The chicken CdxA homeobox gene and axial positioning during gastrulation

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 553-562 ◽  
Author(s):  
A. Frumkin ◽  
R. Haffner ◽  
E. Shapira ◽  
N. Tarcic ◽  
Y. Gruenbaum ◽  
...  
Keyword(s):  
Marginal Zone ◽  
Homeobox Gene ◽  
Primitive Streak ◽  
Immunohistochemical Localization ◽  
Tail Bud ◽  
E Coli ◽  
Tissue Sections ◽  
Whole Mount ◽  
Anterior Posterior

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord

Development ◽  
1994 ◽  
Vol 120 (7) ◽  
pp. 1817-1828 ◽  
Author(s):  
P. Spann ◽  
M. Ginsburg ◽  
Z. Rangini ◽  
A. Fainsod ◽  
H. Eyal-Giladi ◽  
...  
Keyword(s):  
Temporal Dynamics ◽  
Homeobox Gene ◽  
Primitive Streak ◽  
Neural Plate ◽  
Transverse Plane ◽  
Posterior Border ◽  
Anterior Border ◽  
Spatial And Temporal Dynamics ◽  
Specific Localization

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.

scholarly journals The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 707-720 ◽  
Author(s):  
M. Pannese ◽  
C. Polo ◽  
M. Andreazzoli ◽  
R. Vignali ◽  
B. Kablar ◽  
...  
Keyword(s):  
Marginal Zone ◽  
Homeobox Gene ◽  
Cell Stage ◽  
Rnase Protection ◽  
Glutamic Acid Residue ◽  
Body Regions ◽  
Early Embryos ◽  
Anterior Body ◽  
Low Levels

In this paper we study Xotx2, a Xenopus homeobox gene related to orthodenticle, a gene expressed in the developing head of Drosophila. The murine cognate, Otx2, is first expressed in the entire epiblast of prestreak embryos and later in very anterior regions of late-gastrulae, including the neuroectoderm of presumptive fore- and mid-brain. In Xenopus, RNase protection experiments reveal that Xotx2 is expressed at low levels throughout early development from unfertilized egg to late blastula, when its expression level significantly increases. Whole-mount in situ hybridization shows a localized expression in the dorsal region of the marginal zone at stage 9.5. At stage 10.25 Xotx2 is expressed in dorsal bottle cells and in cells of the dorsal deep zone fated to give rise to prechordal mesendoderm, suggesting a role in the specification of very anterior structures. In stage 10.5 gastrulae, Xotx2 transcripts start to be detectable also in presumptive anterior neuroectoderm, where they persist in subsequent stages. Various treatments of early embryos cause a general reorganization of Xotx2 expression. In particular, retinoic acid treatment essentially abolishes Xotx2 expression in neuroectoderm. Microinjection of Xotx2 mRNA in 1-, 2- and 4-cell stage embryos causes the appearance of secondary cement glands and partial secondary axes in embryos with reduced trunk and tail structures. The presence of the Xotx2 homeodomain is required to produce these effects. In particular, this homeodomain contains a specific lysine residue at position 9 of the recognition helix. Microinjected transcripts of Xotx2 constructs containing a homeodomain where this lysine is substituted by a glutamine or a glutamic acid residue fail to cause these effects.

Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression

Development ◽  
1991 ◽  
Vol 112 (3) ◽  
pp. 791-806 ◽  
Author(s):  
S. Mackem ◽  
K.A. Mahon
Keyword(s):  
Expression Patterns ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Limb Bud ◽  
Limb Buds ◽  
Limb Morphogenesis ◽  
Degenerate Oligonucleotide ◽  
Polymerase Chain ◽  
Anterior Posterior

Homeobox genes play a key role in specifying the segmented body plan of Drosophila, and recent work suggests that at least several homeobox genes may play a regulatory role during vertebrate limb morphogenesis. We have used degenerate oligonucleotide primers from highly conserved domains in the homeobox motif to amplify homeobox gene segments from chick embryo limb bud cDNAs using the polymerase chain reaction. Expression of a large number of homeobox genes (at least 17) is detected using this approach. One of these genes contains a novel homeobox loosely related to the Drosophila Abdominal B class, and was further analyzed by determining its complete coding sequence and evaluating its expression during embryogenesis by in situ hybridization. Based on sequence and expression patterns, we have designated this gene as Ghox 4.7 and believe that it is the chick homologue of the murine Hox 4.7 gene (formerly Hox 5.6). Ghox 4.7 is expressed primarily in limb buds during development and shows a striking spatial restriction to the posterior zone of the limb bud, suggesting a role in specifying anterior-posterior pattern formation. In chick, this gene also displays differences in expression between wing and leg buds, raising the possibility that it may participate in specifying limb-type identity.

scholarly journals PDGFRα Expression During Mouse Embryogenesis: Immunolocalization Analyzed by Whole-mount Immunohistostaining Using the Monoclonal Anti-mouse PDGFRα Antibody APA5

1997 ◽  
Vol 45 (6) ◽  
pp. 883-893 ◽  
Author(s):  
Nobuyuki Takakura ◽  
Hisahiro Yoshida ◽  
Yasunori Ogura ◽  
Hiroshi Kataoka ◽  
Satomi Nishikawa ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Growth Factor Receptor ◽  
Paraxial Mesoderm ◽  
Mouse Embryogenesis ◽  
Intense Staining ◽  
Tissue Sections ◽  
Whole Mount ◽  
And Migration ◽  
Glial Precursor

We investigated the cells that express platelet-derived growth factor receptor α (PDGFRα) during mouse embryogenesis. PDGFRα expression has been identified by in situ hybridization or immunohistochemistry using polyclonal antibodies on tissue sections. Because no immunostaining study using whole-mount specimens has been published to date, we established a new monoclonal antibody (MAb), APA5, for this purpose. Our results differed in that APA5 stained only the paraxial mesoderm, whereas other investigators concluded that most if not all mesodermal cells expressed PDGFRα. Moreover, we did not find PDGFRα expression in embryonic erythrocytes, which have been previously suggested to express PDGFRα. On the basis of our present results, we wish to revise the proposed PDGFRα expression as follows. At the pregastrulation stage, PDGFRα is expressed only in primitive endoderm, particularly that in the ectoplacental cone. On gastrulation, it is expressed at high levels in the paraxial mesoderm. This expression continues after its differentiation into the somite. Along with the differentiation and migration of the sclerotome, PDGFRα+ cells begin to become distributed throughout the embryonal mesenchyme. During organogenesis, particularly intense staining is detected in regions of epithelial and mesenchymal interaction, such as the tooth bud and bronchi. In addition to mesodermal derivatives, the developing lens, apical ectodermal ridge, glial precursor, cardiac valves, and choroid plexus express PDGFRα. Our results with whole-mount immunostaining show that PDGFRα is abundantly expressed and may play important roles during embryogenesis.

scholarly journals A Quick, Economical and Gentle Method for Applying Small Volumes of Solutions to Specimens on Glass Slides

1997 ◽  
Vol 5 (7) ◽  
pp. 20-21
Author(s):  
Clare Hasenkampf
Keyword(s):  
Tissue Sections ◽  
Immunocytochemical Detection ◽  
Glass Slides ◽  
Whole Mount ◽  
The One ◽  
Secondary Antibodies ◽  
Detection Schemes ◽  
Antibody Solution ◽  
Whole Mounts

I use immunocytochemical detection schemes that employ the use of primary and secondary antibodies for either tissue sections or whole mount preparations in order to study the in situ distribution of proteins and labeled nucleic acids. As the antibodies are either laboriously generated, or represent costly purchases. I have sought a protocol that minimizes the volume of antibody solution needed. Additionally I need a protocol that involves a quick, thorough, and gentle application and removal of the antibody solutions (mm the specimen with no danger of the specimen drying out during the one or two hour incubations. After trying many various schemes 1 have settled into the use of a procedure which very effectively retains the antibody solution on the specimen without damaging the delicate sections or whole mounts, and which is easy to add and remove.

Neural expression of the Xenopus homeobox gene Xhox3: evidence for a patterning neural signal that spreads through the ectoderm

Development ◽  
1990 ◽  
Vol 108 (4) ◽  
pp. 595-604 ◽  
Author(s):  
A. Ruiz i Altaba
Keyword(s):  
In Situ Hybridization ◽  
Neural Development ◽  
Organizer Region ◽  
Homeobox Gene ◽  
Neural Tissue ◽  
Neural Signal ◽  
Tailbud Stage ◽  
Neural Ectoderm ◽  
Anterior Posterior

The Xenopus laevis homeobox gene Xhox3 is expressed in the axial mesoderm of gastrula and neurula stage embryos. By the late neurula-early tailbud stage, mesodermal expression is no longer detectable and expression appears in the growing tailbud and in neural tissue. In situ hybridization analysis of the expression of Xhox3 in neural tissue shows that it is restricted within the neural tube and the cranial neural crest during the tailbud-early tadpole stages. In late tadpole stages, Xhox3 is only expressed in the mid/hindbrain area and can therefore be considered a marker of anterior neural development. To investigate the mechanism responsible for the anterior-posterior (A-P) regionalization of the neural tissue, the expression of Xhox3 has been analysed in total exogastrula. In situ hybridization analyses of exogastrulated embryos show that Xhox3 is expressed in the apical ectoderm of total exogastrulae, a region that develops in the absence of anterior axial mesoderm. The results provide further support for the existence of a neuralizing signal, which originates from the organizer region and spreads through the ectoderm. Moreover, the data suggest that this neural signal also has a role in A-P patterning the neural ectoderm.

Whole-mount in situ hybridization: minimizing the folding problem of thin-sheet tissue-like crayfish haematopoietic tissue

Crustaceana ◽  
2018 ◽  
Vol 91 (1) ◽  
pp. 1-15
Author(s):  
Aleksandra Zečić ◽  
Chadanat Noonin
Keyword(s):  
In Situ Hybridization ◽  
Histological Study ◽  
Thin Sheet ◽  
Hybridization Technique ◽  
Tissue Sections ◽  
Specific Transcript ◽  
Optimized Protocol ◽  
Whole Mount ◽  
Haematopoietic Tissue

Crayfish haematopoietic tissue (HPT) has a thin-sheet-like structure with a thickness of 100-160 μm and a width of approximately 1-2 cm. This structure makes HPT extremely easy to fold after removal from the animal. Therefore, it is difficult to handle the tissue without folding when processing for sectioning and histological study. The degree of tissue folding reflects the size of the tissue sections obtained, how complicated it is to interpret the location of each tissue section, and the accuracy of the interpretation of the location of a specific transcript. To facilitate the interpretation of a specific transcript location in the HPT, we optimized a whole-mount in situ hybridization technique to minimize tissue folding. This optimized protocol effectively reduced the tissue folding. Therefore, the location of a specific transcript in the HPT was easily and accurately defined. This protocol will be useful for whole-mount staining of other tissues with similar structure.

scholarly journals Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol

2001 ◽  
Vol 49 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Antoon F. M. Moorman ◽  
Arjan C. Houweling ◽  
Piet A. J. de Boer ◽  
Vincent M. Christoffels
Keyword(s):  
In Situ Hybridization ◽  
Tissue Sections ◽  
Whole Mount ◽  
Hybridization Protocol
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