Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Evolution of the DAN gene family in vertebrates.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost.

CERBERUS is critical for stabilization of VAPYRIN during rhizobial infection in Lotus japonicus.

CERBERUS (also known as LIN) and VAPYRIN (VPY) are essential for infection of legumes by rhizobia and arbuscular mycorrhizal fungi (AMF). Medicago truncatula LIN (MtLIN) was reported to interact with MtVPY, but the significance of this interaction is unclear and the function of VPY in Lotus japonicus has not been studied. We demonstrate that CERBERUS has auto-ubiquitination activity in vitro and is localized within distinct motile puncta in L. japonicus root hairs and in Nicotiana benthamiana leaves. CERBERUS colocalized with the trans-Golgi network/early endosome markers. In L. japonicus, two VPY orthologs (LjVPY1 and LjVPY2) were identified. CERBERUS interacted with and colocalized with both LjVPY1 and LjVPY2. Co-expression of CERBERUS with LjVPY1 or LjVPY2 in N. benthamiana led to increased protein levels of LjVPY1 and LjVPY2, which accumulated as mobile punctate bodies in the cytoplasm. Conversely, LjVPY2 protein levels decreased in cerberus roots after rhizobial inoculation. Mutant analysis indicates that LjVPY1 and LjVPY2 are required for rhizobial infection and colonization by AMF. Our data suggest that CERBERUS stabilizes LjVPY1 and LjVPY2 within the trans-Golgi network/early endosome, where they might function to regulate endocytic trafficking and/or the formation or recycling of signaling complexes during rhizobial and AMF symbiosis.

Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression.

Correct patterning of left-right (LR) asymmetry is essential during the embryonic development of bilaterians. Hedgehog (Hh) signaling is known to play a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we report a novel regulatory mechanism for Hh in LR asymmetry development of amphioxus embryos. Our results revealed that Hh-/- embryos abolish Cerberus (Cer) transcription, with bilaterally symmetric expression of Nodal, Lefty and Pitx In consequence, Hh-/- mutants duplicated left-side structures and lost right-side characters, displaying an abnormal bilaterally symmetric body plan. These LR defects in morphology and gene expression could be rescued by Hh mRNA injection. Our results indicate that Hh participates in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signaling failed to alter the Cer expression pattern or LR morphology in amphioxus embryos, indicating that Hh might not provide an asymmetric cue for Cer expression. In addition, Hh is required for mouth opening in amphioxus, hinting at a homologous relationship between amphioxus and vertebrate mouth development.

Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis.

The earliest event in Xenopus development is the dorsal accumulation of nuclear ß-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of ß-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 ß-catenin targets were enriched on the dorsal side and defined an early dorsal ß-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of ß-catenin/Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to ß-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal ß-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

Structure of neuroblastoma suppressor of tumorigenicity 1 (NBL1): insights for the functional variability across bone morphogenetic protein (BMP) antagonists.

Bone morphogenetic proteins (BMPs) are antagonized through the action of numerous extracellular protein antagonists, including members from the differential screening-selected gene aberrative in neuroblastoma (DAN) family. In vivo, misregulation of the balance between BMP signaling and DAN inhibition can lead to numerous disease states, including cancer, kidney nephropathy, and pulmonary arterial hypertension. Despite this importance, very little information is available describing how DAN family proteins effectively inhibit BMP ligands. Furthermore, our understanding for how differences in individual DAN family members arise, including affinity and specificity, remains underdeveloped. Here, we present the structure of the founding member of the DAN family, neuroblastoma suppressor of tumorigenicity 1 (NBL1). Comparing NBL1 to the structure of protein related to Dan and Cerberus (PRDC), a more potent BMP antagonist within the DAN family, a number of differences were identified. Through a mutagenesis-based approach, we were able to correlate the BMP binding epitope in NBL1 with that in PRDC, where introduction of specific PRDC amino acids in NBL1 (A58F and S67Y) correlated with a gain-of-function inhibition toward BMP2 and BMP7, but not GDF5. Although NBL1(S67Y) was able to antagonize BMP7 as effectively as PRDC, NBL1(S67Y) was still 32-fold weaker than PRDC against BMP2. Taken together, this data suggests that alterations in the BMP binding epitope can partially account for differences in the potency of BMP inhibition within the DAN family.

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-ß (TGF-ß) 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.

Brain enlargement with rostral bias in larvae from a spontaneously occurring female variant line of Xenopus; role of aberrant embryonic Wnt/ß-catenin signaling.

Increased brain size and its rostral bias are hallmarks of vertebrate evolution, but the underlying developmental and genetic basis remains poorly understood. To provide clues to understanding vertebrate brain evolution, we investigated the developmental mechanisms of brain enlargement observed in the offspring of a previously unrecognized, spontaneously occurring female variant line of Xenopus that appears to reflect a genetic variation. Brain enlargement in larvae from this line showed a pronounced rostral bias that could be traced back to the neural plate, the primordium of the brain. At the gastrula stage, the Spemann organizer, which is known to induce the neural plate from the adjacent dorsal ectoderm and give it the initial rostrocaudal patterning, was expanded from dorsal to ventral in a large proportion of the offspring of variant females. Consistently, siamois expression, which is required for Spemann organizer formation, was expanded laterally from dorsal to ventral at the blastula stage in variant offspring. This implies that the active region of the Wnt/ß-catenin signaling pathway was similarly expanded in advance on the dorsal side, as siamois is a target gene of this pathway. Notably, the earliest detectable change in variant offspring was in fertilized eggs, in which maternal wnt11b mRNA, a candidate dorsalizing factor responsible for activating Wnt/ß-catenin signaling in the dorsal embryonic region, had a wider distribution in the vegetal cortical cytoplasm. Since lateral spreading of wnt11b mRNA, and possibly that of other potential maternal dorsalizing factors in these eggs, is expected to facilitate lateral expansion of the active region of the Wnt/ß-catenin pathway during subsequent embryonic stages, we concluded that aberrant Wnt/ß-catenin signaling could cause rostral-biased brain enlargement via expansion of siamois expression and consequent expansion of the Spemann organizer in Xenopus. Our studies of spontaneously occurring variations in brain development in Xenopus would provide hints for uncovering genetic mutations that drive analogous morphogenetic variations during vertebrate brain evolution.

Rise of the BQ.1.1.37 SARS-CoV-2 Sublineage, Italy.

BQ.1.1 has dominated the Europe and Americas COVID-19 wave across the 2022-2023 winter, and further viral evolution is expected to escape the consolidating immune responses. We report here the emergence of the BQ.1.1.37 variant in Italy, peaking in January 2022 before suffering competition by XBB.1.*. We attempted to correlate the potential fitness of BQ.1.1.37 with a unique two-amino acid insertion within the Spike protein.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Geometry and Segmentation of Cerberus Fossae, Mars: Implications for Marsquake Properties.

The NASA InSight mission to Mars successfully landed on 26 November 2018 in Elysium Planitia. It aims to characterize the seismic activity and aid in the understanding of the internal structure of Mars. We focus on the Cerberus Fossae region, a giant fracture network ∼1,200 km long situated east of the InSight landing site where M ∼3 marsquakes were detected during the past 2 years. It is formed of five main fossae located on the southeast of the Elysium Mons volcanic rise. We perform a detailed mapping of the entire system based on high-resolution satellite images and Digital Elevation Models. The refined cartography reveals a range of morphologies associated with dike activity at depth. Width and throw measurements of the fossae are linearly correlated, suggesting a possible tectonic control on the shapes of the fossae. Widths and throws decrease toward the east, indicating the long-term direction of propagation of the dike-induced graben system. They also give insights into the geometry at depth and how the possible faults and fractures are rooted in the crust. The exceptional preservation of the fossae allows us to detect up to four scales of segmentation, each formed by a similar number of 3-4 segments/subsegments. This generic distribution is comparable to continental faults and fractures on Earth. We anticipate higher stress and potential marsquakes within intersegment zones and at graben tips.

Construction and regulation of aqueous-based Cerberus droplets by vortex mixing.

HYPOTHESIS: The emerging aqueous-based Cerberus emulsion droplets with multi-domains behave as an excellent platform to design cyto-mimetic compartmentalization for fabrication of anisotropic biomimetic materials and microreactors. However, the ultralow water/water interfacial tension impedes fabrication of aqueous Cerberus droplets in batch-scale and precisely topology regulation especially under lack of deep understanding of w/w interface properties. EXPERIMENTS: Aqueous-based ternary phase diagram composed by salt, hydrophilic polymer and fluorocarbon compound is determined. Different emulsions employing the multiple aqueous solutions as internal phase and vegetable oil as continuous phase, are prepared by traditional vortex mixing based on the diagram. The construction mechanism of (W1 + W2 + W3)/O Cerberus droplets and relationship between droplet topology and the diagram are investigated. FINDINGS: Diverse categories of aqueous-based emulsions from single emulsions, Janus emulsions, to Cerberus emulsions are delicately controlled in the same system based on the diagram. Various morphologies of Cerberus droplets such as linear and fan-like configurations are obtained, although spreading coefficient based on interfacial tension indicates preference of onion-like configuration. The viscosity plays an unexpected role in the construction of Cerberus droplets due to highly sensitive water/water interfaces within the droplets. Moreover, an empirical equation is perfectly applied, which endows quantitative prediction and control of lobe volume ratio within Cerberus droplets.

BMP antagonists in tissue development and disease.

Bone morphogenic proteins (BMPs) are important growth regulators in embryogenesis and postnatal homeostasis. Their tight regulation is crucial for successful embryonic development as well as tissue homeostasis in the adult organism. BMP inhibition by natural extracellular biologic antagonists represents the most intensively studied mechanistic concept of BMP growth factor regulation. It was shown to be critical for numerous developmental programs, including germ layer specification and spatiotemporal gradients required for the establishment of the dorsal-ventral axis and organ formation. The importance of BMP antagonists for extracellular matrix homeostasis is illustrated by the numerous human connective tissue disorders caused by their mutational inactivation. Here, we will focus on the known functional interactions targeting BMP antagonists to the ECM and discuss how these interactions influence BMP antagonist activity. Moreover, we will provide an overview about the current concepts and investigated molecular mechanisms modulating BMP inhibitor function in the context of development and disease.

New Ligand Binding Function of Human Cerberus and Role of Proteolytic Processing in Regulating Ligand-Receptor Interactions and Antagonist Activity.

Cerberus is a key regulator of vertebrate embryogenesis. Its biological function has been studied extensively in frog and mouse embryos. Its ability to bind and antagonize the transforming growth factor-ß (TGF-ß) family ligand Nodal is well established. Strikingly, the molecular function of Cerberus remains poorly understood. The underlying reason is that Cerberus is a complex, multifunctional protein: It binds and inhibits multiple TGF-ß family ligands, it may bind and inhibit some Wnt family members, and two different forms with distinct activities have been described. In addition, sequence homology between frog and mammalian Cerberus is low, suggesting that previous studies, which analyzed frog Cerberus function, may not accurately describe the function of mammalian Cerberus. We therefore undertook to determine the molecular activities of human Cerberus in TGF-ß family signaling. Using purified proteins, surface plasmon resonance, and reporter gene assays, we discovered that human Cerberus bound and inhibited the TGF-ß family ligands Activin B, BMP-6, and BMP-7, but not the frog Cerberus ligand BMP-2. Notably, full-length Cerberus successfully blocked ligand binding to type II receptors, but the short form was less effective. In addition, full-length Cerberus suppressed breast cancer cell migration but the short form did not. Thus, our findings expand the roles of Cerberus as TGF-ß family signaling inhibitor, provide a molecular rationale for the function of the N-terminal region, and support the idea that Cerberus could have regulatory activities beyond direct inhibition of TGF-ß family signaling.

Anthropogenic impacts drive niche and conservation metrics of a cryptic rattlesnake on the Colorado Plateau of western North America.

Ecosystems transition quickly in the Anthropocene, whereas biodiversity adapts more slowly. Here we simulated a shifting woodland ecosystem on the Colorado Plateau of western North America by using as its proxy over space and time the fundamental niche of the Arizona black rattlesnake (Crotalus cerberus). We found an expansive (= end-of-Pleistocene) range that contracted sharply (= present), but is blocked topographically by Grand Canyon/Colorado River as it shifts predictably northwestward under moderate climate change (= 2080). Vulnerability to contemporary wildfire was quantified from available records, with forested area reduced more than 27% over 13 years. Both 'ecosystem metrics' underscore how climate and wildfire are rapidly converting the Plateau ecosystem into novel habitat. To gauge potential effects on C. cerberus, we derived a series of relevant 'conservation metrics' (i.e. genetic variability, dispersal capacity, effective population size) by sequencing 118 individuals across 846 bp of mitochondrial (mt)DNA-ATPase8/6. We identified five significantly different clades (net sequence divergence = 2.2%) isolated by drainage/topography, with low dispersal (F ST = 0.82) and small sizes (2N ef = 5.2). Our compiled metrics (i.e. small-populations, topographic-isolation, low-dispersal versus conserved-niche, vulnerable-ecosystem, dispersal barriers) underscore the susceptibility of this woodland specialist to a climate and wildfire tandem. We offer adaptive management scenarios that may counterbalance these metrics and avoid the extirpation of this and other highly specialized, relictual woodland clades.

An In vitro Study on Chick Somite Ability to Express Cerberus, Chordin, FGF8, Follistatin, and Noggin Transcripts.

BACKGROUND: In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. METHODS: Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. RESULTS: Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. CONCLUSION: We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin.

Post-zygotic breakage of a dicentric chromosome results in mosaicism for a telocentric 9p marker chromosome in a boy with developmental delay.

Chromosomal rearrangements resulting in an inverted duplication and a terminal deletion (inv dup del) can occur due to three known mechanisms, two of them resulting in a normal copy region between the duplicated regions. These mechanisms involve the formation of a dicentric chromosome, which undergo breakage during cell division resulting in cells with either an inverted duplication and deletion or a terminal deletion. We describe a mosaic 3 year old patient with two cell lines carrying a chromosome 9p deletion where one of the cell lines contains an additional telocentric marker chromosome. Our patient is mosaic for the product of a double breakage of a dicentric chromosome including a centric fission. Mosaicism involving different rearrangements of the same chromosome is rare and suggests an early mitotic breakage event. Chr9p terminal deletions associated with duplications have previously been reported in 11 patients. We compare the clinical features of all 12 patients including the patient that we report here. To the best to our knowledge this is a first case reported where the double breakage occurred in the dicentric derivative chromosome 9.

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-ß (TGF-ß) 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.