153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 194
Author(s):  
K. Schauser ◽  
T. S. Sundkvist ◽  
M. Vejlsted ◽  
P. Maddox-Hyttel
Keyword(s):  
Transcription Factor ◽  
Neural Tube ◽  
Neural Development ◽  
Primitive Streak ◽  
Immunofluorescent Staining ◽  
Santa Cruz ◽  
Neuronal Marker ◽  
Somite Stage ◽  
Anterior Posterior ◽  
Β Tubulin

In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 �m) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.

The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins

Development ◽  
1993 ◽  
Vol 119 (4) ◽  
pp. 1301-1315 ◽  
Author(s):  
S.L. Ang ◽  
A. Wierda ◽  
D. Wong ◽  
K.A. Stevens ◽  
S. Cascio ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neural Tube ◽  
Critical Role ◽  
Primitive Streak ◽  
Cell Types ◽  
Definitive Endoderm ◽  
Gut Development ◽  
Binding Assays ◽  
Anterior Portion ◽  
Endoderm Development

Little is known about genes that govern the development of the definitive endoderm in mammals; this germ layer gives rise to the intestinal epithelium and various other cell types, such as hepatocytes, derived from the gut. The discovery that the rat hepatocyte transcription factor HNF3 is similar to the Drosophila forkhead gene, which plays a critical role in gut development in the fly, led us to isolate genes containing the HNF3/forkhead (HFH) domain that are expressed in mouse endoderm development. We recovered mouse HNF3 beta from an embryo cDNA library and found that the gene is first expressed in the anterior portion of the primitive streak at the onset of gastrulation, in a region where definitive endoderm first arises. Its expression persists in axial structures derived from the mouse equivalent of Hensen's node, namely definitive endoderm and notochord, and in the ventral region of the developing neural tube. Expression of the highly related gene, HNF3 alpha, appears to initiate later than HNF3 beta and is first seen in midline endoderm cells. Expression subsequently appears in notochord, ventral neural tube, and gut endoderm in patterns similar to HNF3 beta. Microscale DNA binding assays show that HNF3 proteins are detectable in the midgut at 9.5 days p.c. At later stages HNF3 mRNAs and protein are expressed strongly in endoderm-derived tissues such as the liver. HNF3 is also the only known hepatocyte-enriched transcription factor present in a highly de-differentiated liver cell line that retains the capacity to redifferentiate to the hepatic phenotype. Taken together, these studies suggest that HNF3 alpha and HNF3 beta are involved in both the initiation and maintenance of the endodermal lineage. We also discovered a novel HFH-containing gene, HFH-E5.1, that is expressed transiently in posterior ectoderm and mesoderm at the primitive streak stage, and later predominantly in the neural tube. HFH-E5.1 is highly similar in structure and expression profile to the Drosophila HFH gene FD4, suggesting that HFH family members have different, evolutionarily conserved roles in development.

scholarly journals Aberrant Gcm1 expression mediates Wnt/β-catenin pathway activation in folate deficiency involved in neural tube defects

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Jianting Li ◽  
Qiu Xie ◽  
Jun Gao ◽  
Fang Wang ◽  
Yihua Bao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neural Tube ◽  
Neural Development ◽  
Folate Deficiency ◽  
Early Embryo ◽  
Neural Tube Closure ◽  
Anteroposterior Patterning ◽  
T Cell Factor ◽  
Pathway Activation ◽  
Tube Closure

AbstractWnt signaling plays a major role in early neural development. An aberrant activation in Wnt/β-catenin pathway causes defective anteroposterior patterning, which results in neural tube closure defects (NTDs). Changes in folate metabolism may participate in early embryo fate determination. We have identified that folate deficiency activated Wnt/β-catenin pathway by upregulating a chorion-specific transcription factor Gcm1. Specifically, folate deficiency promoted formation of the Gcm1/β-catenin/T-cell factor (TCF4) complex formation to regulate the Wnt targeted gene transactivation through Wnt-responsive elements. Moreover, the transcription factor Nanog upregulated Gcm1 transcription in mESCs under folate deficiency. Lastly, in NTDs mouse models and low-folate NTDs human brain samples,Gcm1and Wnt/β-catenin targeted genes related to neural tube closure are specifically overexpressed. These results indicated that low-folate level promoted Wnt/β-catenin signaling via activating Gcm1, and thus leaded into aberrant vertebrate neural development.

scholarly journals Competence, specification and induction of Pax-3 in the trigeminal placode

Development ◽  
1999 ◽  
Vol 126 (1) ◽  
pp. 147-156 ◽  
Author(s):  
C.V. Baker ◽  
M.R. Stark ◽  
C. Marcelle ◽  
M. Bronner-Fraser
Keyword(s):  
Nervous System ◽  
Transcription Factor ◽  
Molecular Marker ◽  
Sensory Neurons ◽  
Neural Tube ◽  
Collagen Gel ◽  
Paired Domain ◽  
Detailed Model ◽  
Somite Stage ◽  
Inductive Interaction

Placodes are discrete regions of thickened ectoderm that contribute extensively to the peripheral nervous system in the vertebrate head. The paired-domain transcription factor Pax-3 is an early molecular marker for the avian ophthalmic trigeminal (opV) placode, which forms sensory neurons in the ophthalmic lobe of the trigeminal ganglion. Here, we use collagen gel cultures and heterotopic quail-chick grafts to examine the competence, specification and induction of Pax-3 in the opV placode. At the 3-somite stage, the whole head ectoderm rostral to the first somite is competent to express Pax-3 when grafted to the opV placode region, though competence is rapidly lost thereafter in otic-level ectoderm. Pax-3 specification in presumptive opV placode ectoderm occurs by the 8-somite stage, concomitant with robust Pax-3 expression. From the 8-somite stage onwards, significant numbers of cells are committed to express Pax-3. The entire length of the neural tube has the ability to induce Pax-3 expression in competent head ectoderm and the inductive interaction is direct. We propose a detailed model for Pax-3 induction in the opV placode.

scholarly journals Transcription factor TEAD2 is involved in neural tube closure

genesis ◽  
2007 ◽  
Vol 45 (9) ◽  
pp. 577-587 ◽  
Author(s):  
Kotaro J. Kaneko ◽  
Matthew J. Kohn ◽  
Chengyu Liu ◽  
Melvin L. DePamphilis
Keyword(s):  
Transcription Factor ◽  
Neural Tube ◽  
Neural Tube Closure ◽  
Tube Closure

Control of dorsoventral pattern in vertebrate neural development: induction and polarizing properties of the floor plate

Development ◽  
1991 ◽  
Vol 113 (Supplement_2) ◽  
pp. 105-122 ◽  
Author(s):  
Marysia Placzek ◽  
Toshiya Yamada ◽  
Marc Tessier-Lavigne ◽  
Thomas Jessell ◽  
Jane Dodd
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Cell Types ◽  
Floor Plate ◽  
Neural Cells ◽  
Dorsoventral Patterning ◽  
Cellular Mechanisms ◽  
Cell Position

Distinct classes of neural cells differentiate at specific locations within the embryonic vertebrate nervous system. To define the cellular mechanisms that control the identity and pattern of neural cells we have used a combination of functional assays and antigenic markers to examine the differentiation of cells in the developing spinal cord and hindbrain in vivo and in vitro. Our results suggest that a critical step in the dorsoventral patterning of the embryonic CNS is the differentiation of a specialized group of midline neural cells, termed the floor plate, in response to local inductive signals from the underlying notochord. The floor plate and notochord appear to control the pattern of cell types that appear along the dorsoventral axis of the neural tube. The fate of neuroepithelial cells in the ventral neural tube may be defined by cell position with respect to the ventral midline and controlled by polarizing signals that originate from the floor plate and notochord.

scholarly journals Transcriptional response of Hoxb genes to retinoid signalling is regionally restricted along the neural tube rostrocaudal axis

2017 ◽  
Vol 4 (4) ◽  
pp. 160913 ◽  
Author(s):  
Nicoletta Carucci ◽  
Emanuele Cacci ◽  
Paola S. Nisi ◽  
Valerio Licursi ◽  
Yu-Lee Paul ◽  
...  
Keyword(s):  
Neural Tube ◽  
Neural Development ◽  
Transcriptional Response ◽  
Neural Tissue ◽  
Positional Information ◽  
Multiple Roles ◽  
Chromatin Activation ◽  
Neural Stem Progenitor Cells ◽  
Rostrocaudal Axis

During vertebrate neural development, positional information is largely specified by extracellular morphogens. Their distribution, however, is very dynamic due to the multiple roles played by the same signals in the developing and adult neural tissue. This suggests that neural progenitors are able to modify their competence to respond to morphogen signalling and autonomously maintain positional identities after their initial specification. In this work, we take advantage of in vitro culture systems of mouse neural stem/progenitor cells (NSPCs) to show that NSPCs isolated from rostral or caudal regions of the mouse neural tube are differentially responsive to retinoic acid (RA), a pivotal morphogen for the specification of posterior neural fates. Hoxb genes are among the best known RA direct targets in the neural tissue, yet we found that RA could promote their transcription only in caudal but not in rostral NSPCs. Correlating with these effects, key RA-responsive regulatory regions in the Hoxb cluster displayed opposite enrichment of activating or repressing histone marks in rostral and caudal NSPCs. Finally, RA was able to strengthen Hoxb chromatin activation in caudal NSPCs, but was ineffective on the repressed Hoxb chromatin of rostral NSPCs. These results suggest that the response of NSPCs to morphogen signalling across the rostrocaudal axis of the neural tube may be gated by the epigenetic configuration of target patterning genes, allowing long-term maintenance of intrinsic positional values in spite of continuously changing extrinsic signals.

scholarly journals Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells

Development ◽  
1987 ◽  
Vol 100 (2) ◽  
pp. 339-349 ◽  
Author(s):  
L. Pardanaud ◽  
C. Altmann ◽  
P. Kitos ◽  
F. Dieterlen-Lievre ◽  
C.A. Buck
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Single Cells ◽  
Primitive Streak ◽  
Vascular Network ◽  
Vascular Tree ◽  
Somite Stage ◽  
Area Vasculosa ◽  
Haemopoietic Cells ◽  
Area Pellucida

QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.

scholarly journals BMP4-induced symmetry breaking in a 3D model of the human epiblast

2019 ◽  
Author(s):  
Mijo Simunovic ◽  
Ali H. Brivanlou ◽  
Eric D. Siggia
Keyword(s):  
Live Cell Imaging ◽  
3D Model ◽  
Epithelial To Mesenchymal Transition ◽  
Cell Imaging ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Mesenchymal Transition ◽  
Pluripotency Markers ◽  
Anterior Posterior

Abstract We describe the protocol of generating a 3D stem-cell-based model of the human pre-gastrulation epiblast by culturing human embryonic stem cells in a mix of hydrogel and Matrigel. Much like the epiblast of an in vitro attached day-10 human embryo, this model is an epithelial sphere with a cavity at its center, it is expressing key pluripotency markers, and it displays apico-basal polarity. The 3D colonies can further be differentiated with morphogens and in the case of intermediate concentrations of BMP4, they break the anterior-posterior symmetry characterized by an asymmetric expression of a primitive streak marker and showing signs of epithelial to mesenchymal transition. The protocol described here is suitable for immunofluorescence staining and for live-cell imaging.

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