Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.     

Cerberus-like is a secreted BMP and nodal antagonist not essential for mouse development

Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural-inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l.     

A mouse cerberus/Dan-related gene family

The Xenopus cerberus gene is able to induce ectopic heads in Xenopus embryos. At the time of its identification, cerberus shared significant homology with only one other protein, the putative rat tumor suppressor protein Dan. Sequence analysis has revealed that cerberus and Dan are members of a family of predicted secreted proteins, here called the can family. The identification of a can-family member in the nematode Caenorhabditis elegans, CeCan1, suggests that this family is of ancient origin. In the mouse, there are at least five family members: Cer1, Drm, PRDC, Dan, and Dte. These genes are expressed in patterns that suggest that they may play important roles in patterning the developing embryo. Cer1 marks the anterior visceral endoderm at E6.5. Dte is expressed asymmetrically in the developing node. Dan is first seen in the head mesoderm of early head fold stage embryos and Drm is expressed in the lateral paraxial mesoderm at E8.5. The region of homology shared by these genes, here called the can domain, closely resembles the cysteine knot motif found in a number of signaling molecules, such as members of the TGFbeta superfamily. Epitope-tagged versions of Cer1 show that, unlike in TGFbeta superfamily members, the cysteine knot motif is not processed away from a proprotein. Recent experiments in Xenopus have suggested that cerberus may act as an inhibitor of BMP signaling. To examine this further, the ability of Dan, Cer1, and human DRM to attenuate Bmp4 signaling has been assessed in P19 cells using pTlx-Lux, a BMP-responsive reporter. All three genes are able to inhibit Bmp4 signaling. These data suggest that the different family members may act to modulate the action of TGFbeta family members during development.     

Cell fate specification and competence by Coco,a maternal BMP,TGFbeta and Wnt inhibitor

Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFbeta/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFbeta signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.     

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.     

Biglycan is a new extracellular component of the Chordin-BMP4 signaling pathway

The BMP4 signaling pathway plays key roles during early embryonic development and for maintenance of adult homeostasis. In the extracellular space, BMP4 activity is regulated by a group of interacting molecules including the BMP antagonist Chordin, the metalloproteinase Tolloid and Twisted gastrulation (Tsg). In this study, we identified Biglycan (Bgn), a member of the small leucine-rich proteoglycan family, as a new extracellular modulator of BMP4 signaling. Xenopus Bgn (xBgn) is expressed uniformly in the ectoderm and mesoderm and their derivatives during development. Microinjection of Bgn mRNA induced secondary axes, dorsalized the mesoderm and inhibited BMP4 activity in Xenopus embryos. Biochemical experiments showed that Bgn binds BMP4 and Chordin, interaction that increased binding of BMP4 to Chordin. Bgn was also able to improve the efficiency of Chordin-Tsg complexes to block BMP4 activity. Using antisense morpholinos, we demonstrated that Bgn required Chordin to induce double axes in Xenopus. This work unveiled a new function for Bgn, its ability to regulate BMP4 signaling through modulation of Chordin anti-BMP4 activity.     

BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left-right asymmetry in the chick embryo

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.     

A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.     

Inhibitory control of neural differentiation in mammalian cells

 In Xenopus embryos, a truncated type II activin receptor (Δ1XAR1), capable of blocking signals by several transforming growth factor (TGF)-β family members, can induce neural tissue suggesting neural fate is under inhibitory control. Activin and bone morphogenetic protein 4 (BMP4) can act as neural inhibitors but only BMP4 can induce epidermis in Xenopus ectodermal cells. We have used the pluripotent mouse embryonal carcinoma cell line P19 to examine whether the mechanisms of ectodermal cell fate decisions are conserved among vertebrates. We show that a P19 cell line expressing Δ1XAR1 will differentiate into neurons. In addition, BMP4 inhibits retinoic acid (RA)-induced neural differentiation of P19 cells and induces keratin expression. These results suggest that in mammals as in amphibians neural fate is under inhibitory control and BMP4 can alter ectodermal differentiation. Received: 23 September 1996 / Accepted: 8 January 1997     

Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus

We report here the identification of four members of T-box family genes, Xltbx2-Xltbx5, in Xenopus. Two of them are probable pseudovariant genes of XTbx5 and ET, a putative Xenopus ortholog of Tbx3. We compared their expression patterns in both embryos and limbs. In embryos, expression of Xltbx2 and Xltbx3 showed novel diversities, such as Xltbx2 in the neural crest cells and Xltbx3 in the ventral spinal cord, together with mutual similarities in the following regions: dorsal retina, proctoderm, lateral line organ, cement gland and cranial ganglia. The patterns in limbs were highly conserved with mouse and chick orthologs, including the limb-type specific expression of Xltbx4 and Xltbx5. In addition, RT-PCR analysis showed that they are expressed weakly even in adult limbs as previously reported in the newt.     

Brorin, a novel secreted bone morphogenetic protein antagonist, promotes neurogenesis in mouse neural precursor cells

We identified a gene encoding a novel secreted protein in mice and humans and named it Brorin. Mouse Brorin consists of 324 amino acids with a putative secreted signal sequence at its amino terminus and two cysteine-rich domains in its core region. Positions of 10 cysteine residues in the domains of Brorin are similar to those in the cysteine-rich domains of members of the Chordin family. However, the amino acid sequence of Brorin is not significantly similar to that of any other member of the Chordin family, indicating that Brorin is a unique member of the family. Mouse Brorin protein produced in cultured cells was efficiently secreted into the culture medium. The protein inhibited the activity of bone morphogenetic protein 2 (BMP2) and BMP6 in mouse preosteoblastic MC3T3-E1 cells. Mouse Brorin was predominantly expressed in neural tissues in embryos and also predominantly expressed in the adult brain. In the brain, the expression was detected in neurons, but not glial cells. The neural tissue-specific expression profile of Brorin is quite distinct from that of any other member of the Chordin family. Brorin protein promoted neurogenesis, but not astrogenesis, in mouse neural precursor cells. The present findings indicate that Brorin is a novel secreted BMP antagonist that potentially plays roles in neural development and functions.     

Xenopus GDF6, a new antagonist of noggin and a partner of BMPs.

In Xenopus, ectodermal cell fates are determined by antagonistic interaction between the BMP subfamily of TGF-(beta) ligands and the organizer-specific secreted factors (e.g. noggin, chordin and follistatin). Inhibition of BMP function by these factors can convert cells from an epidermal to a neural cell fate. In this study, we report that GDF6, a new member of the Xenopus TGF-(beta) family, can function in antagonistic interaction with neural inducers. GDF6 induces epidermis and inhibits neural tissue in dissociated cells, and this activity is blocked by the presence of noggin. We demonstrate that GDF6 binds directly to the neural inducer noggin. Furthermore, we find that GDF6 and BMP2 can form heterodimers and the process seems to require cotranslation of the proteins in the same cells. In normal embryos, GDF6 and BMP2 are coexpressed in several places, including the edge of the neural plate at early neurula stages, suggesting that GDF6 may synergize with BMPs to regulate patterning of the ectoderm. Our data show for the first time that noggin can bind directly to and inhibit another TGF-(beta) family member: GDF6. In addition, BMP and GDF6 heterodimers may play an important role in vivo to regulate cell fate determination and patterning.     

Oocytes and embryos of Xenopus laevis express two different isoforms of germ cell nuclear factor (GCNF,NR6A1)

The germ cell nuclear factor (GCNF) is a nuclear orphan receptor and a putative regulator of the pluripotent state of cells. Although it was first described in mouse germ cells, GCNF is also expressed in mouse and Xenopus embryos. By means of 5'RACE we have identified a novel isoform of Xenopus laevis GCNF that is predominantly expressed in germ cells, whereas both the oocyte and embryonic forms are expressed during Xenopus embryogenesis. EST database search revealed that the homologues of both isoforms are also transcribed in Xenopus tropicalis.     

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.     

Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.     

A family of Xenopus BTB-Kelch repeat proteins related to ENC-1: new markers for early events in floorplate and placode development

We report the expression of a family of Xenopus genes encoding BTB-Kelch repeat proteins related to mouse ENC-1. Xenopus ENC-related-1 (Xencr-1) is expressed throughout the dorsal midline in dorsal endoderm, notochord, and deep neuroectoderm cells during early neurula stages. Later, Xencr-1 expression is downregulated in the notochord in an anterior-to-posterior progression but is maintained in the floorplate and dorsal endoderm. Xencr-3 is expressed strongly in several cranial placodes during the early neurula stages, placing it among the earliest molecular markers of placode precursor populations.