Jixiang Ding

TAM Receptors Affect Adult Brain Neurogenesis by Negative Regulation of Microglial Cell Activation

TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3−/−Axl−/−Mertk−/− triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl−/−Mertk−/− double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

Analysis of Meox-2 mutant mice reveals a novel postfusion-based cleft palate

Cleft palate represents a common human congential disease involving defects in the development of the secondary palate. Major steps in mammalian palatogenesis include vertical growth, elevation, and fusion of the palate shelves. Our current study with the homeobox gene Meox‐2 during mouse secondary palate development reveals a novel postfusion‐based mechanism for cleft palate. Meox‐1 and Meox‐2 are two functionally related homeobox genes playing important roles in somitogenesis and limb muscle differentiation. We found that the expression of Meox‐2, not Meox‐1, marks the specification of early mouse palatal mesenchymal cells in the maxillary processes at embryonic day 11.5 (E11.5). From E12.5 to E15.5, the expression of Meox‐2 occupies only the posterior part of the palate, providing an early molecular marker for the anterior–posterior polarity in mouse secondary palate formation. A total of 35.3% of Meox‐2−/− (n = 17) and 25.5% of Meox‐2+/− (n = 55) mouse embryos display a cleft palate phenotype at E15.5, indicating that the reduction of Meox‐2 function is associated with susceptibility to cleft palate. Unlike previously reported clefts, none of the clefts found in Meox‐2 mutants contain any epithelial sheets in the medial edge areas, and detailed examination revealed that the clefts resulted from the breakdown of newly fused palates. This article is the first report of a gene required to maintain adherence of the palatal shelves after fusion. Developmental Dynamics 235:539–546, 2006.

Expression and functional analysis of Tgif during mouse midline development

The Tgif gene encodes a homeodomain protein that functions as a transforming growth factor beta (TGF‐β) repressor by binding to Smad2. Mutations in the TGIF gene are associated with human holoprosencephaly, a common birth defect caused by the failure of anterior ventral midline formation. However, Smad2‐mediated TGF‐β signaling in the axial mesendoderm has been demonstrated to be essential for ventral midline formation, and loss of a Smad2 antagonist should in principle promote rather than inhibit ventral midline formation. This suggests a more complex mechanism for the function of TGIF in controlling ventral midline formation. To explore the role of TGIF in ventral forebrain formation and patterning, we investigated Tgif expression and function during mouse development by in situ hybridization and gene targeting. We found that Tgif is highly expressed in the anterior neural plate, consistent with the proposed neural differentiation model in which TGF‐β suppression is required for normal neural differentiation. This result suggests a possible role for Tgif in anterior neural differentiation and patterning. However, targeted disruption of the Tgif gene during mouse development does not cause any detectable defects in development and growth. Both histological examination and gene expression analysis showed that Tgif−/− embryos have a normal ventral specification in the central nervous system, including the forebrain region. One interpretation of these results is that the loss of TGIF function is compensated by other TGF‐β antagonists such as c‐Ski and SnoN during vertebrate anterior neural development. Developmental Dynamics 235:547–553, 2006.

TAM Receptors Support Neural Stem Cell Survival, Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

Mesenchymal cell remodeling during mouse secondary palate reorientation

The formation of mammalian secondary palate requires a series of developmental events such as growth, elevation, and fusion. Despite recent advances in the field of palate development, the process of palate elevation remains poorly understood. The current consensus on palate elevation is that the distal end of the vertical palatal shelf corresponds to the medial edge of the elevated horizontal palatal shelf. We provide evidence suggesting that the prospective medial edge of the vertical palate is located toward the interior side (the side adjacent to the tongue), instead of the distal end, of the vertical palatal shelf and that the horizontal palatal axis is generated through palatal outgrowth from the side of the vertical palatal shelf rather than rotating the pre‐existing vertical axis orthogonally. Because palate elevation represents a classic example of embryonic tissue re‐orientation, our findings here may also shed light on the process of tissue re‐orientation in general. Developmental Dynamics 239:2110–2117, 2010.

Retinoic acid regulates embryonic development of mammalian submandibular salivary glands.

Organogenesis is orchestrated by cell and tissue interactions mediated by molecular signals. Identification of relevant signals, and the tissues that generate and receive them, are important goals of developmental research. Here, we demonstrate that Retinoic Acid (RA) is a critical signaling molecule important for morphogenesis of mammalian submandibular salivary glands (SMG). By examining late stage RA deficient embryos of Rdh10 mutant mice we show that SMG development requires RA in a dose-dependent manner. Additionally, we find that active RA signaling occurs in SMG tissues, arising earlier than any other known marker of SMG development and persisting throughout gland morphogenesis. At the initial bud stage of development, we find RA production occurs in SMG mesenchyme, while RA signaling occurs in epithelium. We also demonstrate active RA signaling occurs in glands cultured ex vivo, and treatment with an inhibitor of RA signaling blocks growth and branching. Together these data identify RA signaling as a direct regulator of SMG organogenesis.

Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

During mouse gastrulation, cells in the primitive streak undergo epithelial-mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8-Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.

Ggnbp2 Is Essential for Pregnancy Success via Regulation of Mouse Trophoblast Stem Cell Proliferation and Differentiation

ABSTRACT The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.

Deciphering TGF-β3 function in medial edge epithelium specification and fusion during mouse secondary palate development.

Background: Transforming growth factor‐β3 (TGF‐β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF‐β3 functions during secondary palate fusion are still poorly understood. Results: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf‐β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down‐regulated in the medial edge epithelium during palatal fusion. Jagged2 down‐regulation was regulated by TGF‐β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf‐β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf‐β3 null mutant palatal shelves. Conclusions: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF‐β3 mediates palate fusion in part by down‐regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF‐β3 downstream target genes in palate medial edge epithelium differentiation. Developmental Dynamics 243:1536–1543, 2014.

GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells

Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.