Unexpected functional redundancy between Twist and Slug (Snail2) and their feedback regulation of NF-κB via Nodal and Cerberus

A NF-κB-Twist-Snail network controls axis and mesoderm formation in Drosophila. Using translation-blocking morpholinos and hormone-regulated proteins, we demonstrate the presence of an analogous network in the early Xenopus embryo. Loss of twist (twist1) function leads to a reduction of mesoderm and neural crest markers, an increase in apoptosis, and a decrease in snail1 (snail) and snail2 (slug) mRNA levels. Injection of snail2 mRNA rescues twist's loss of function phenotypes and visa versa. In the early embryo NF-κB/RelA regulates twist, snail2, and snail1 mRNA levels; similarly Nodal/Smad2 regulate twist, snail2, snail1, and relA RNA levels. Both Twist and Snail2 negatively regulate levels of cerberus RNA, which encodes a Nodal, bone morphogenic protein (BMP), and Wnt inhibitor. Cerberus's anti-Nodal activity inhibits NF-κB activity and decreases relA RNA levels. These results reveal both conserved and unexpected regulatory interactions at the core of a vertebrate's mesodermal specification network.     

Determination of half-maximal inhibitory concentration using biosensor-based protein interaction analysis

Half-maximal inhibitory concentration (IC₅₀) is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research. Most approaches to determine IC₅₀ of a pharmacological compound are based on assays that utilize whole cell systems. While they generally provide outstanding potency information, results can depend on the experimental cell line used and may not differentiate a compound's ability to inhibit specific interactions. Here we show using the secreted Transforming Growth Factor-β (TGF-β) family ligand BMP-4 and its receptors as example that surface plasmon resonance can be used to accurately determine IC₅₀ values of individual ligand-receptor pairings. The molecular resolution achievable wih this approach can help distinguish inhibitors that specifically target individual complexes, or that can inhibit multiple functional interactions at the same time.     

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.     

Cripto-independent Nodal signaling promotes positioning of the A-P axis in the early mouse embryo

During early mouse development, the TGFβ-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto^−/− embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.     

Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro

Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.     

Identification and expression analysis of BMP signaling inhibitors genes of the DAN family in amphioxus

Bone morphogenetic proteins (BMPs) are members of the Transforming Growth Factor-β (TGF-β) family implicated in many developmental processes in metazoans such as embryo axes specification. Their wide variety of actions is in part controlled by inhibitors that impede the interaction of BMPs with their specific receptors. Here, we focused our attention on the Differential screening-selected gene Aberrative in Neuroblastoma (DAN) family of inhibitors. Although they are well-characterized in vertebrates, few data are available for this family in other metazoan species. In order to understand the evolution of potential developmental roles of these inhibitors in chordates, we identified the members of this family in the cephalochordate amphioxus, and characterized their expression patterns during embryonic development. Our data suggest that the function of Cerberus/Dand5 subfamily genes is conserved among chordates, whereas Gremlin1/2 and NBL1 subfamily genes seem to have acquired divergent expression patterns in each chordate lineage. On the other hand, the expression of Gremlin in the amphioxus neural plate border during early neurulation strengthens the hypothesis of a conserved neural plate border gene network in chordates.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Coordination of BMP-3b and cerberus is required for head formation of Xenopus embryos

Bone morphogenetic proteins (BMPs) and their antagonists are involved in the axial patterning of vertebrate embryos. We report that both BMP-3b and BMP-3 dorsalize Xenopus embryos, but act as dissimilar antagonists within the BMP family. BMP-3b injected into Xenopus embryos triggered secondary head formation in an autonomous manner, whereas BMP-3 induced aberrant tail formation. At the molecular level, BMP-3b antagonized nodal-like proteins and ventralizing BMPs, whereas BMP-3 antagonized only the latter. These differences are due to divergence of their pro-domains. Less BMP-3b than BMP-3 precursor is proteolytically processed in embryos. BMP-3b protein associated with a monomeric form of Xnrl, a nodal-like protein, whereas BMP-3 did not. These molecular features are consistent with their expression profiles during Xenopus development. XBMP-3b is expressed in the prechordal plate, while xBMP-3 is expressed in the notochord. Using antisense morpholino oligonucleotides, we found that the depletion of both xBMP-3b and cerberus, a head inducer, caused headless Xenopus embryos, whereas the depletion of both xBMP-3 and cerberus affected the size of the somite. These results revealed that xBMP-3b and cerberus are essential for head formation regulated by the Spemann organizer, and that xBMP-3b and perhaps xBMP-3 are involved in the axial patterning of Xenopus embryos.