Wei Hsu

The Mouse Fused Locus Encodes Axin, an Inhibitor of the Wnt Signaling Pathway That Regulates Embryonic Axis Formation

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians.

The role of Axin2 in calvarial morphogenesis and craniosynostosis

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of β-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of β-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis.

Identification of a Domain of Axin That Binds to the Serine/Threonine Protein Phosphatase 2A and a Self-binding Domain

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and β-catenin. Axin has been shown to have distinct binding sites for GSK-3 and β-catenin and to promote the phosphorylation of β-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through β-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of β-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin·APC·GSK-3·β-catenin complex, where it could modulate the effect of GSK-3 on β-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.

Fos and Jun repress transcription activation by NF-IL6 through association at the basic zipper region.

NF-IL6 and AP-1 family transcription factors are coordinately induced by interleukin-6 (IL-6) in a cell-type-specific manner, suggesting that they mediate IL-6 signals in the nucleus. We show that the basic leucine zipper (bZIP) region of NF-IL6 mediates a direct association with the bZIP regions of Fos and Jun in vitro. This interaction does not depend on the presence of their cognate recognition DNA elements or the posttranslational modification of either partner. NF-IL6 homodimers can bind to both NF-IL6 and AP-1 sites, whereas Fos and Jun cannot bind to most NF-IL6 sites. Cross-family association with Fos or with Jun alters the DNA binding specificity of NF-IL6 and reduced its binding to NF-IL6 sites. NF-IL6 isoforms that differ in the site of translation initiation have distinct transcriptional activities. Activation of a reporter gene linked to the NF-IL6 site by NF-IL6 is repressed by Fos and by Jun in transient transfection assays. Thus, association with AP-1 results in repression of transcription activation by NF-IL6. The repression is NF-IL6 site dependent and may have a role in determining the promoter and cell type specificity in IL-6 signaling.

Reciprocal regulation of Wnt and Gpr177/mouse Wntless is required for embryonic axis formation.

Members of the Wnt family are secreted glycoproteins that trigger cellular signals essential for proper development of organisms. Cellular signaling induced by Wnt proteins is involved in diverse developmental processes and human diseases. Previous studies have generated an enormous wealth of knowledge on the events in signal-receiving cells. However, relatively little is known about the making of Wnt in signal-producing cells. Here, we describe that Gpr177, the mouse orthologue of Drosophila Wls, is expressed during formation of embryonic axes. Embryos with deficient Gpr177 exhibit defects in establishment of the body axis, a phenotype highly reminiscent to the loss of Wnt3. Although many different mammalian Wnt proteins are required for a wide range of developmental processes, the Wnt3 ablation exhibits the earliest developmental abnormality. This suggests that the Gpr177-mediated Wnt production cannot be substituted. As a direct target of Wnt, Gpr177 is activated by β-catenin and LEF/TCF-dependent transcription. This activation alters the cellular distributions of Gpr177 which binds to Wnt proteins and assists their sorting and secretion in a feedback regulatory mechanism. Our findings demonstrate that the loss of Gpr177 affects Wnt production in the signal-producing cells, leading to alterations of Wnt signaling in the signal-receiving cells. A reciprocal regulation of Wnt and Gpr177 is essential for the patterning of the anterior-posterior axis during mammalian development.

Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function

Neurog3 (Neurogenin 3 or Ngn3) is both necessary and sufficient to induce endocrine islet cell differentiation from embryonic pancreatic progenitors. Since robust Neurog3 expression has not been detected in hormone-expressing cells, Neurog3 is used as an endocrine progenitor marker and regarded as dispensable for the function of differentiated islet cells. Here we used 3 independent lines of Neurog3 knock-in reporter mice and mRNA/protein-based assays to examine Neurog3 expression in hormone-expressing islet cells. Neurog3 mRNA and protein are detected in hormone-producing cells at both embryonic and adult stages. Significantly, inactivating Neurog3 in insulin-expressing β cells at embryonic stages or in Pdx1-expressing islet cells in adults impairs endocrine function, a phenotype that is accompanied by reduced expression of several Neurog3 target genes that are essential for islet cell differentiation, maturation, and function. These findings demonstrate that Neurog3 is required not only for initiating endocrine cell differentiation, but also for promoting islet cell maturation and maintaining islet function.

SUMO-Specific Protease 2 Is Essential for Modulating p53-Mdm2 in Development of Trophoblast Stem Cell Niches and Lineages

SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G–S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53–Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2–Mdm2–p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.

NF-IL6 and AP-1 cooperatively modulate the activation of the TSG-6 gene by tumor necrosis factor alpha and interleukin-1.

Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) activate transcription of the TSG-6 gene in normal human fibroblasts through a promoter region (-165 to -58) that encompasses an AP-1 and a NF-IL6 site. We show by deletion analysis and substitution mutagenesis that both sites are necessary for activation by TNF-alpha. Activation by IL-1 requires the NF-IL6 site and is enhanced by the AP-1 site. These results suggest that the NF-IL6 and AP-1 family transcription factors functionally cooperate to mediate TNF-alpha and IL-1 signals. Consistent with this possibility, IL-1 and TNF-alpha markedly increase the binding of Fos and Jun to the AP-1 site, and NF-IL6 activates the native TSG-6 promoter. Activation by NF-IL6 requires an intact NF-IL6 site and is modulated by the ratio of activator to inhibitor NF-IL6 isoforms that are translated from different in-frame AUGs. However, the inhibitor isoform can also bind to the AP-1 site and repress AP-1 site-mediated transcription. The finding that the inhibitor isoform antagonizes activation of the native TSG-6 promoter by IL-1 and TNF-alpha suggests that NF-IL6 has a physiologic role in these cytokine responses. Thus, the functionally distinct NF-IL6 isoforms cooperate with Fos and Jun to positively and negatively regulate the native TSG-6 promoter by TNF-alpha and IL-1.

The Balance of WNT and FGF Signaling Influences Mesenchymal Stem Cell Fate During Skeletal Development

Imbalance of WNT and FGF signaling promotes premature closure of skull bones by inducing bone formation through chondrogenesis. A Delicate Balance in Skull Development When skull bones initially form, they are separated by sites called sutures, and, in humans, the skull bones fuse after birth. Skull bone growth occurs through a process called intramembranous ossification, in which mesenchymal cells differentiate directly into bone-forming osteoblasts that deposit the bone matrix. Maruyama et al. found that, in mice, one layer of the posterior frontal suture closed through a process called endochondral ossification in which skeletal precursors differentiate into cartilage cells called chondrocytes before bone matrix deposition. Furthermore, they found that, when β-catenin signaling was increased and fibroblast growth factor signaling was simultaneously reduced, aberrant closure of another suture occurred through a process involving chondrogenesis. Their data suggest that, in addition to excessive osteoblastogenesis, aberrant chondrogenesis may be a mechanism by which premature closure of the skull bones, causing the disorder craniosynostosis, can occur. Craniosynostosis, a developmental disorder resulting from premature closure of the gaps (sutures) between skull bones, can be caused by excessive intramembranous ossification, a type of bone formation that does not involve formation of a cartilage template (chondrogenesis). Here, we show that endochondral ossification, a type of bone formation that proceeds through a cartilage intermediate, caused by switching the fate of mesenchymal stem cells to chondrocytes, can also result in craniosynostosis. Simultaneous knockout of Axin2, a negative regulator of the WNT–β-catenin pathway, and decreased activity of fibroblast growth factor (FGF) receptor 1 (FGFR1) in mice induced ectopic chondrogenesis, leading to abnormal suture morphogenesis and fusion. Genetic analyses revealed that activation of β-catenin cooperated with FGFR1 to alter the lineage commitment of mesenchymal stem cells to differentiate into chondrocytes, from which cartilage is formed. We showed that the WNT–β-catenin pathway directly controlled the stem cell population by regulating its renewal and proliferation, and indirectly modulated lineage specification by setting the balance of the FGF and bone morphogenetic protein pathways. This study identifies endochondral ossification as a mechanism of suture closure during development and implicates this process in craniosynostosis.

Cartilage-specific β-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development.

The WNT/β‐catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of β‐catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β‐catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β‐catenin in committed growth‐plate chondrocytes has been less robust. To identify the specific role of cartilage‐derived β‐catenin in regulating cartilage and bone development, we studied chondrocyte‐specific gain‐ and loss‐of‐function genetic mouse models using the tamoxifen‐inducible Col2CreERT2 transgene in combination with β‐cateninfx(exon3)/wt or β‐cateninfx/fx floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage‐specific β‐catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)‐mediated mechanism. Second, cartilage‐specific β‐catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage‐specific β‐catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage‐derived β‐catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.