scholarly journals Cryosectioning and Immunostaining of Xenopus Embryonic Tissues

2021 ◽  
Vol 2021 (9) ◽  
pp. pdb.prot107151
Author(s):  
Olga Ossipova ◽  
Sergei Y. Sokol
Keyword(s):  
Developmental Biology ◽  
Protein Function ◽  
Egg Size ◽  
Tissue Sections ◽  
Embryonic Tissues ◽  
Vertebrate Model ◽  
External Development

The Xenopus embryo is a classical vertebrate model for molecular, cellular, and developmental biology. Despite many advantages of this organism, such as large egg size and external development, imaging of early embryonic stages is challenging because of nontransparent cytoplasm. Staining and imaging of thin tissue sections is one way to overcome this limitation. Here we describe a step-by-step protocol that combines cryosectioning of gelatin-embedded embryos with immunostaining and imaging. The purpose of this protocol is to examine various cellular and tissue markers after the manipulation of protein function. This protocol can be performed within a 2-d period and allows detection of many antigens by immunofluorescence.

scholarly journals Reflections on the use of protein binders to study protein function in developmental biology

2019 ◽  
Vol 8 (6) ◽  
Author(s):  
Gustavo Aguilar ◽  
M. Alessandra Vigano ◽  
Markus Affolter ◽  
Shinya Matsuda
Keyword(s):  
Developmental Biology ◽  
Protein Function ◽  
Protein Binders

scholarly journals N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization.

1988 ◽  
Vol 107 (4) ◽  
pp. 1325-1335 ◽  
Author(s):  
G Mugrauer ◽  
F W Alt ◽  
P Ekblom
Keyword(s):  
In Situ Hybridization ◽  
Hair Follicles ◽  
Early Differentiation ◽  
Cell Lineages ◽  
Tissue Sections ◽  
Embryonic Tissues ◽  
Differentiated Cells ◽  
The Central Nervous System ◽  
Developing Kidney

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.

scholarly journals Unintended inhibition of protein function using GFP nanobodies in human cells

2019 ◽  
Author(s):  
Cansu Küey ◽  
Gabrielle Larocque ◽  
Nicholas I. Clarke ◽  
Stephen J. Royle
Keyword(s):  
Developmental Biology ◽  
Genome Editing ◽  
Cell Lines ◽  
Protein Function ◽  
Human Cells ◽  
Popular Approach ◽  
Inactive State ◽  
Dynamin 2 ◽  
Gfp Tag

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

scholarly journals Using Nanobodies to Study Protein Function in Developing Organisms

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 16 ◽  
Author(s):  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
M. Alessandra Vigano ◽  
Markus Affolter
Keyword(s):  
Developmental Biology ◽  
Protein Function ◽  
Dependent Manner ◽  
Single Chain ◽  
Functional Studies ◽  
Extracellular Milieu ◽  
The Past ◽  
Biological Studies ◽  
Living Organisms

Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms.

scholarly journals Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Satoru Ken Nishimoto ◽  
Miyako Nishimoto
Keyword(s):  
Binding Site ◽  
Matrix Protein ◽  
Cell Attachment ◽  
Matrix Gla Protein ◽  
Extracellular Matrix Protein ◽  
Tissue Sections ◽  
Embryonic Tissues ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions

Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions.Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin.Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections.Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions.

scholarly journals Genotype–phenotype mapping and the end of the ‘genes as blueprint’ metaphor

2010 ◽  
Vol 365 (1540) ◽  
pp. 557-566 ◽  
Author(s):  
Massimo Pigliucci
Keyword(s):  
Developmental Biology ◽  
Protein Function ◽  
Rna Folding ◽  
Theoretical Underpinning ◽  
Algorithmic Approach ◽  
Mapping Problem ◽  
Genetic Encoding ◽  
Evo Devo ◽  
Classic Paper ◽  
Genetic Programme

In a now classic paper published in 1991, Alberch introduced the concept of genotype–phenotype (G→P) mapping to provide a framework for a more sophisticated discussion of the integration between genetics and developmental biology that was then available. The advent of evo-devo first and of the genomic era later would seem to have superseded talk of transitions in phenotypic space and the like, central to Alberch's approach. On the contrary, this paper shows that recent empirical and theoretical advances have only sharpened the need for a different conceptual treatment of how phenotypes are produced. Old-fashioned metaphors like genetic blueprint and genetic programme are not only woefully inadequate but positively misleading about the nature of G→P, and are being replaced by an algorithmic approach emerging from the study of a variety of actual G→P maps. These include RNA folding, protein function and the study of evolvable software. Some generalities are emerging from these disparate fields of analysis, and I suggest that the concept of ‘developmental encoding’ (as opposed to the classical one of genetic encoding) provides a promising computational–theoretical underpinning to coherently integrate ideas on evolvability, modularity and robustness and foster a fruitful framing of the G→P mapping problem.

scholarly journals A phylogenomic framework and timescale for comparative studies of tunicates

2017 ◽  
Author(s):  
Frédéric Delsuc ◽  
Hervé Philippe ◽  
Georgia Tsagkogeorga ◽  
Paul Simion ◽  
Marie-Ka Tilak ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Phylogenetic Analyses ◽  
Nuclear Gene ◽  
Sister Group ◽  
Evolutionary Developmental Biology ◽  
Sequence Evolution ◽  
Precise Position ◽  
Representative Species ◽  
Vertebrate Model ◽  
Phylogenetic Framework

AbstractBackgroundTunicates are the closest relatives of vertebrates and are widely used as models to study the evolutionary developmental biology of chordates. Their phylogeny, however, remains poorly understood and to date, only the 18S rRNA nuclear gene and mitogenomes have been used to delineate the major groups of tunicates. To resolve their evolutionary relationships and provide a first estimate of their divergence times, we used a transcriptomic approach to build a phylogenomic dataset including all major tunicate lineages, consisting of 258 evolutionarily conserved orthologous genes from representative species.ResultsPhylogenetic analyses using site-heterogeneous CAT mixture models of amino acid sequence evolution resulted in a strongly supported tree topology resolving the relationships among four major tunicate clades: 1) Appendicularia, 2) Thaliacea + Phlebobranchia + Aplousobranchia, 3) Molgulidae, and 4) Styelidae + Pyuridae. Notably, the morphologically derived Thaliacea are confirmed as the sister-group of the clade uniting Phlebobranchia + Aplousobranchia within which the precise position of the model ascidian genus Ciona remains uncertain. Relaxed molecular clock analyses accommodating the accelerated evolutionary rate of tunicates reveal ancient diversification (~450-350 million years ago) among the major groups and allow comparing their evolutionary age with respect to the major vertebrate model lineages.ConclusionsOur study represents the most comprehensive phylogenomic dataset for the main tunicate lineages. It offers a reference phylogenetic framework and first tentative timescale for tunicates, allowing the direct comparison with vertebrate model species in comparative genomics and evolutionary developmental biology studies.

The 1-MeV Electron Microscope Installation at the University of Colorado

1973 ◽  
Vol 31 ◽  
pp. 8-9 ◽  
Author(s):  
Mircea Fotino
Keyword(s):  
Electron Microscopy ◽  
Developmental Biology ◽  
Transmission Electron Microscopy ◽  
Electron Microscope ◽  
National Institutes Of Health ◽  
Experimental Program ◽  
University Of Colorado ◽  
Transmission Electron ◽  
The University ◽  
Research Resources

A new 1-MeV transmission electron microscope (Model JEM-1000) was installed at the Department of Molecular, Cellular and Developmental Biology of the University of Colorado in Boulder during the summer and fall of 1972 under the sponsorship of the Division of Research Resources of the National Institutes of Health. The installation was completed in October, 1972. It is installed primarily for the study of biological materials without many of the limitations hitherto unavoidable in standard transmission electron microscopy. Only the technical characteristics of the installation are briefly reviewed here. A more detailed discussion of the experimental program under way is being published elsewhere.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Sign in / Sign up

Export Citation Format

Share Document