spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade

pou5f1, also known as Oct4, is required to establish the pluripotent cell population necessary for embryogenesis in mouse. Additional roles during development, including endoderm formation, have been proposed. In zebrafish, the zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate patterning defects and reduced endoderm at the tailbud stage. To investigate the function of maternal and early zygotic pou5f1 expression, we rescued zygotic spg(m793) mutants by injecting pou5f1 mRNA at the one-cell stage and raised them into fertile homozygous spg(m793) adults that mate to produce maternal-zygotic spg (MZspg) mutant embryos. Although neurectoderm, mesoderm, and germ cells develop in MZspg mutants, gastrulation is delayed and proceeds abnormally. Further, MZspg mutants do not maintain expression of sox32/casanova, express little or no sox17, and fail to develop endodermal tissue. Constitutively active Nodal receptor TARAM-A or sox32 overexpression induces ubiquitous sox17 expression in wild-type embryos, but not in MZspg mutants. Overexpression of a Pou5f1-VP16 activator fusion protein can rescue gastrulation and endodermal tissues in MZspg mutants. We propose that pou5f1 plays an activating role in zebrafish endodermal development, where it maintains sox32 expression during gastrulation and acts with sox32 to induce sox17 expression in endodermal precursor cells.     

Tgfbr2 is required for development of the skull vault

Transforming growth factor β (TGFβ) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre⁺/Tgfbr2^f/f embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre⁺/Tgfbr2^f/f embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre⁺/Tgfbr2^f/f embryos suggesting that osteoblast differentiation was also altered in Prx1Cre⁺/Tgfbr2^f/f embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2^f/f embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.     

Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish

Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-β signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis.     

Inhibition of Cancer Cell Migration and Invasion through Suppressing the Wnt1-mediating Signal Pathway by G-quadruplex Structure Stabilizers

WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of β-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.     

Valproic acid silencing of ascl1b/Ascl1 results in the failure of serotonergic differentiation in a zebrafish model of fetal valproate syndrome

Fetal valproate syndrome (FVS) is caused by in utero exposure to the drug sodium valproate. Valproate is used worldwide for the treatment of epilepsy, as a mood stabiliser and for its pain-relieving properties. In addition to birth defects, FVS is associated with an increased risk of autism spectrum disorder (ASD), which is characterised by abnormal behaviours. Valproate perturbs multiple biochemical pathways and alters gene expression through its inhibition of histone deacetylases. Which, if any, of these mechanisms is relevant to the genesis of its behavioural side effects is unclear. Neuroanatomical changes associated with FVS have been reported and, among these, altered serotonergic neuronal differentiation is a consistent finding. Altered serotonin homeostasis is also associated with autism. Here we have used a chemical-genetics approach to investigate the underlying molecular defect in a zebrafish FVS model. Valproate causes the selective failure of zebrafish central serotonin expression. It does so by downregulating the proneural gene ascl1b, an ortholog of mammalian Ascl1, which is a known determinant of serotonergic identity in the mammalian brainstem. ascl1b is sufficient to rescue serotonin expression in valproate-treated embryos. Chemical and genetic blockade of the histone deacetylase Hdac1 downregulates ascl1b, consistent with the Hdac1-mediated silencing of ascl1b expression by valproate. Moreover, tonic Notch signalling is crucial for ascl1b repression by valproate. Concomitant blockade of Notch signalling restores ascl1b expression and serotonin expression in both valproate-exposed and hdac1 mutant embryos. Together, these data provide a molecular explanation for serotonergic defects in FVS and highlight an epigenetic mechanism for genome-environment interaction in disease.KEY WORDS: Serotonin, Fetal valproate syndrome, Zebrafish, Notch, Proneural gene, Hdac1