scholarly journals An intrinsic cell cycle timer terminates limb bud outgrowth

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Joseph Pickering ◽  
Constance A Rich ◽  
Holly Stainton ◽  
Cristina Aceituno ◽  
Kavitha Chinnaiya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Sequencing ◽  
Late Stage ◽  
Limb Development ◽  
Limb Bud ◽  
Limb Outgrowth ◽  
Bmp Signalling ◽  
Mesenchyme Cells

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

scholarly journals An intrinsic cell cycle timer terminates limb bud outgrowth

2018 ◽  
Author(s):  
Joseph Pickering ◽  
Kavitha Chinnaiya ◽  
Constance A. Rich ◽  
Patricia Saiz-Lopez ◽  
Maria A. Ros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Sequencing ◽  
Late Stage ◽  
Limb Development ◽  
Limb Bud ◽  
Limb Outgrowth ◽  
Bmp Signalling ◽  
Mesenchyme Cells

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the mesenchyme and the overlying epithelium (e-m signalling). However, by grafting mesenchyme cells from late stage chick wing buds to an early epithelial environment we show that this mechanism is not required. RNA sequencing reveals that mesenchyme cells terminate growth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4729-4736
Author(s):  
L. Lettice ◽  
J. Hecksher-Sorensen ◽  
R.E. Hill
Keyword(s):  
Gene Expression ◽  
Pattern Formation ◽  
Limb Development ◽  
Limb Bud ◽  
Mouse Mutation ◽  
Number Of Factors ◽  
Limb Outgrowth ◽  
Gene Functions ◽  
Regulatory Loop ◽  
Limb Initiation

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.

The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4417-4425 ◽  
Author(s):  
M. Takahashi ◽  
K. Tamura ◽  
D. Buscher ◽  
H. Masuya ◽  
S. Yonei-Tamura ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Limb Development ◽  
Feedback Loop ◽  
Limb Bud ◽  
Negative Regulator ◽  
Negative Feedback Loop ◽  
Vertebrate Limb ◽  
Limb Outgrowth ◽  
Vertebrate Limb Development

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds

Development ◽  
1992 ◽  
Vol 116 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M.A. Ros ◽  
G. Lyons ◽  
R.A. Kosher ◽  
W.B. Upholt ◽  
C.N. Coelho ◽  
...  
Keyword(s):  
Pattern Formation ◽  
Limb Development ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Rapid Decline ◽  
Limb Buds ◽  
Limb Outgrowth ◽  
Removal Experiments ◽  
The Relationship

The homeobox-containing genes GHox-7 and GHox-8 have been proposed to play fundamental roles in limb development. The expression of GHox-8, by the apical ridge cells, and GHox-7, in the subridge mesoderm, suggests the involvement of these two genes in limb outgrowth and proximo-distal pattern formation. A straightforward way to test this is to remove the apical ridge. Here we report the relationship between the mesodermal expression of GHox-7 and GHox-8 and the apical ectodermal ridge in the chick limb bud. The data from ridge removal experiments indicate that there are at least two domains of GHox-7 expression in the apical limb bud mesoderm. The posterior subridge GHox-7 domain in the progress zone requires the influence of the apical ridge for continued expression, while the anterior GHox-7 domain continues expression after ridge removal. Posterior subridge mesoderm is exquisitely sensitive to the loss of the ridge in that GHox-7 expression by these cells is reduced in only two hours and undetectable by three hours after ridge removal. It would appear that one of the ways progress zone cells respond to the apical ridge signal is by expressing GHox-7. The loss of ridge influence whether by growth at the apex or by ridge removal is followed by an unusually rapid decline in detectable GHox-7 transcripts. Maintenance of GHox-8 expression by the anterior mesoderm appears to be independent of the presence of the apical ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal limb development in conditional mutants of Fgf4

Development ◽  
2000 ◽  
Vol 127 (5) ◽  
pp. 989-996 ◽  
Author(s):  
A.M. Moon ◽  
A.M. Boulet ◽  
M.R. Capecchi
Keyword(s):  
Limb Development ◽  
Expression Patterns ◽  
Large Body ◽  
Embryonic Lethality ◽  
Limb Bud ◽  
Recombination Reaction ◽  
Site Specific ◽  
Early Embryonic Lethality ◽  
Limb Outgrowth

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.

The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5515-5522 ◽  
Author(s):  
R. Merino ◽  
J. Rodriguez-Leon ◽  
D. Macias ◽  
Y. Ganan ◽  
A.N. Economides ◽  
...  
Keyword(s):  
Cell Death ◽  
Soft Tissue ◽  
Programmed Cell Death ◽  
Limb Development ◽  
Limb Bud ◽  
Central Core ◽  
Limb Outgrowth ◽  
Bmp Antagonists ◽  
And Function ◽  
Bmp Antagonist

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.

scholarly journals Effects of Aspergillus fumigatus Conidia on Apoptosis and Proliferation in an In Vitro Model of the Lung Microenvironment

2021 ◽  
Vol 9 (7) ◽  
pp. 1435
Author(s):  
Hisako Kushima ◽  
Toshiyuki Tsunoda ◽  
Taichi Matsumoto ◽  
Yoshiaki Kinoshita ◽  
Koichi Izumikawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Pulmonary Fibrosis ◽  
Aspergillus Fumigatus ◽  
Rna Sequencing ◽  
A549 Cells ◽  
Western Blots ◽  
Mesenchymal Transition ◽  
Expression Of Genes ◽  
Nih 3T3

Background/Aim: Aspergillus is often detected in respiratory samples from patients with chronic respiratory diseases, including pulmonary fibrosis, suggesting that it can easily colonize the airways. To determine the role of Aspergillus colonization in pulmonary fibrosis, we cultured human lung epithelial A549 cells or murine embryo fibroblast NIH/3T3 cells with Aspergillus conidia in 3D floating culture representing the microenvironment. Materials and Methods: Cells were cultured in two-dimensional (2D) and three-dimensional floating (3DF) culture with heat-inactivated Aspergillus fumigatus (AF) 293 conidia at an effector-to-target cell ratio of 1:10 (early-phase model) and 1:100 (colonization model), and RNA-sequencing and Western blots (WB) were performed. Results: AF293 conidia reduced A549 cell growth in 2D and 3DF cultures and induced apoptosis in A549 spheroids in 3DF culture. RNA-sequencing revealed the increased expression of genes associated with interferon-mediated antiviral responses including MX dymamin-like GTPase 1 (MX1). Interestingly, the decreased expression of genes associated with the cell cycle was observed with a high concentration of AF293 conidia. WB revealed that epithelial-mesenchymal transition was not involved. Notably, AF293 conidia increased NIH/3T3 growth only in 3DF culture without inducing an apoptotic reaction. RNA-sequencing revealed the increased expression of genes associated with interferon signalling, including MX2; however, the decreased expression of genes associated with the cell cycle was not observed. Conclusions: AF affects both apoptosis of epithelial cells and the growth of fibroblasts. A deeper understanding of the detailed mechanisms underlying Aspergillus-mediated signaling pathway in epithelial cells and fibroblasts will help us to understand the lung microenvironment.

Retinoic acid and chick limb bud development

Development ◽  
1991 ◽  
Vol 113 (Supplement_1) ◽  
pp. 113-121 ◽  
Author(s):  
C. Tickle
Keyword(s):  
Retinoic Acid ◽  
Target Genes ◽  
Homeobox Gene ◽  
Experimental System ◽  
Shoulder Girdle ◽  
Limb Bud ◽  
Local Concentration ◽  
Anterior Position ◽  
Mesenchyme Cells ◽  
Vitamin A Derivative

The chick limb bud is a powerful experimental system in which to study pattern formation in vertebrate embryos. Exogenously applied retinoic acid, a vitamin A derivative, can bring about changes in pattern and, on several grounds, is a good candidate for an endogenous morphogen. As such, the local concentration of retinoic acid might provide cells with information about their position in relation to one axis of the limb. Alternatively, retinoic acid may be part of a more complex signalling system. Homeobox genes are possible target genes for regulation by retinoic acid in the limb. In particular, one homeobox gene, XlHbox 1 is expressed locally in the mesenchyme of vertebrate forelimbs and might code for an anterior position. When the pattern of the chick wing is changed by retinoic acid or by grafts of signalling tissue such that anterior cells now form posterior structures, the domain of XlHbox 1 expression expands rather than contracts. The expansion of XlHbox 1 expression correlates with shoulder girdle abnormalities. Retinoic acid application leads to visible changes in bud shape and this allows dissection of the way in which patterning is co-ordinated with morphogenesis. Results of recombination experiments and studies of changes in the apical ridge and proliferation in the mesenchyme suggest the following scheme: retinoic acid is involved in specification of position of mesenchyme cells; this specification determines their local interaction with the ridge that controls ridge morphology; the thickened apical ridge permits local proliferation in the underlying mesenchyme. The recent advances in molecular biology that permit analysis of the expression of various interesting genes in developing limbs hold out the promise that further investigation may soon allow a complete account of the patterning process in one part of the vertebrate embryo.

Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development

Development ◽  
2000 ◽  
Vol 127 (23) ◽  
pp. 5133-5144
Author(s):  
A.T. Tavares ◽  
T. Tsukui ◽  
J.C. Izpisua Belmonte
Keyword(s):  
Limb Development ◽  
Limb Bud ◽  
Wing Margin ◽  
Lateral Plate ◽  
Posterior Limb ◽  
Downstream Target ◽  
Notch Pathways ◽  
Specialized Region ◽  
Dorsoventral Boundary ◽  
Cycle Regulation

In vertebrates, the apical ectodermal ridge (AER) is a specialized epithelium localized at the dorsoventral boundary of the limb bud that regulates limb outgrowth. In Drosophila, the wing margin is also a specialized region located at the dorsoventral frontier of the wing imaginal disc. The wingless and Notch pathways have been implicated in positioning both the wing margin and the AER. One of the nuclear effectors of the Notch signal in the wing margin is the transcription factor cut. Here we report the identification of two chick homologues of the Cut/Cux/CDP family that are expressed in the developing limb bud. Chick cux1 is expressed in the ectoderm outside the AER, as well as around ridge-like structures induced by (β)-catenin, a downstream target of the Wnt pathway. cux1 overexpression in the chick limb results in scalloping of the AER and limb truncations, suggesting that Cux1 may have a role in limiting the position of the AER by preventing the ectodermal cells around it from differentiating into AER cells. The second molecule of the Cut family identified in this study, cux2, is expressed in the pre-limb lateral plate mesoderm, posterior limb bud and flank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. The polarizing activity is determined by the ability of a certain region to induce digit duplications when grafted into the anterior margin of a host limb bud. Several manipulations of the chick limb bud show that cux2 expression is regulated by retinoic acid, Sonic hedgehog and the posterior AER. These results suggest that Cux2 may have a role in generating or mediating polarizing activity. Taking into account the probable involvement of Cut/Cux/CDP molecules in cell cycle regulation and differentiation, our results raise the hypothesis that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively.

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