Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration

Development ◽  
1988 ◽  
Vol 103 (3) ◽  
pp. 567-573
Author(s):  
D.M. Fekete ◽  
J.P. Brockes
Keyword(s):  
Monoclonal Antibody ◽  
Progenitor Cells ◽  
Limb Regeneration ◽  
Growth Zone ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Limb Buds ◽  
Molecular Identity

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the ‘aneurogenic’ limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).

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 Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody.

1984 ◽  
Vol 99 (5) ◽  
pp. 1856-1866 ◽  
Author(s):  
J Sasse ◽  
A Horwitz ◽  
M Pacifici ◽  
H Holtzer
Keyword(s):  
Monoclonal Antibody ◽  
Keratan Sulfate ◽  
Precursor Cells ◽  
Limb Bud ◽  
Myogenic Cells ◽  
Limb Buds ◽  
Normal Medium ◽  
Cell Lineages ◽  
A Cell ◽  
Multinucleated Myotubes

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells--those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle-specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.

Altered expression of the chicken homeobox-containing genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1487-1493 ◽  
Author(s):  
C.N. Coelho ◽  
K.M. Krabbenhoft ◽  
W.B. Upholt ◽  
J.F. Fallon ◽  
R.A. Kosher
Keyword(s):  
Proper Time ◽  
Blot Hybridization ◽  
Development Stage ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Chick Embryos ◽  
Dot Blot ◽  
Limb Buds ◽  
Altered Expression

It has been suggested that the reciprocal expression of the chicken homeobox-containing genes GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm might be involved in regulating the proximodistal outgrowth of the developing chick limb bud. In the present study the expression of GHox-7 and GHox-8 has been examined by in situ and dot blot hybridization in the developing limb buds of limbless mutant chick embryos. The limb buds of homozygous mutant limbless embryos form at the proper time in development (stage 17/18), but never develop an apical ectodermal ridge, fail to undergo normal elongation, and eventually degenerate. At stage 18, which is shortly following the formation of the limb bud, the expression of GHox-7 is considerably reduced (about 3-fold lower) in the mesoderm of limbless mutant limb buds compared to normal limb bud mesoderm. By stages 20 and 21, as the limb buds of limbless embryos cease outgrowth, GHox-7 expression in limbless mesoderm declines to very low levels, whereas GHox-7 expression increases in the mesoderm of normal limb buds which are undergoing outgrowth. In contrast to GHox-7, expression of GHox-8 in limbless mesoderm at stage 18 is quantitatively similar to its expression in normal limb bud mesoderm, and in limbless and normal mesoderm GHox-8 expression is highly localized in the anterior mesoderm of the limb bud. In normal limb buds, GHox-8 is also expressed in high amounts by the apical ectodermal ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals BMP-2/-4 mediate programmed cell death in chicken limb buds

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 3725-3734 ◽  
Author(s):  
Y. Yokouchi ◽  
J. Sakiyama ◽  
T. Kameda ◽  
H. Iba ◽  
A. Suzuki ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mesenchymal Cells ◽  
Dominant Negative ◽  
Limb Bud ◽  
Death Process ◽  
Signal Molecules ◽  
Opaque Zone ◽  
Limb Buds ◽  
Bmp Receptor

During limb development, the mesenchymal cells in restricted areas of limb bud, anterior necrotic zone, posterior necrotic zone, opaque zone and interdigital necrotic zones, are eliminated by programmed cell death. The transcripts of bone morphogenetic protein (Bmp)-2 and −4 were first detected in the areas where cell death was observed, then showed overlapping expression with the programmed cell death zones except the opaque zone. To investigate the function of BMP-2 and BMP-4 during limb pattern formation, the dominant negative form of BMP receptor was overexpressed in chick leg bud via a replication-competent retrovirus to block the endogenous BMP-2/-4 signaling pathway. This resulted in excess web formation at the anterior and posterior regions of limb buds in addition to marked suppression of the regression of webbing at the interdigital regions. Significant reductions in the number of apoptotic cells in these three necrotic zones were found in the limb buds which received the virus carrying dominant negative BMP receptor. This indicates that extra tissue formation is due to suppression of programmed cell death in the three necrotic zones. Moreover, BMP-2/-4 protein induced apoptosis of mesenchymal cells isolated from the interdigital region in vitro. Other TGFbeta family proteins as TGFbeta1 and Activin did not show this effect. These results suggest that BMP-2 and BMP-4 are the apoptotic signal molecules of the programmed cell death process in the chick limb buds.

scholarly journals Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 431-444 ◽  
Author(s):  
Y. Yokouchi ◽  
K. Ohsugi ◽  
H. Sasaki ◽  
A. Kuroiwa
Keyword(s):  
Retinoic Acid ◽  
Early Stage ◽  
Local Treatment ◽  
Cell Lineage ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Mirror Image ◽  
Limb Bud ◽  
Northern Analysis ◽  
Limb Buds

A chicken gene carrying a homeobox highly homologous to the Drosophila muscle segment homeobox (msh) gene was isolated and designated as Msx-1. Conceptual translation from the longest ORF gave a protein of 259 amino acids lacking the conserved hexapeptide. Northern analysis detected a single 2.6 kb transcript. As early as day 2 of incubation, the transcript was detected but was not found in adult tissue. In situ hybridization analysis revealed that Msx-1 expression is closely related to a particular mesenchymal cell lineage during limb bud formation. In early stage embryos, Msx-1 was expressed in the somatopleure. When primordial mesenchyme cells for limb bud were generated from the Wolffian ridge of the somatopleure, Msx-1 expression began to diminish in the posterior half of the limb bud then in the presumptive cartilage-forming mesenchyme. In developing limb buds, remarkable expression was seen in the apical ectodermal ridge (AER), which is responsible for the sustained outgrowth and development of the limb. The Msx-1 transcripts were found in the limb mesenchymal cells in the region covering the necrotic zone and ectodermal cells overlying such mesenchymal cells. Both ectodermal and mesenchymal expression in limb bud were rapidly suppressed by local treatment of retinoic acid which can generate mirror-image duplication of digits. This indicates that retinoic acid alters the marginal presumptive non-cartilage forming mesenchyme cell lineage through suppression of Msx-1 expression.

Expression pattern of homeobox-containing genes during chick embryogenesis

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 639-650 ◽  
Author(s):  
S.E. Wedden ◽  
K. Pang ◽  
G. Eichele
Keyword(s):  
Expression Pattern ◽  
Northern Blotting ◽  
Amino Acid Sequences ◽  
Limb Bud ◽  
Northern Analysis ◽  
Rnase Protection ◽  
Limb Buds ◽  
Chick Embryogenesis ◽  
Spinal Chord

We have isolated, sequenced and examined the expression pattern of two tandemly arranged homeobox-containing genes from the chicken. The predicted amino acid sequences of the homeodomain and the adjacent carboxyterminal portion of the protein of the first gene is virtually identical (99%) to that of murine homeobox 2.1 and hence we refer to it as Ghox 2.1 (Gallus homeobox). The closest mouse homologue of the second homeodomain is Hox 2.2 (95% identical within the homeobox), and hence referred to as Ghox 2.2. Northern analysis of embryonic RNA reveals major transcripts of 2 kb for Ghox 2.1 and 1.7 kb for Ghox 2.2. To investigate the transcript pattern, embryos of various stages were dissected into heads, trunks and limb buds and the RNA was analysed by Northern blotting and RNase protection assays. Ghox 2.1 transcripts are present in all three regions. Ghox 2.2 RNA is found in trunks and limb buds, but it is strikingly absent from the developing head. In situ hybridization with 35S-labelled antisense riboprobes derived from Ghox 2.1 demonstrates that this gene is expressed at high levels in spinal chord, myelencephalon and mesonephros. Dorsal root ganglia and the lung rudiment also contain Ghox 2.1 message, but in somewhat lower amounts. Mid- and forebrain, the heart, presomitic mesenchyme and notochord do not contain detectable levels of Ghox 2.1 mRNA. Of particular interest is the expression of Ghox 2.1 in a well-defined patch of mesenchymal tissue situated in an anterioproximal region of the limb bud.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

Cell cycle controls and the role of nerves and the regenerate epithelium in urodele forelimb regeneration: possible modifications of basic concepts

1987 ◽  
Vol 65 (8) ◽  
pp. 739-749 ◽  
Author(s):  
Roy A. Tassava ◽  
David J. Goldhamer ◽  
Bruce L. Tomlinson
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Limb Regeneration ◽  
Blastema Formation ◽  
Basic Concepts ◽  
Regenerate Epithelium ◽  
Early Wound ◽  
Skin Epidermis ◽  
Wound Epithelium

Data from pulse and continuous labeling with [3H]thymidine and from studies with monoclonal antibody WE3 have led to the modification of existing models and established concepts pertinent to understanding limb regeneration. Not all cells of the adult newt blastema are randomly distributed and actively progressing through the cell cycle. Instead, many cells are in a position that we have designated transient quiescence (TQ) and are not actively cycling. We postulate that cells regularly leave the TQ population and enter the actively cycling population and vice versa. The size of the TQ population may be at least partly determined by the quantity of limb innervation. Larval Ambystoma may have only a small or nonexisting TQ, thus accounting for their rapid rate of regeneration. Examination of reactivity of monoclonal antibody WE3 suggests that the early wound epithelium, which is derived from skin epidermis, is later replaced by cells from skin glands concomitant with blastema formation. WE3 provides a useful tool to further investigate the regenerate epithelium.

Abstract 305: Single Cell RNA Sequencing of Adult Cardiac c-kit+ Cells in a Murine Lineage Tracing Model

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Bryan D Maliken ◽  
Onur Kanisicak ◽  
Jeffery D Molkentin
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Cardiac Injury ◽  
Mesenchymal Cells ◽  
Lineage Tracing ◽  
Tyrosine Kinase Receptor ◽  
Early Differentiation ◽  
Gfp Reporter ◽  
Single Cell Rna Sequencing

A subset of adult cardiac resident cells defined by the stem cell factor tyrosine kinase receptor termed c-kit, are believed to have myogenic potential and are now being delivered via intracoronary infusion to presumably promote cardiac regeneration and improve ventricular function after ischemic cardiac injury. However, recent studies have shown that despite these benefits, c-kit+ progenitor cells in the adult murine heart are more inclined to take on an endothelial rather than cardiomyocyte lineage. To better define the factors involved in early differentiation of these resident cardiac progenitor cells and to distinguish distinct cell subpopulations, we performed single cell RNA sequencing on c-kit+ cells from Kit-Cre lineage traced GFP reporter mice versus total mesenchymal cells from the heart that were CD31- and CD45-. Cells were isolated by cardiac digestion and FACS was performed, positively sorting for the c-kit+ lineage while negatively sorting for CD31 and CD45 to eliminate endothelial and leukocyte progenitor contamination, respectively. Following this isolation, cells were examined to determine GFP reporter status and then submitted for single cell RNA sequencing using the Fluidigm A1 system. Clustering of 654 genes from this data identified 6 distinct subpopulations indicating various stages of early differentiation among CD31- and CD45-negative cardiac interstitial cells. Furthermore, comparison of GFP+ c-kit cells to the total non-GFP mesenchymal cells identified 75 differentially expressed transcripts. These unique gene signatures may help parse the genes that underlie cellular plasticity in the heart and define the best molecular lineages for transdifferentiation into cardiac myocytes.

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