FoxD3 is a forkhead-related transcriptional regulator that isThe Fox gene family is a diverse group of forkhead-related transcriptional regulators, many of which play essential roles in metazoan embryogenesis and physiology (1-3). FoxD3 is required for multiple developmental processes in the vertebrate embryo, including neural crest development and maintenance of mammalian stem cell lineages. In Xenopus, zebrafish, chick, and mouse, FoxD3 orthologs are expressed in pre-migratory and migrating neural crest cells (4 -12), and functional studies indicate that FoxD3 regulates the determination, migration, and/or differentiation of neural crest lineages (13)(14)(15)(16)(17)(18)(19)(20). FoxD3 is also expressed in the preimplantation mouse embryo, as well as mammalian embryonic and trophoblast stem cells (9, 21-23). FoxD3 null embryos do not form a primitive streak, fail to undergo gastrulation or form mesoderm, and die by 6.5 days postcoitum with greatly reduced epiblast cell number (21). Extraembryonic defects are also observed in FoxD3 nulls due to a failure of trophoblast progenitors to self-renew and differentiate (23). Furthermore, embryonic and trophoblast stem cell lines cannot be established from FoxD3 null embryos (21, 23). This requirement for FoxD3 in multiple progenitor populations, including embryonic stem cells, trophoblast stem cells, and possibly neural crest stem cells, suggests that FoxD3 may play a conserved role in maintaining cellular multipotency. Whether FoxD3 has similar transcriptional activity and target genes in these distinct progenitor populations remains to be determined.In the Xenopus gastrula, FoxD3 is expressed in the Spemann organizer (17,18,24), a signaling center that controls germ layer patterning, morphogenesis, and axis formation (25-27). Organizer-restricted expression of FoxD3 is conserved in the zebrafish shield and the chick Hensen node, whereas in the mouse, FoxD3 is expressed throughout the gastrula, including the node (8,10,21). In cells of the organizer, FoxD3 is coexpressed with a variety of developmentally important genes, including Nodal-related members of the transforming growth factor-␤ superfamily that are essential for the induction and patterning of dorsal mesoderm (28,29). Recently, we found that FoxD3 is essential in the Xenopus gastrula for dorsal mesodermal development, and subsequent formation of the body axis (30). FoxD3 is necessary for the maintenance of Nodal expression in the organizer, and is sufficient for induction of ectopic Nodal expression outside of the organizer. Consistent with a regulatory interaction of FoxD3 with the Nodal pathway, mesoderm induction in response to FoxD3 gain-of-function was dependent on Nodal, and the developmental defects resulting from FoxD3 knockdown were rescued by activation of Nodal signaling. These studies indicate that FoxD3 function is required in the Spemann organizer to maintain Nodal expression, thus promoting dorsal mesoderm induction and axis formation in Xenopus.Interestingly, a fusion protein containing the ...

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Induction and patterning of the mesodermal germ layer is a key early step of vertebrate embryogenesis. We report that FoxD3 function in the Xenopus gastrula is essential for dorsal mesodermal development and for Nodal expression in the Spemann organizer. In embryos and explants, FoxD3 induced mesodermal genes, convergent extension movements and differentiation of axial tissues. Engrailed-FoxD3, but not VP16-FoxD3, was identical to native FoxD3 in mesoderm-inducing activity, indicating that FoxD3 functions as a transcriptional repressor to induce mesoderm. Antagonism of FoxD3 with VP16-FoxD3 or morpholinoknockdown of FoxD3 protein resulted in a complete block to axis formation, a loss of mesodermal gene expression, and an absence of axial mesoderm, indicating that transcriptional repression by FoxD3 is required for mesodermal development. FoxD3 induced mesoderm in a non-cell-autonomous manner, indicating a role for secreted inducing factors in the response to FoxD3. Consistent with this mechanism, FoxD3 was necessary and sufficient for the expression of multiple Nodal-related genes, and inhibitors of Nodal signaling blocked mesoderm induction by FoxD3. Therefore, FoxD3 is required for Nodal expression in the Spemann organizer and this function is essential for dorsal mesoderm formation. KEY WORDS:Xenopus, FoxD3, Forkhead, Nodal, Mesoderm, Transcription Development 133, 4827-4838 (2006) DEVELOPMENT 4828 formation, and antagonism or knockdown of FoxD3 results in severe axial defects and loss of dorsal mesodermal gene expression. FoxD3 induction of mesoderm is non-cell-autonomous and requires the Nodal signaling pathway. Consistent with the co-expression of FoxD3 and Nodal genes in the organizer, FoxD3 is necessary and sufficient for the expression of several Nodal-related genes. Taken together, our results demonstrate a novel mode of Nodal regulation in the Spemann organizer, where transcriptional repression by FoxD3 maintains Nodal expression to promote mesoderm induction and axial development. MATERIALS AND METHODS Embryos and microinjectionEmbryos were collected, fertilized, injected and cultured as previously described (Yao and Kessler, 1999), and embryonic stage was determined according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Dorsal and ventral blastomeres were identified by pigmentation differences (Klein, 1987). Explants were prepared using a Gastromaster microsurgery instrument (Xenotek Engineering). Capped, in vitro transcribed RNA for microinjection was synthesized from linearized template DNA using the Message Machine kit (Ambion) and 10 nl of RNA solution was injected.Templates for in vitro transcription were pCS2-FoxD3, pCS2-mFoxD3, pCS2-Eng-FoxD3, pCS2-VP16-FoxD3, pCS2-FoxD3(N140A/H144A), pCS2-Eng-FoxD3(N140A/H144A), pCS2-VP16-FoxD3(N140A/H144A), pCS2-NLS-FoxD3WH, pCS2-FoxD3-utr (this study), pCS2-Eng, pCS2-VP16 (Kessler, 1997), pCS2-MT-SID (Chen et al., 1997), pCS2-Cer-S (Piccolo et al., 1999), pCS2-Xnr1 (Sampath et al., 1997), and pCS2-VegT⌬UTR (Engleka et al., 2001). FoxD3 expression ...

Background: The Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes.