[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Immunology 11/2013; · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vertebrate cardiac progenitor cells are initially allocated in two distinct domains, the first and second heart fields. It has been demonstrated that first heart field cells give rise to the myocardial cells in the left ventricle and part of the atria, whereas second heart field cells move into the developing heart tube and contribute to the myocardium of the outflow tract and right ventricle and the majority of atria. In this study, we have examined the expression of the mouse Cripto gene and the lineage of Cripto-expressing cells, focusing on its relationship with cardiac myocyte differentiation. The mouse Cripto gene is initially expressed at late head fold (LHF) stages in the cardiac crescent region, known as the first heart field; later in the medial region of the early heart tube, and by embryonic day 8.5, it is localized to the outflow tract. Using a Cripto-LacZ allele, we found that Cripto- expressing progeny cells contribute to the myocardium of the entire outflow tract and right ventricle, as well as to a majority of cells within the left ventricle. In contrast, no Cripto- expressing progeny cells were found in the atria or atrio-ventricular canal. Therefore, Cripto is transiently expressed in early differentiating myocardial cells of the left ventricle, right ventricle and outflow tract between LHF stages and E8.5. Cripto expression is subsequently downregulated as cells undergo further differentiation.
The International journal of developmental biology 10/2013; · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 is 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Cleft palate is a common birth defect that involves disruptions in multiple developmental steps such as growth, differentiation, elevation, and fusion. Medial edge epithelial (MEE) differentiation is essential for palate fusion. An important question is whether the MEE differentiation that occurs during fusion is induced by palate shelf contact or is programmed intrinsically by the palate shelf itself. Here, we report that the loss of Zfhx1a function in mice leads to a cleft palate phenotype that is mainly attributable to a delay in palate elevation. Zfhx1a encodes a transcription regulatory protein that modulates several signaling pathways including those activated by members of the transforming growth factor-beta (TGF-beta) superfamily. Loss of Zfhx1a function in mice leads to a complete cleft palate with 100% penetrance. Zfhx1a mutant palatal shelves display normal cell differentiation and proliferation and are able to fuse in an in vitro culture system. The only defect detected was a delay of 24-48 h in palatal shelf elevation. Using the Zfhx1a mutant as a model, we studied the relationship between MEE differentiation and palate contact/adhesion. We found that down-regulation of Jag2 expression in the MEE cells, a key differentiation event establishing palate fusion competence, was independent of palate contact/adhesion. Moreover, the expression of several key factors essential for fusion, such as TGF-beta3 and MMP13, was also down-regulated at embryonic stage 16.5 in a contact-independent manner, suggesting that differentiation of the medial edge epithelium was largely programmed through an intrinsic mechanism within the palate shelf.
Cell and Tissue Research 08/2008; 333(1):29-38. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cleft palate is a common birth defect caused by disruptions in secondary palate development. Anterior-posterior (A-P) regional specification plays a critical role in palate development and fusion. Previous studies have shown that at the molecular level, the anterior palate can be defined by the expression of Shox-2 and the posterior palate by Meox-2 expression in certain mouse strains. Here, we have extended previous studies by performing a more detailed analysis of these genes during mouse palate development. We found that the expression patterns of Shox-2 and Meox-2 are dynamic during palate development. At embryonic day 12.5 (E12.5), Shox-2 expression is localized to the anterior end and its expression domain covers less than 25% of the length of the palate shelf. The Shox-2 expression domain then gradually expands towards the posterior end and ultimately occupies more than 60% of the palate shelf by E14.5. The expansion of the Shox-2 domain may involve induction of Shox2 expression in additional cells. Reciprocally, the Meox-2 expression domain at E12.5 covers a large portion of the palate shelf, a region more than 70% of the entire palate, but then regresses to the posterior 25% by E14.5. This regression is likely caused by the repression of Meox-2 expression in certain Meox2 expressing cells, rather than the cessation of cell proliferation. Therefore, certain Meox-2 positive "primitive posterior cells" are differentiated/converted into Shox-2 positive "definitive anterior cells" during A-P regional specification.
The International Journal of Developmental Biology 02/2007; 51(2):167-72. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malformations in secondary palate fusion will lead to cleft palate, a common human birth defect. Palate fusion involves the formation and subsequent degeneration of the medial edge epithelial seam. The cellular mechanisms underlying seam degeneration have been a major focus in the study of palatogenesis. Three mechanisms have been proposed for seam degeneration: lateral migration of medial edge epithelial cells; epithelial-mesenchymal trans-differentiation; and apoptosis of medial edge epithelial cells. However, there is still a great deal of controversy over these proposed mechanisms. In this study, we established a [Rosa26<-->C57BL/6] chimeric culture system, in which a Rosa26-originated ;blue' palatal shelf was paired with a C57BL/6-derived ;white' palatal shelf. Using this organ culture system, we observed the migration of medial edge epithelial cells to the nasal side, but not to the oral side. We also observed an anteroposterior migration of medial edge epithelial cells, which may play an important role in posterior palate fusion. To examine epithelial-mesenchymal transdifferentiation during palate fusion, we bred a cytokeratin 14-Cre transgenic line into the R26R background. In situ hybridization showed that the Cre transgene is expressed exclusively in the epithelium. However, beta-galactosidase staining gave extensive signals in the palatal mesenchymal region during and after palate fusion, demonstrating the occurrence of an epithelial-mesenchymal transdifferentiation mechanism during palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are able to complete palate fusion without DNA fragmentation-mediated programmed cell death, indicating that this is not essential for palate fusion in vivo.
Development 09/2006; 133(17):3341-7. · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The Tgif gene encodes a homeodomain protein that functions as a transforming growth factor beta (TGF-beta) 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-beta 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-beta 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-beta antagonists such as c-Ski and SnoN during vertebrate anterior neural development.