Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex

Regulace exprese provirus RSV v savcich hostitelskych bunkach.

Available from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

The Mandibular and Hyoid Arches—From Molecular Patterning to Shaping Bone and Cartilage

The mandibular and hyoid arches collectively make up the facial skeleton, also known as the viscerocranium. Although all three germ layers come together to assemble the pharyngeal arches, the majority of tissue within viscerocranial skeletal components differentiates from the neural crest. Since nearly one third of all birth defects in humans affect the craniofacial region, it is important to understand how signalling pathways and transcription factors govern the embryogenesis and skeletogenesis of the viscerocranium. This review focuses on mouse and zebrafish models of craniofacial development. We highlight gene regulatory networks directing the patterning and osteochondrogenesis of the mandibular and hyoid arches that are actually conserved among all gnathostomes. The first part of this review describes the anatomy and development of mandibular and hyoid arches in both species. The second part analyses cell signalling and transcription factors that ensure the specificity of individual structures along the anatomical axes. The third part discusses the genes and molecules that control the formation of bone and cartilage within mandibular and hyoid arches and how dysregulation of molecular signalling influences the development of skeletal components of the viscerocranium. In conclusion, we notice that mandibular malformations in humans and mice often co-occur with hyoid malformations and pinpoint the similar molecular machinery controlling the development of mandibular and hyoid arches

Tcf7L2 is essential for neurogenesis in the developing mouse neocortex

Abstract Generation of neurons in the embryonic neocortex is a balanced process of proliferation and differentiation of neuronal progenitor cells. Canonical Wnt signalling is crucial for expansion of radial glial cells in the ventricular zone and for differentiation of intermediate progenitors in the subventricular zone. We detected abundant expression of two transcrtiption factors mediating canonical Wnt signalling, Tcf7L1 and Tcf7L2, in the ventricular zone of the embryonic neocortex. Conditional knock-out analysis showed that Tcf7L2, but not Tcf7L1, is the principal Wnt mediator important for maintenance of progenitor cell identity in the ventricular zone. In the absence of Tcf7L2, the Wnt activity is reduced, ventricular zone markers Pax6 and Sox2 are downregulated and the neuroepithelial structure is severed due to the loss of apical adherens junctions. This results in decreased proliferation of radial glial cells, the reduced number of intermediate progenitors in the subventricular zone and hypoplastic forebrain. Our data show that canonical Wnt signalling, which is essential for determining the neuroepithelial character of the neocortical ventricular zone, is mediated by Tcf7L2

Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.

During mouse eye development, all retinal cell types are generated from the population of retina-committed progenitors originating from the neuroepithelium of the optic vesicle. Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited. Here we report generation of the mRx-Cre BAC transgenic mouse line in which the expression of Cre recombinase is controlled by regulatory sequences of the mouse Rx gene, one of the earliest determinants of retinal development. When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup. Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development. Moreover, since eye organogenesis is dependent on the inductive signals between the optic vesicle and head surface ectoderm, the inductive ability of the optic vesicle can be analyzed using mRx-Cre transgenic mice

A dynamic gradient of Wnt signaling controls initiation of neurogenesis in the mammalian cortex and cellular specification in the hippocampus

AbstractNeurogenesis in the developing neocortex is a strictly regulated process of cell division and differentiation. Here we report that a gradual retreat of canonical Wnt signaling in the cortex from lateral-to-medial and anterior-to-posterior is a prerequisite of neurogenesis. Ectopic expression of a β-catenin/LEF1 fusion protein maintains active canonical Wnt signaling in the developing cortex and delays the expression onset of the neurogenic factors Pax6, Ngn2 and Tbr2 and subsequent neurogenesis. Contrary to this, conditional ablation of β-catenin accelerates expression of the same neurogenic genes. Furthermore, we show that a sustained canonical Wnt activity in the lateral cortex gives rise to cells with hippocampal characteristics in the cortical plate at the expense of the cortical fate, and to cells with dentate gyrus characteristics in the hippocampus. This suggests that the dose of canonical Wnt signaling determines cellular fate in the developing cortex and hippocampus, and that recession of Wnt signaling acts as a morphogenetic gradient regulating neurogenesis in the cortex

The <i>mRx-Cre</i> activity in the eye, forebrain and hypothalamus analyzed using the <i>ROSA26R</i> reporter line.

<p>Whole-mounts or sections were stained with X-gal at indicated stages to show the <i>mRx-Cre</i>-mediated Cre activity. (A) The X-gal⁺ cells were first observed in the optic sulcus of E8.5 embryo. (B–D’) The Cre activity in developing brain. Whole-mounts (C, D, D’), coronal sections (C’, D’) and transversal section (C’) showing Cre activity in embryonic brain. (F) Coronal section of adult brain showing Cre activity in the hypothalamus and cortex. (E–E’) Sections through the adult eye showing strong uniform Cre activity in all layers of the retina. OS-optic sulcus; F-forebrain; GE-ganglionic eminences; H-hypothalamus; OB-olfactory bulbs, C-cortex; RPE-retinal pigmented epithelium; ONL-outer nuclear layer; INL-inner nuclear layer; GCL-ganglion cell layer.</p

Additional file 2: of Tcf7L2 is essential for neurogenesis in the developing mouse neocortex

Figure S2. Radial glial cells, cortical neurons and structure are not altered in in D6-Cre/Tcf7L1fl/fl/Tcf7L2fl/fl mutants at E15. a-c Hematoxylin-eosin staining of coronal sections from controls, Tcf7L2 single and Tcf7L1/Tcf7L2 double mutants. d-f Pax6 and Ctip2 double immunofluorescence with DAPI showing RGC marker Pax6. d‘-f‘Ctip2 immunofluorescence showing neuronal marker Ctip2 in the cortical plate (CP) at E15 in Tcf7L1/Tcf7L2 double mutants. g-i Tuj1 and Tbr2 double immunofluorescence counterstained with DAPI showing the cortical plate and intermediate neuronal progenitors in the SVZ at E15. g‘-i‘a higher magnification of Tbr2+ cells in the SVZ. . j-l PH3 and ZO1 double immunofluorescence with DAPI showing normal adherens junctions and normally dividing PH3+ progenitors at E15. j‘-l‘PH3 and ZO1 double immunofluorescence without DAPI. (JPG 2237 kb