12.4 myc sox11 sox15 sox2 sox3 st13 st14 szl tfap2a zic1 zic2 zic3
Antibodies: Smad1 Ab7
Morpholinos: foxd4l1.1 MO1 foxd4l1.2 MO1

Article Images: [+] show captions

	Fig. 2. FoxD5 is required for the normal expression of NE genes. (A) Myc-tagged FoxD5 (foxD5-MT) is highly expressed in both the cytoplasm and nucleus (arrowheads); this expression is greatly reduced in the presence of foxD5-MOs (foxMOs). Myc-tagged N-terminal deleted FoxD5 (δN-foxD5) is strongly expressed even in the presence of foxMOs. (B) Control MO (cMO) does not alter the expression of sox2 on the injected side () of the embryo (st14, dorsal view, anterior is down). (C) Co-injection of δNfoxD5 mRNA prevents the foxMOs reduction of sox2 expression (cf. E) on the injected side () of the embryo (st15, same view as in B). (D) The percentage of embryos exhibiting the phenotypes shown in E (reduced expression above x-axis; expanded expression below x-axis) after injection of foxD5-MOs (analyzed at four different stages), cMO (analyzed at st14) or foxD5-MOs + δNfoxD5 mRNA (analyzed at st14). These data indicate that the effects of foxD5-MOs are specific and can be rescued in a high percentage of cases by δNfoxD5. Sample sizes are indicated above each bar. (E) Arrows denote patches of reduced gene expression at the site of foxD5-MOs injection for each NE gene at gastrulation (st10.5–12) and neural plate (st13–15) stages. For zic1 and zic3, foxD5-MOs expand their expression domains at neural plate stages (lines denote width of neural plate on injected sides). For gem, sox2, zic2, zic3, Xiro1 and Xiro2: dorsal views with anterior to the bottom; for sox3, anterior view with dorsal to the top; for sox11, soxD, and zic1: gastrula stages are dorsal views (as for gem) and neural plate stages are anterior views (as for sox3); for Xiro3: st10.5 is vegetal view with dorsal to the top and st13 is dorsal view (as for Xiro1, 2).
	Fig. 3. foxD5 mRNA injected into D1.1 lineages affects all 11 NE genes. (A) foxD5-expressing cells (red nuclei) showed increased expression of gem at both gastrulation (left) and neural plate (right) stages. For gastrula, small arrow denotes blastopore lip and inset shows cells at a higher magnification. Dorsal views with anterior to the bottom. sox2 (B) and sox3 (C) were weakly reduced (small arrows) compared to adjacent NE cells () at gastrula stages (dorsal views with anterior to the bottom). This effect waned by neural plate stages, when their expression domains were expanded on the injected side (black lines); anterior views with dorsal to the top. (D) foxD5-expressing cells showed reduced sox11 expression (small arrows) compared to adjacent NE cells () at early gastrula (dorsal view with anterior to the bottom), but later (by st12) showed highly increased expression in both the neural plate (above white arrows) and in the adjacent ectoderm (red asterisk). Black bar indicates the expanded sox11 domain on the injected side. Anterior view with dorsal to the top. (E) soxD expression was unaffected at early gastrulation (dorsal view with anterior to the bottom, and top inset), began to be reduced (small arrows) compared to adjacent NE cells () by stage 12 (bottom inset). The reduced phenotype was maintained at neural plate (anterior-lateral view) stages; higher magnification shows reduced soxD in foxD5-expressing cells (red nuclei) compared to adjacent NE cells (). For zic2 (G), foxD5-expressing cells (dark blue) showed increased expression at both gastrula (bracket and inset) and neural plate stages (arrow depicts ectopic expression along the midline). Both are dorsal views with anterior to the bottom. zic1 (F) and zic3 (H) showed reduced expression in foxD5-expressing cells (red nuclei) compared to adjacent NE cells () at gastrulation. Gaps in their expression (large arrows) coinciding with foxD5-expressing cells (red nuclei) also were observed at neural plate stages. Xiro1 (I), Xiro2 (J) and Xiro3 (K) were repressed (arrows) at both gastrulation (not shown) and neural plate stages. For A–K, the percentage of embryos affected and sample sizes are indicated. (L) These results were confirmed for several of the NE genes by RT-PCR comparing uninjected (Un) and foxD5-injected ACs. H4 is internal control.
	Fig. 4. foxD5 ectopically induces a subset of NE genes and represses the BMP pathway. (A) Left side: foxD5-expressing cells (arrows, red nuclei) express zic2 (blue cytoplasm) in the st11 ventral epidermis, but do not express sox2; inset shows higher magnification of sox2 to demonstrate lack of blue cytoplasm surrounding red nuclei. Asterisks denote endogenous gene expression on the dorsal sides of the embryos. Right side: high magnifications show strong induction () of gem, zic2 and sox11 in the st11 ventral epidermis; the red nuclei are partially masked by the blue signal in the cytoplasm. A few cells (arrows) weakly express sox3. (B) The percentage of embryos showing ectopic ventral induction of each NE gene (collected at st10.5–13). Sample sizes are above each bar. (C) foxD5 expression in the ventral epidermis at gastrulation (st10–11) reduces the expression of two epidermal genes downstream of BMP signaling. AP2 is expressed throughout the control ectoderm (left: animal view of whole embryo; right: higher magnification). foxD5-expressing cells (red nuclei) show reduced AP2 expression. Asterisk denotes adjacent unaffected cells. Epidermis-specific keratin (epi-keratin) is expressed throughout the ventral ectoderm (left: ventral view with animal hemisphere to the top of whole embryo; right: higher magnification). foxD5-expressing cells (red nuclei) show reduced epi-keratin expression. Asterisk denotes adjacent unaffected cells. The frequencies of the phenotypes and sample sizes are given. (D) Injection of foxD5 mRNA into a neural progenitor cell-autonomously induces szl (arrow) in the neural plate (st13, anterior view). The frequency of the phenotype and sample size are given. (E) Injection of foxD5 mRNA cell-autonomously reduces the number of cells displaying nuclear phospho-SMAD1/5/8 staining (brown nuclei) in the st11 ventral epidermis. Top panel illustrates staining in ventral epidermis of uninjected control embryo. Bottom panel illustrates staining in a region of foxD5-expressing cells stained blue by expression of the cytoplasmic βGal lineage marker (asterisk) and in an adjacent region (right side) that does not contain foxD5-expressing cells. Graph on right indicates the mean number of cells displaying nuclear phospho-SMAD1/5/8 staining in foxD5-expressing ventral epidermis clones compared to the uninjected side (control) of the same embryos (n = 16 per group). Bars indicate s.e.m. and indicates p < 0.0001 (paired t-test).
	Fig. 6. foxD5 regulates transcription by both activation and repression. (A) The percentage of embryos showing either increased (above x-axis) or decreased (below x-axis) expression after injection of wt foxD5, an activating construct (foxD5VP) or a repressing construct (EnRfoxD5); embryos were collected at st10.5–11 (gem, sox2, sox3, sox11, zic1–3) or st12 (soxD, Xiro1–3). Sample sizes for wt foxD5 are in Fig. 3, and for other constructs are above each bar. (B) foxD5VP strongly induced gem in st11 ventral epidermis, whereas EnRfoxD5 induced only a few cells (arrows in both low and high magnification pictures). foxD5VP induced zic2 expression in the st12 neural plate midline (bracket), distant from the endogenous expression () along the lateral neural plate border, whereas EnRfoxD5 induced only a few patches of cells (arrows in both low and high magnification pictures). (C) EnRfoxD5 reduced zic1 expression in the st11 NE (arrow).
	Fig. 7. When foxD5-hGR was activated by Dex in the presence of Chx (+ Chx/+ Dex), the cells expressed high levels of gem, sox11 or zic2 (arrows), indicating that protein synthesis is not required. This induction did not occur in the absence of Dex (+ Chx/− Dex) (arrows, red nuclei). Dex treatment alone (− Chx/+ Dex) induced each gene (arrows) either above their endogenous levels of gene expression (, gem) or outside the normal endogenous domains of gene expression (, sox11, zic2). The frequencies of the phenotypes and sample sizes are indicated below each example. Embryos were fixed when siblings reached st12.
	Fig. 8. In combination, gem, sox11 and zic2 account for the foxD5 effects. (A) The percentage of embryos in which NE genes showed increased (above x-axis) or decreased (below x-axis) expression after injection of wt foxD5, n-gem, sox11 or zic2 (analyzed at st10–11). Sample sizes for n-gem, sox11 and zic2 are above each bar. (B) The percentage of embryos showing the st14 foxD5-MOs phenotype (cf. Fig. 2) after injection of foxD5-MOs alone or in combination with n-gem, sox11 or zic2 mRNAs. Asterisks denote significant differences (i.e., rescue of the phenotype) compared to foxD5-MOs alone (p < 0.05, Chi-squared test). Sample sizes for rescue experiments are above each bar. (C) Injection of n-gem mRNA induced midline expression (arrows) of both sox11 and zic2 in the st13 neural plate (dorsal views, anterior to the bottom). The asterisks on the zic2 sample denote endogenous expression domains; the sox11 sample was developed only until the induced expression was detected, so endogenous levels are not visible. (D) Co-expression of n-gem rescues the foxD5-MOs reduction in zic2 on the injected side (arrow, anterior view at st12, dorsal to the top) but does not rescue the effect on sox2 (arrow, dorsal view at st11, anterior to top). cf. Fig. 2E.
	Fig. 10. Effects of NE genes on foxD5 expression at gastrulation (st10.5 unless otherwise noted). (A) Cells (red nuclei) expressing n-gem do not alter foxD5 expression (blue); black arrow points to blastopore lip. Cells expressing (B) sox11 or (F) zic2 show weak repression of foxD5 (clear area surrounding red nuclei noted by small red arrows) in a low percentage of embryos; cells expressing zic1 usually do not show altered foxD5 expression (E), but in a few cases (12%) there were phenotypes similar to that shown for zic2 (F). Cells expressing (C) sox2 or (D) sox3 show weak repression of foxD5 (small red arrows) in a majority of embryos. Cells expressing (G) zic3, (H) soxD, (I) Xiro1 or (L) Xiro3 show strong repression of foxD5 (clear area surrounding red nuclei). Cells expressing Xiro2 rarely showed repression at stage 10.5 (J) but repression is strong by stage 12 (K). Only sox3 and Xiro3 induce ectopic foxD5 expression (arrows) in the ventral epidermis (inserts, blue cytoplasm surrounding the red nuclei). The frequencies of the phenotypes and sample sizes are indicated in each panel.

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