SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in up-regulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are up-regulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

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SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

10.1101/2021.09.08.459466 ◽

2021 ◽

Author(s):

Julie Gamart ◽

Iros Barozzi ◽

Frédéric Laurent ◽

Robert Reinhardt ◽

Laurène Ramos Martins ◽

...

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Transcriptional Regulation ◽

Target Genes ◽

Cholesterol Biosynthesis ◽

Limb Bud ◽

Positive Role ◽

Shh Signaling ◽

Bud Development ◽

Repressive Effect

ABSTRACTSMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

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Sonic hedgehog is not required for polarising activity in the Doublefoot mutant mouse limb bud

Development ◽

10.1242/dev.125.3.351 ◽

1998 ◽

Vol 125 (3) ◽

pp. 351-357 ◽

Cited By ~ 2

Author(s):

C. Hayes ◽

J.M. Brown ◽

M.F. Lyon ◽

G.M. Morriss-Kay

Keyword(s):

Gene Expression ◽

Limb Development ◽

Target Genes ◽

Anterior Part ◽

Expression Patterns ◽

Ectopic Expression ◽

Mutant Gene ◽

Limb Bud ◽

Active Constituent ◽

Limb Buds

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly of all four limbs. We have analysed limb development in this mutant with respect to morphogenesis, gene expression patterns and ectopic polarising activity. The results reveal a gain-of-function mutation at a locus that mediates pattern formation in the developing limb. Shh expression is identical with that of wild-type embryos, i.e. there is no ectopic expression. However, mesenchyme from the anterior aspects of Dbf/+ mutant limb buds, when transplanted to the anterior side of chick wing buds, induces duplication of the distal skeletal elements. Mid-distal mesenchymal transplants from early, but not later, Dbf/+ limb buds are also able to induce duplication. This demonstration of polarising activity in the absence of Shh expression identifies the gene at the Dbf locus as a new genetic component of the Shh signalling pathway, which (at least in its mutated form) is able to activate signal transduction independently of Shh. The mutant gene product is sufficient to fulfil the signalling properties of Shh including upregulation of the direct Shh target genes Ptc and Gli, and induction of the downstream target genes Bmp2, Fgf4 and Hoxd13. The expression domains of all these genes extend from their normal posterior domains into the anterior part of the limb bud without being focused on a discrete ectopic site. These observations dissociate polarising activity from Shh gene expression in the Dbf/+ limb bud. We suggest that the product of the normal Dbf gene is a key active constituent of the polarising region, possibly acting in the extracellular compartment.

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Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms

Development ◽

10.1242/dev.115.2.553 ◽

1992 ◽

Vol 115 (2) ◽

pp. 553-560 ◽

Cited By ~ 8

Author(s):

J.C. Izpisua-Belmonte ◽

J.M. Brown ◽

A. Crawley ◽

D. Duboule ◽

C. Tickle

Keyword(s):

Gene Expression ◽

Gene Network ◽

Primitive Streak ◽

Mouse Embryos ◽

Limb Bud ◽

Chick Embryos ◽

Mouse Limb ◽

Mouse Cells ◽

Species Specific ◽

Genital Tubercle

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5′-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.

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The chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation

Development ◽

10.1242/dev.122.12.3851 ◽

1996 ◽

Vol 122 (12) ◽

pp. 3851-3861 ◽

Cited By ~ 18

Author(s):

U. Grieshammer ◽

G. Minowada ◽

J.M. Pisenti ◽

U.K. Abbott ◽

G.R. Martin

Keyword(s):

Gene Expression ◽

Limb Development ◽

Positional Information ◽

Limb Bud ◽

Local Application ◽

Chick Embryos ◽

Limb Buds ◽

Early Stages ◽

Potential Link

In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.

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Distribution of nestin in the developing mouse limb bud in vivo and in micro-mass cultures of cells isolated from limb buds

Differentiation ◽

10.1046/j.1432-0436.1997.6130151.x ◽

1997 ◽

Vol 61 (3) ◽

pp. 151-159 ◽

Cited By ~ 33

Author(s):

Joanna Wroblewski ◽

Marianne Engström ◽

Caroline Edwall-Arvidsson ◽

Gunnar Sjöberg ◽

Thomas Sejersen ◽

...

Keyword(s):

Limb Bud ◽

Limb Buds ◽

Mass Cultures ◽

Mouse Limb

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Copper Response Element and Crr1-Dependent Ni2+-Responsive Promoter for Induced, Reversible Gene Expression in Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.2.5.995-1002.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 995-1002 ◽

Cited By ~ 42

Author(s):

Jeanette M. Quinn ◽

Janette Kropat ◽

Sabeeha Merchant

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Chlamydomonas Reinhardtii ◽

Target Genes ◽

Response Regulator ◽

Signal Transduction Pathway ◽

Transduction Pathway ◽

Nickel Ions ◽

Cobalt Ions ◽

Inducible Gene

ABSTRACT The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni2+-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.

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Epithelial Cell Gene Expression Induced by IntracellularStaphylococcus aureus

International Journal of Microbiology ◽

10.1155/2009/753278 ◽

2009 ◽

Vol 2009 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Xianglu Li ◽

William G. Fusco ◽

Keun S. Seo ◽

Kenneth W. Bayles ◽

Erin E. Mosley ◽

...

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Stress Responses ◽

Dna Microarrays ◽

Cholesterol Biosynthesis ◽

Transcriptional Level ◽

Cell Monolayers ◽

Cell Gene Expression ◽

Cellular Stress Responses ◽

Cell Gene

HEp-2 cell monolayers were cocultured with intracellularStaphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. IntracellularS. aureusaffected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes includingatf3, sgk, map2k1, map2k3, arhb, andarhewas increased. In addition, elevated transcription of proinflammatory genes was observed fortnfa, il1b, il6, il8, cxcl1, ccl20, cox2,andpai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellularS. aureus. Notably, intracellularS. aureusinduced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellularS. aureusby inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

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Interleukin-6–dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family–independent survival pathway closely associated with Stat3 activation

Blood ◽

10.1182/blood-2003-04-1048 ◽

2004 ◽

Vol 103 (1) ◽

pp. 242-251 ◽

Cited By ~ 94

Author(s):

Katja Brocke-Heidrich ◽

Antje K. Kretzschmar ◽

Gabriele Pfeifer ◽

Christian Henze ◽

Dennis Löffler ◽

...

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Multiple Myeloma ◽

Interleukin 6 ◽

Target Genes ◽

B Lymphocyte ◽

Expression Profiles ◽

B Cell Lymphoma ◽

Myeloma Cells ◽

Survival Pathway

Abstract Interleukin 6 (IL-6) is a growth and survival factor for multiple myeloma cells. As we report here, the IL-6–dependent human myeloma cell line INA-6 responds with a remarkably rapid and complete apoptosis to cytokine withdrawal. Among the antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of apoptosis regulators, only myeloid cell factor-1 (Mcl-1) was slightly induced by IL-6. Overexpression studies demonstrated, however, that IL-6 does not exert its survival effect primarily through this pathway. The IL-6 signal transduction pathways required for survival and the target genes controlled by them were analyzed by using mutated receptor chimeras. The activation of signal transducer and activator of transcription 3 (Stat3) turned out to be obligatory for the survival of INA-6 cells. The same held true for survival and growth of XG-1 myeloma cells. Gene expression profiling of INA-6 cells by using oligonucleotide microarrays revealed many novel IL-6 target genes, among them several genes coding for transcriptional regulators involved in B-lymphocyte differentiation as well as for growth factors and receptors potentially implicated in autocrine or paracrine growth control. Regulation of most IL-6 target genes required the activation of Stat3, underscoring its central role for IL-6 signal transduction. Taken together, our data provide evidence for the existence of an as yet unknown Stat3-dependent survival pathway in myeloma cells.

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GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling

eLife ◽

10.7554/elife.50670 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

Author(s):

Rachel K Lex ◽

Zhicheng Ji ◽

Kristin N Falkenstein ◽

Weiqiang Zhou ◽

Joanna L Henry ◽

...

Keyword(s):

Transcriptional Repression ◽

Hedgehog Signaling ◽

Target Genes ◽

Limb Bud ◽

Enhancer Activity ◽

Tissue Specific ◽

Major Mechanism ◽

Gli Proteins ◽

Repression Complex ◽

Mouse Limb

Transcriptional repression needs to be rapidly reversible during embryonic development. This extends to the Hedgehog pathway, which primarily serves to counter GLI repression by processing GLI proteins into transcriptional activators. In investigating the mechanisms underlying GLI repression, we find that a subset of GLI binding regions, termed HH-responsive enhancers, specifically loses acetylation in the absence of HH signaling. These regions are highly enriched around HH target genes and primarily drive HH-specific transcriptional activity in the mouse limb bud. They also retain H3K27ac enrichment in limb buds devoid of GLI activator and repressor, indicating that their activity is primarily regulated by GLI repression. Furthermore, the Polycomb repression complex is not active at most of these regions, suggesting it is not a major mechanism of GLI repression. We propose a model for tissue-specific enhancer activity in which an HDAC-associated GLI repression complex regulates target genes by altering the acetylation status at enhancers.

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Ultrastructural aspects of early development of the fore-limb buds in the chick and the mouse

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1965.0045 ◽

1965 ◽

Vol 162 (988) ◽

pp. 387-405 ◽

Cited By ~ 50

Keyword(s):

Endoplasmic Reticulum ◽

Early Development ◽

Secretory Granules ◽

Free Cell ◽

Mouse Embryos ◽

Limb Bud ◽

Chick Embryos ◽

Limb Buds ◽

Mouse Limb ◽

A Chain

Light microscope investigations of the early development of the fore-limb buds in chick and mouse were made to guide electron microscope studies with these tissues. At the time of maximal development of the ectodermal apical ridge there is a higher concentration of cytoplasmic RNA in the apical ridge cells than in the other cells of the limb bud. Ultrastructural investigations showed that, in the mesoblast cells at the earliest stages, profiles of endoplasmic reticulum are often found attached to the outer nuclear membrane. Some what later, discontinuities of nuclear envelope occur by which the content of the nucleus may communicate with the endoplasmic reticulum. In the cytoplasm of the mesoblast cells at these stages there were many granules similar in form and size to secretory granules of gland cells. Ribosomes are in the polysomal condition. At stages later than 20 in chick and in 11-day-old mouse embryos, the mesoblast shows the character of a syncytial tissue. Epiblast cells possess all the characters of an epithelium with well developed junctional complexes. The desmosomes form a chain consisting of units equipped with individual dense plaques, but connected by continuous bundles of fibres running parallel to the chain. The free cell membrane of the epiblastic cells, particularly at early stages, forms numerous microvilli and single cilia. In later stages during the form action of the ectodermal apical ridge, cilia have been found between the cells. This fact indicates that when the apical ridge is formed ectodermal cells migrate towards the margin of the limb bud. At these stages microvilli are also found between the apical ridge cells where they contribute to the cell-to-cell adhesion. Beginning at stage 22 in chick embryos and from the 12th day in mouse embryos there are in cells of the apical ridge numerous and extensive Golgi systems spread throughout the cytoplasm. Some what later there appear successively lysosomes, cytolysomes and extranuclear necrotic centres. All these organelles manifest acid phosphatase activity and are thoughtto initiate the involutive process in the apical ridge. Pycnosis and karyorrhexis appear as the last stage of cellular degeneration. Degenerating cells undergo phagocytosis by neighbouring epithelial cells. A basement membrane is present at all stages of development of the chick and mouse limb buds. It is an acellular continuous structure lining the internal (basal) surface of the epiblast, but in chick embryos it shows discontinuities immediately under the apical ectodermal ridge at the time of its maximum development.

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