A gene regulatory network for neural induction.

During early vertebrate development, signals from a special region of the embryo, the organizer, can redirect the fate of non-neural ectoderm cells to form a complete, patterned nervous system. This is called neural induction and has generally been imagined as a single signalling event, causing a switch of fate. Here, we undertake a comprehensive analysis, in very fine time course, of the events following exposure of competent ectoderm of the chick to the organizer (the tip of the primitive streak, Hensen's node). Using transcriptomics and epigenomics we generate a gene regulatory network comprising 175 transcriptional regulators and 5614 predicted interactions between them, with fine temporal dynamics from initial exposure to the signals to expression of mature neural plate markers. Using in situ hybridization, single-cell RNA-sequencing, and reporter assays, we show that the gene regulatory hierarchy of responses to a grafted organizer closely resembles the events of normal neural plate development. The study is accompanied by an extensive resource, including information about conservation of the predicted enhancers in other vertebrates.

WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

OBJECTIVE: Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. METHODS: Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. RESULTS: WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. CONCLUSIONS: In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

ADP-ribosyl cyclases regulate early development of the sea urchin.

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca2+ signalling such as cADPR and NAADP. Although Ca2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

An essential role for LPA signalling in telencephalon development.

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

The signaling protein CD38 is essential for early embryonic development.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.

A single residue in a novel ADP-ribosyl cyclase controls production of the calcium-mobilizing messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.

Molecular characterization of a novel cell surface ADP-ribosyl cyclase from the sea urchin.

The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.

Molecular determinants of Xolloid action in vivo.

Xld (Xolloid) is a member of the Tolloid family of metalloproteases found in embryos of the frog Xenopus laevis. It cleaves Chordin, an inhibitory binding protein for BMP2/4, releasing fragments with reduced affinity for these important ventralizing signals. As a consequence, increasing Xld activity ventralizes Xenopus embryos. We have used this phenotype as an assay to determine the requirement for the C-terminal, nonprotease component of Xld for in vivo activity. This part of the protein is composed of five complement C1r/C1s-sea urchin epidermal growth factor-BMP1 (CUB) and two epidermal growth factor domains, which are thought to be involved in protein-protein interactions and may confer substrate specificity. Our results show that the protease coupled to CUB1 and CUB2 is the minimum domain structure required to ventralize Xenopus embryos and to block the dorsal axis-inducing activity of Chordin. Xld-CUB1-CUB2 cleaves Chordin, and a protease-inactive version co-precipitates Chordin. Our results indicate that the first and second CUB domains bind Chordin and present it to the protease domain. Protease-inactive Xld blocks the cleavage of Chordin by wild-type Xld and dorsalizes injected Xenopus embryos. We find that protease-inactive Xld-CUB1-CUB2 does not share this activity and that all of the C-terminal domains are required to generate the dorsalized phenotype.

A novel nucleotide receptor in Xenopus activates the cAMP second messenger pathway.

We describe a Xenopus P2Y receptor that shares only weak homology with members of the mammalian P2Y family, being most similar to human P2Y(11). When activated by nucleotide analogs, it stimulates both calcium and cAMP mobilization pathways, a feature unique, among mammalian P2Y receptors, to P2Y(11). Activity can be blocked by compounds known to act as antagonists of mammalian P2Y(11). Genomic synteny between Xenopus and mammals suggests that the novel gene is a true ortholog of P2Y(11). Xenopus P2Y(11) is transcribed during embryonic development, beginning at gastrulation, and is enriched in the developing nervous system.

Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

BACKGROUND: ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. CONCLUSIONS/SIGNIFICANCE: Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development.

We have characterized a cDNA encoding a Xenopus laevis apyrase (XAPY) that is expressed during embryogenesis. XAPY is highly homologous to two recently described mammalian apyrases, human SCAN-1 and rat Ca2+-NDPase, and to a lesser extent the salivary apyrase of the blood-feeding arthropod Cimex lectularis. RT-PCR analysis shows that Xapy is expressed at all the developmental stages tested, from oocytes through to tadpoles. Xapy transcripts are widely distributed in the embryo, but from late neurulae through to late tailbud stages they are highly enriched in the cement gland, an adhesive organ in the epidermis of the head. When expressed in HEK 293 cells, XAPY is largely retained in the endoplasmic reticulum, although some is also secreted. XAPY conditioned media hydrolyses UDP and UTP, confirming that it is a functional apyrase.

Members of the lysyl oxidase family are expressed during the development of the frog Xenopus laevis.

Lysyl oxidase (Lox) is a copper-dependent amine oxidase that catalyzes the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM). In mammals, four closely related Lox-like enzymes have been described that share a highly conserved catalytic domain with Lox. We have characterized Xenopus laevis cDNAs for Lox, Loxl-1, and Loxl-3, and show that they are expressed during early embryonic development. Using RT-PCR we detected maternal transcripts for Xloxl-1, but levels remained low until tailbud stages. Transcripts for Xlox and Xloxl-3 were not detected until early neurulae, although transcripts for Xlox remained at low levels until tailbud stages. Whole mount in situ hybridization showed that transcripts for Xloxl-1 and Xloxl-3 are localized in the notochord, while transcripts for Xlox are found in the notochord, somites, and head. X. laevis Lox-like enzymes were inhibited by incubating embryos, from cleavage stages to tadpole stages, in beta-aminopropionitrile, a specific inhibitor of the catalytic domain. The resulting embryos appeared to differentiate normally but suffered from poor collagen fiber formation. Defects included kinks in the notochord, a posterior shift of the somites, abnormal gut coiling, and the formation of edemas. Our data suggest that Lox-related enzymes are required for the proper formation of the ECM during X. laevis development.

Xolloid-related: a novel BMP1/Tolloid-related metalloprotease is expressed during early Xenopus development.

We have identified a novel Tolloid-like metalloprotease, called Xolloid-related (Xlr), that is expressed during early Xenopus development. Transcripts for xlr are localized to the marginal zone of mid-gastrulae and are most abundant in ventral and lateral sectors. At neurula stages xlr is strongly expressed around the blastopore and in the pharyngeal endoderm, and more weakly expressed throughout the ventral half of the embryo. Transcripts are detected in the nervous system, particularly the hindbrain and spinal cord, and tailbud of tailbud stage embryos, with weaker expression in the anterior nervous system, otic vesicle, heart, and pronephric duct. Transcription of xlr is increased by BMP4 and decreased by Noggin and tBR, indicating that xlr is regulated by BMP signalling. Injection of xlr mRNA inhibits dorsoanterior development and the dorsal axis inducing ability of coinjected chordin, but not noggin or tBR, mRNA. Xlr conditioned media cleaves Chordin in vitro, indicating that this protease may regulate the availability of Chordin in vivo.

Wound healing and regeneration in the imaginal wing disc ofDrosophila.

When complementary fragments of an imaginal disc ofDrosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In "06" fragments (Bryant 1975a) wound healing has occurred after 1-2 days of culture and the regeneration of missing structures after 2-4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days.When disc fragments are cultured in female abdomens and then exposed to3H-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication.

Is regeneration inDrosophila the result of epimorphic regulation?

It has been known for many years that when a wing disc ofDrosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed to3H-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic.