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Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1–3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions.

Experiments in Xenopus have illustrated the importance of extracellular morphogens for embryonic gene regulation in vertebrates. Much less is known about how induction leads to the correct positioning of boundaries; for example, between germ layers. Here we report that the neuroectoderm/mesoderm boundary is controlled by the chromatin remodeling ATPase CHD4/Mi-2β. Gain and loss of CHD4 function experiments shifted this boundary along the animal–vegetal axis at gastrulation, leading to excess mesoderm formation at the expense of neuroectoderm, or vice versa. This phenotype results from specific alterations in gene transcription, notably of the neural-promoting gene Sip1 and the mesodermal regulatory gene Xbra. We show that CHD4 suppresses Sip1 transcription by direct binding to the 5′ end of the Sip1 gene body. Furthermore, we demonstrate that CHD4 and Sip1 expression levels determine the “ON” threshold for Nodal-dependent but not for eFGF-dependent induction of Xbra transcription. The CHD4/Sip1 epistasis thus constitutes a regulatory module, which balances mesoderm and neuroectoderm formation.

In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermalFgf10acting redundantly with neuralFgf3is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, we took advantage of the unique strengths of the two model organisms. We show that endoderm is necessary for otic induction in the chick and thatFgf8, expressed in the chick endoderm subjacent toFgf19, is both sufficient and necessary for the expression ofFgf19in the mesoderm. In the mouse,Fgf8is also expressed in endoderm as well as in other germ layers in the periotic placode region. We show that otic induction fails in embryos null forFgf3and hypomorphic forFgf8and expression of mesodermalFgf10is reduced. Thus,Fgf8plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse.

Nieuwkoop's two-signal model proposed that induced neural tissue is inherently anterior (forebrain) in character and that a graded transforming (or posteriorizing) signal specifies posterior identity to the anterior neuroectoderm (Nieuwkoop 1952). It has been suggested that during vertebrate head development, the level of Wnt activity may specify posterior-to-anterior fates within the neural plate (Niehrs 1999; Heisenberg et al. 2001; Kiecker and Niehrs 2001). Wnt signaling must be inhibited to allow the development of the rostral telencephalon, or the prospective forebrain will acquire a caudal diencephalic identity (Niehrs 1999; Heisenberg et al. 2001; Kiecker and Niehrs 2001). This anterior Wnt-signaling-free zone is maintained by Wnt antagonists secreted by the anterior neuroectoderm and adjacent anterior mesendoderm (Niehrs 1999; Houart et al. 2002). Head truncations occur when genes that are required for the development of the anterior visceral endoderm (AVE; i.e., Hex, Lim1, and Otx2) are mutated (Thomas and Beddington 1996; Shawlot et al. 1999; Martinez-Barbera and Beddington 2001; Perea-Gomez et al. 2001). The lack of anterior head structures also occurs in mice that are double-homozygous for chordin and noggin, which encode secreted bone morphogenetic protein antagonists (Bachiller et al. 2000). In addition, mouse embryos lacking Dickkopf1 (Dkk1), a secreted protein that acts as an inhibitor of the Wnt coreceptor LRP-6, lack head structures anterior to the midbrain; Dkk1 activity is required in the axial mesendoderm (Mukhopadhyay et al. 2001). Variable forebrain truncations are also observed in mice with inactivating mutations in the homeobox gene Hesx1, whose activity is required in the anterior neural ectoderm (Martinez-Barbera and Beddington 2001). We have previously shown that in mice, Six3 is expressed in the most anterior part of the developing neural plate (Oliver et al. 1995). To determine the role of Six3 during vertebrate development, we inactivated the mouse Six3 locus. We find that Six3 is required for development of the mammalian rostral forebrain. The absence of Six3 results in forebrain truncations and posteriorization of the remaining mutant head. We demonstrate that Six3 binds to the Wnt1 promoter region in vivo and represses Wnt1 expression in the most anterior neuroectoderm. Work recently performed in zebrafish embryos has suggested that telencephalic induction, as well as the subsequent patterning of the forebrain into telencephalic, eye, and diencephalic regions, is the result of the graded expression of Wnt signaling in the anterior neural plate (Houart et al. 2002). Thus, during vertebrate head regional specification, the maintenance and refinement of anterior neural fates requires that Wnt signaling is transcriptionally repressed in the anterior neuroectoderm, and Six3 is a key player during this process. We also show that Six3 is sufficient to suppress the loss of forebrain resulting from excess Wnt1 signaling in headless (Tlc3) zebrafish mutants. Taken together, these results not only identified Six3 as a key player in vertebrate head development, but also demonstrated the existence of another regulatory step in the complex Wnt signaling pathway, the direct repression of Wnt1 expression by a transcription factor in the mammalian anterior neural plate at the late headfold–early somite stage, a step that is probably required for the maintenance of the anterior neural fates.

Animal development involves a complex progression of tissue induction and morphogenesis, expansion of precursor cell populations, and the death or terminal differentiation of these cells into specific functional types. Bone morphogenetic protein (BMP) signaling is repeatedly used in this dynamic process for both vertebrates and invertebrates. In gastrulating Xenopus and Drosophila, BMPs and the BMP2/4 homolog Decapentaplegic (Dpp) contribute to specifying the embryonic dorsoventral axis. High activity of BMP/Dpp induces blood/amnioserosa, whereas maximal inhibition of BMP/Dpp leads to neural ectoderm induction (Dale 2000; Nakayama et al. 2000). Subsequently, BMP/Dpp signaling contributes to the specification and/or expansion of many tissues such as vertebrate limb and Drosophila wing (Vogt and Duboule 1999; Day and Lawrence 2000) and neural ectoderm (Cornell and Ohlen 2000). Vertebrate neural ectoderm undergoes a period of rapid proliferation and morphogenetic movements to form a neural tube, as signals from adjacent tissues induce dorsal–ventral and anterior-posterior identities on precursors (Altmann and Brivanlou 2001). BMPs are expressed at high levels in nonneural ectoderm and then in the roof plate of the neural tube (Liem et al. 1995; Furuta et al. 1997). A BMP activity gradient induces dorsal identity markers indicative of neural crest or dorsal interneuron precursors (Liem et al. 1997; Nguyen et al. 1998, 2000; Barth et al. 1999). However, BMPs also cause apoptosis in early central nervous system (CNS) precursor cells (Graham et al. 1996; Furuta et al. 1997), neuronal differentiation in mid-gestation CNS precursors (Li et al. 1998; Mehler et al. 2000), and glial differentiation in late embryonic or adult CNS precursors (Gross et al. 1996). It is unclear how BMPs mediate such a wide variety of responses, but it may be a result of diversity in the components of signal transduction. Some BMP-related ligands are known to be involved in the generation of specific cell types (Shah et al. 1996; Lee et al. 1998), but there is evidence that many act in a redundant manner (Dudley and Robertson 1997; Solloway et al. 1998; Zhao et al. 1999). BMPs exert their effects by activating a complex of type I and type II receptors. The type II receptor is the primary ligand-binding component; both BMPRII and ActRIIB are functional type II receptors for BMPs. Type I receptors also have BMP-binding properties but are primarily responsible for transducing the signal into the cell: BMPR-IA (Alk3), BMPR-IB (Alk6), and ActR-I (Alk2) are all known to transduce BMP signals (Kawabata et al. 1998). The activated type I receptors in turn phosphorylate the DNA-binding proteins Smad1, Smad5, and Smad8, each of which can then heteromerize with Smad4 and translocate to the nucleus to regulate transcription of downstream target genes (Itoh et al. 2000). The expression patterns of Bmpr-1a and Bmpr-1b in the CNS (Dewulf et al. 1995; Zhang et al. 1998) suggest that they have distinct roles in the transduction of BMP signals. In an effort to understand their role in neural development, we used multipotent CNS stem cells as a defined system to understand signals that control proliferation and fate choice (Johe et al. 1996; Studer et al. 1998). We have also developed a vector using POU-regulated genomic elements that control expression of the intermediate filament gene nestin in CNS precursor cells (Josephson et al. 1998). This vector, called pNERV, expresses mutant BMP receptors selectively in CNS stem cells in vitro and in transgenic mice. Using these tools, we show that most of the known actions of BMPs during neural precursor development can be attributed to the distinct actions of BMPR-IA and BMPR-IB. Furthermore, the activities of these receptors are linked sequentially, with BMPR-IA acting first and inducing the expression of Bmpr-1b. These results identify a feed-forward mechanism by which BMPs can control both the production and fate of precursor cells.

We report a new role for Wnt signaling in the vertebrate embryo: the induction of neural tissue from ectoderm. Early expression of mouse wnt8, Xwnt8, beta-catenin, or dominant-negative GSK3 induces the expression of neural-specific markers and inhibits the expression of Bmp4 in Xenopus ectoderm. We show that Wnt8, but not the BMP antagonist Noggin, can inhibit Bmp4 expression at early gastrula stages. Furthermore, inhibition of beta-catenin activity in the neural ectoderm of whole embryos by a truncated TCF results in a decrease in neural development. Therefore, we suggest that a cleavage-stage Wnt signal normally contributes to an early repression of Bmp4 on the dorsal side of the embryo and sensitizes the ectoderm to respond to neural inducing signals from the organizer. The Wnt targets Xnr3 and siamois have been shown previously to have neuralizing activity when overexpressed. However, antagonists of Wnt signaling, dnXwnt8 and Nxfrz8, inhibit Wnt-mediated Xnr3 and siamois induction, but not neural induction, suggesting an alternative mechanism for Bmp repression and neuralization. Conversely, dnTCF blocks both Wnt-mediated Xnr3 and neural induction, suggesting that both pathways require this transcription factor.

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. We have analyzed the phenotype of Fgf8(-/-) embryos and discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate their expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-βB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show thatNoggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog(Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.

Bone morphogenetic proteins (BMPs), which have been implicated in the patterning of mesoderm, are members of the transforming growth factor-beta (TGF-beta) superfamily. We have investigated the roles of Xenopus BMP-7 (XBMP-7) and BMP-4 (XBMP-4), and activin (another TGF-beta-related molecule) in early development by generating dominant-negative versions of these growth factors. Mutations were generated by altering the cleavage sites that are required for maturation of the active dimeric forms of XBMP-7, XBMP-4, and activin. These mutant constructs, designated Cm-XBMP-7, Cm-XBMP-4, and Cm-activin, result in polypeptides that allow for dimerization of the subunits, but are incapable of maturation. Expression of Cm-XBMP-7 and Cm-XBMP-4, but not Cm-activin, in the ventral marginal zone of the Xenopus embryo results in the development of a secondary axis, similar to that seen by ectopic expression of the truncated BMP receptor. These results suggest that the cleavage mutants interfere with BMP signaling during mesodermal patterning. We also found that expression of Cm-XBMP-7 or Cm-XBMP-4 in animal cap ectoderm directly induces neuroectoderm. The neural induction was specific for Cm-XBMP-7 and Cm-XBMP-4 because ectopic expression of Cm-activin or Vg-1 did not mimic the same phenotype. Molecular study of neural patterning by Cm-XBMP-7 and Cm-XBMP-4 revealed that only anterior neuroectodermal markers are expressed in response to these Cm-XBMPs. These results suggest that the BMPs are involved in the specification of ectoderm in Xenopus development, and that neural induction requires the removal of BMP signals in the ectoderm. We propose that neural induction occurs by a default mechanism, whereby the inhibition of BMP signaling is required for the conversion of ectoderm to neuroectoderm in the developing Xenopus embryo.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

The dorsal (dl) nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. Here we present evidence that a second signaling pathway, controlled by the torso (tor) receptor tyrosine kinase, also modulates dl activity. Evidence is presented that the tor pathway selectively masks the ability of dl to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of tor, such as D-raf and the rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and tor pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the tor pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor.

The first step in the differentiation of the Drosophila mesoderm is the activation of two regulatory genes, twist (twi) and snail (sna), in ventral regions of early embryos. sna is a transcriptional repressor that is uniformly expressed throughout the presumptive mesoderm. Its sharp lateral limits help to establish the boundary between the mesoderm and neuroectoderm. Genetic studies suggest that sna is a target of the dorsal (dl) morphogen, and this interaction provides a model for determining how a morphogen gradient establishes a sharp, on/off threshold response. We present evidence that dl and twi directly activate sna expression. Site-directed mutagenesis of dl- and twi-binding sites within defined regions of the sna promoter suggest that the two proteins (containing the Rel and helix-loop-helix domains, respectively) function multiplicatively to ensure strong, uniform expression of sna, particularly in ventral-lateral regions where there are diminishing amounts of dl. These results are consistent with the possibility that the sharp sna borders are formed by multiplying the shallow dl gradient and the steeper twi gradient.