XB-ART-35222Development. February 1, 2007; 134 (4): 779-88.
The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus, where the process is best understood, the endoderm is specified by a vegetally localized transcription factor, VegT, which releases nodal signals that specify the adjacent marginal zone of the blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this paper, we show that the forkhead protein FoxI1e (also known as Xema) is required at the blastula stage for normal formation of both the central nervous system and epidermis, the two early derivatives of the ectoderm. In addition, FoxI1e is required to maintain the regional identity of the animal cells of the blastula, the cells that are precursors of ectodermal structures. In its absence, they lose contact with the animal cap, mix with cells of other germ layers and differentiate according to their new positions. Because FoxI1e is initially expressed in the animal region of the embryo and is rapidly downregulated in the neural plate, its role in neural and epidermal gene expression must precede the division of the ectoderm into neural and epidermal. The work also shows that FoxI1e plays a role in the embryo in the poorly understood process of differential adhesion, which limits cell mixing as primary germ layers become specified.
PubMed ID: 17229765
Article link: Development.
Grant support: R01 HD45737 NICHD NIH HHS , T32 HD046387 NICHD NIH HHS
Genes referenced: a2m cdh1 dnai1 foxi1 nodal snai2 sox2 tfap2a vegt xk81a1
Morpholinos referenced: foxi1 MO4 foxi1 MO5 foxi1 MO7 foxi1 MO8 foxi1 MO9
Article Images: [+] show captions
|Fig. 2. FoxI1e is not expressed in all ectodermal cells and does not co-localize with epidermal cilia. (A) In situ hybridization for FoxI1e at stage 10 showing salt and pepper expression. (B,C) Coimmunostaining for α-tubulin (brown, arrows) and in situ hybridization for FoxI1e (purple, arrowheads) demonstrates that these two markers are expressed in different cell populations. Staining was done in whole mount (B), and stained embryos were then embedded and sectioned. A high-power picture is shown in C. (D) Twenty high-power fields were analyzed for expression of the two markers. Sixty-one FoxI1e-positive cells and 83 ciliated cells were counted. There were five instances of overlapping expression.|
|Fig. 3. FoxI1e expression is sufficient for ectoderm formation. RTPCR analysis of vegetal explants injected with 300-600 pg FoxI1e mRNA and cultured to stage 11. (A) Expression of ectoderm-specific markers is increased, including E-cadherin, epidermal markers epidermal cytokeratin and AP-2, the neural marker Sox-2 and the neural crest marker slug. (B) Endodermal markers Sox17α and endodermin were reduced. (C) Schematic of experimental design to determine behavior of vegetal hemispheres ectopically expressing FoxI1e. (D) In control vegetal hemispheres, pigmented mesodermal cells formed aggregates that extended from bases, whereas in FoxI1e-positive hemispheres, pigmented mesodermal cells formed a layer around the explant (E). Immunostaining for α-tubulin to mark cilia was negative in control embryos (F), but demonstrated surface ciliation on FoxI1e-positive hemispheres (G).|
|Fig. 6. Loss of FoxI1e causes a rescuable loss of epidermal cilia.Control embryos (A,B) injected with RLDX (red) in an animal, ventral cell at the eight-cell stage and stained for α-tubulin (green) have a normal cilia pattern in injected cells. (C,D) Co-injection of SBMO resulted in loss of cilia in cells that received the morpholino. (E,F) SBMO-resistant FoxI1e mRNA was injected after the SBMO, restoring normal cilia formation.|
|Fig. 8. FoxI1e controls regional position of ectodermal cells. (A,B) Stage 25 embryos injected at the 32-cell stage in the A4 blastomere, a precursor of epidermis, with RLDX (red) alone (A) or with RLDX + SBMO (B) and then stained for α-tubulin (green). With RLDX alone, injected cells co-localize with cilia (A). With SBMO, the cells are located in the interior of the embryo (B). At stage 47, embryos were sectioned and embedded. (C-E) DIC and fluorescent images of a control embryo injected with RLDX in A4. RLDX is localized to the epidermis. (F-H) Co-injection with SBMO causes cells to localize to the gut. These cells give rise to morphologically normal endodermal structures, including the intestinal epithelium.|
|Fig. 9. FoxI1e is required for normal ectodermal cell adhesion in the gastrula. (A,B) Brightfield and fluorescent images of a hemisected control stage 11 embryo injected with FLDX alone into A4 at the 32-cell stage. (C,D) Co-injection of SBMO causes the cells to disaggregate and fall into the blastocoel. (E-G) This effect is rescued by morpholino-resistant mRNA injected at the two-cell stage. *P=8.8×10-5, **P=0.03.|