scholarly journals EGF controls the in vivo developmental potential of a mammary epithelial cell line possessing progenitor properties

2002 ◽  
Vol 159 (3) ◽  
pp. 453-463 ◽  
Author(s):  
Marie-Ange Deugnier ◽  
Marisa M. Faraldo ◽  
Bassam Janji ◽  
Patricia Rousselle ◽  
Jean Paul Thiery ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Basal Layer ◽  
Mammary Epithelial ◽  
Secretory Cells ◽  
Molecular Characteristics ◽  
Epithelial Cell Line ◽  
Basal Cells ◽  
Developmental Potential ◽  
Cells In Culture

The bilayered mammary epithelium comprises a luminal layer of secretory cells and a basal layer of myoepithelial cells. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells; however, their molecular characteristics and differentiation pathways are largely unknown. BC44 mammary epithelial cells in culture, display phenotypic characteristics of basal epithelium, i.e., express basal cytokeratins 5 and 14 and P-cadherin, but no smooth muscle markers. In vivo, after injection into the cleared mammary fat pad, these cells gave rise to bilayered, hollow, alveolus-like structures comprising basal cells expressing cytokeratin 5 and luminal cells positive for cytokeratin 8 and secreting β-casein in a polarized manner into the lumen. The persistent stimulation of EGF receptor signaling pathway in BC44 cells in culture resulted in the loss of the in vivo morphogenetic potential and led to the induction of active MMP2, thereby triggering cell scattering and motility on laminin 5. These data (a) suggest that BC44 cells are capable of asymmetric division for self-renewal and the generation of a differentiated progeny restricted to the luminal lineage; (b) clarify the function of EGF in the control of the BC44 cell phenotypic plasticity; and (c) suggest a role for this phenomenon in the mammary gland development.

Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium

1989 ◽  
Vol 94 (3) ◽  
pp. 403-413 ◽  
Author(s):  
J. Taylor-Papadimitriou ◽  
M. Stampfer ◽  
J. Bartek ◽  
A. Lewis ◽  
M. Boshell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Basal Layer ◽  
Reduction Mammoplasty ◽  
Mammary Epithelial ◽  
Luminal Cell ◽  
Breast Cancers ◽  
Basal Cells ◽  
Cells Cultured

The luminal and basal epithelial cells in the human mammary gland can be distinguished in tissue sections on the basis of the pattern of keratins they express. Moreover, the invasive cells in primary carcinomas show a keratin profile that corresponds to that of the dominant luminal cell (7, 8, 18, 19). When homogeneous populations of luminal epithelial cells from milk or from breast cancer metastases are cultured the profile of keratin expression seen in vivo is maintained. We have therefore used monospecific antibodies reactive with individual keratins to examine the phenotype of cells cultured in three different media from reduction mammoplasty tissue that contains both luminal and basal cells. The phenotype of cells cultured from primary breast cancers in one of these media (MCDB170) has also been examined. In characterizing cell phenotypes, antibodies to a polymorphic epithelial mucin (PEM) expressed in vivo by luminal cells, and to smooth muscle (a) actin, expressed in vivo by basal cells, have also been used. Our results show that proliferation of different cell phenotypes is selected for in different media. In milk mix (MX) developed for growth of luminal cells from milk, only the luminal cell phenotype proliferates (for only 1 or 2 passages). In medium MCDB 170, which was developed for long-term growth of human mammary epithelial cells from reduction mammoplasty organoids, cells from the basal layer proliferate, while in MM medium the basal phenotype dominates, but a few cells with the luminal phenotype are found. Around passage 3, in medium MCDB 170, most cells senesce and a subpopulation of cells proliferates on further passage. These cells retain expression of the basal epithelial keratins but also express some features characteristic of luminal epithelial cells, suggesting that the basal layer may contain a stem cell that can develop along the luminal lineage. In culture, however, they do not express keratin 19, which in vivo is a feature of the fully differentiated luminal cell. The cells cultured from primary breast cancer in medium MCDB 170 have a similar keratin profile to that of the normal cells cultured in this medium. They do not express keratin 19, even though the invasive cells in primary cancers homogeneously express this keratin in vivo. The invasive phenotype, which in its keratin profile corresponds to the differentiated luminal cell and that of the metastatic cancer lines, cannot be cultured from primary breast cancers using MX, which supports proliferation of the corresponding normal cell.

scholarly journals Fine-tuning of Epithelial EGFR signals Supports Coordinated Mammary Gland Development

2020 ◽  
Author(s):  
Alexandr Samocha ◽  
Hanna M. Doh ◽  
Vaishnavi Sitarama ◽  
Quy H. Nguyen ◽  
Oghenekevwe Gbenedio ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelium ◽  
Gene Signature ◽  
Mammary Epithelial ◽  
Fine Tuning ◽  
Basal Cells ◽  
Stem And Progenitor Cells

SummaryDuring puberty, robust morphogenesis occurs in the mammary gland; stem- and progenitor-cells develop into mature basal- and luminal-cells to form the ductal tree. The receptor signals that govern this process in mammary epithelial cells (MECs) are incompletely understood. The EGFR has been implicated and here we focused on EGFR’s downstream pathway component Rasgrp1. We find that Rasgrp1 dampens EGF-triggered signals in MECs. Biochemically and in vitro, Rasgrp1 perturbation results in increased EGFR-Ras-PI3K-AKT and mTORC1-S6 kinase signals, increased EGF-induced proliferation, and aberrant branching-capacity in 3D cultures. However, in vivo, Rasgrp1 perturbation results in delayed ductal tree maturation with shortened branches and reduced cellularity. Rasgrp1-deficient MEC organoids revealed lower frequencies of basal cells, the compartment that incorporates stem cells. Molecularly, EGF effectively counteracts Wnt signal-driven stem cell gene signature in organoids. Collectively, these studies demonstrate the need for fine-tuning of EGFR signals to properly instruct mammary epithelium during puberty.

137 SPLITTING OF IVP BOVINE BLASTOCYST AFFECTS MORPHOLOGY AND GLOBAL GENE EXPRESSION OF RESULTING DEMI-EMBRYOS DURING ELONGATION

2015 ◽  
Vol 27 (1) ◽  
pp. 161
Author(s):  
A. E. Velásquez ◽  
D. Veraguas ◽  
J. F. Cox ◽  
F. O. Castro ◽  
L. l. Rodriguez
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Average Length ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Control Group ◽  
Molecular Characteristics ◽  
Bovine Embryo ◽  
Developmental Potential

Embryo splitting has been used since the early 1980s to produce identical twins and increase the pregnancy rate per available embryo. However, very little is known about the effect of splitting on embryo development and competence. Indeed splitting could provoke a negative effect on embryo survival and it can be presumed that each demi-embryo might respond differently to the injury. In this sense, even when embryos are genetically and morphologically identical at the moment of splitting, their developmental potential and molecular characteristics might change as a consequence of the intense manipulation or epigenetic differences due to the interaction with the environment. We have proposed an approach to evaluate the effect of blastocyst splitting on the morphological and gene expression in in vivo development up to the filamentous stage. For that, the effect of splitting on bovine embryo development was evaluated during the elongation period by transferring split and nonsplit IVF-derived blastocysts to cattle recipients and collecting them at Day 17 of development. The number of collected embryos, embryo size, and global gene expression was compared between both groups. Collected elongated embryos derived from split blastocyst were compared with time matched collected control embryos. From 14 transferred hemi-embryos, 5 (35.7%) were collected while 9 elongated from 17 controls were recovered (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between the two treatments. However, when embryos were rated depending on their size, more than 50% of embryos from the control group had a length surpassing 100 mm, while only 33% of the split embryos reached that size. Global gene expression was performed using 2-colour microarray-based gene expression analysis. This was a whole-genome microarray study comparing 10 individual elongated embryos derived from split and nonsplit IVF blastocysts. Genes were considered differentially expressed if the fold change is greater than 2 (up or down-regulation) with P ≤ 0.05. A total of 29 585 transcripts were detected in all embryos. From those, 449 (1.5%) were differentially expressed between elongated embryos derived from split and nonsplit IVF blastocysts, among them, 248 (0.83%) genes were down-regulated and 201 (0.67%) genes were up-regulated in split embryos. Gene ontology analysis identified deregulated genes related with intrinsic component of membrane (ELOVL7, GJA1, LAPTM4B, LDLR, SLC18A2, SLC1A3, SLC38A5, TSPAN13), lipid transporter activity (RBP4, APOA1, MTTP), and organophosphate ester transport (GJA1, GJB1, ATP9B). In conclusion, we showed that splitting affect the in vivo developmental capability and gene expression profile during the elongation period of bovine embryos. However, further studies are needed to determine the long-term effect of this technique to produce viable offspring. This work was partially supported by Fondecyt No. 11100082 and Fondequip No. EQM12113 from the Ministry of Education of Chile.

Integrin expression during human epidermal development in vivo and in vitro

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 193-206 ◽  
Author(s):  
M.D. Hertle ◽  
J.C. Adams ◽  
F.M. Watt
Keyword(s):  
Basement Membrane ◽  
Spatial Organization ◽  
Basal Layer ◽  
Subsequent Development ◽  
Sweat Glands ◽  
Collagen Type Iv ◽  
Integrin Expression ◽  
Basal Cells

In order to investigate the role of extracellular matrix receptors of the integrin family in establishing the spatial organization of epidermal kerotinocytes, we used immunofluorescence microscopy to examine the expression of a range of integrin subunits during development of human palm and sole skin. All of the integrins expressed during development were also present in mature epidermis and were largely confined to the basal layer of keratinocytes in a pericellular distribution. The alpha 3 and beta 1 subunits were expressed prior to the initiation of stratification and did not change in abundance or distribution during subsequent development. alpha 4 and beta 3 were not detected at any time in the epidermis. Every other subunit examined showed spatial or temporal changes in expression. Staining for alpha 1 was strong before stratification and until mid-development, but was greatly decreased in neonatal epidermis. alpha 2 was first detected in small patches of basal cells prior to stratification, and thereafter was found in the entire basal layer, with greater staining in developing sweat glands. alpha 5 was not expressed until mid-development, and then primarily in developing sweat glands, with faint expression in neonatal epidermis. alpha v was detected following stratification, in developing sweat glands, and occasionally in neonatal epidermis. alpha 6 and beta 4 were peribasally expressed before stratification, but thereafter became concentrated at the basal cell surface in contact with the basement membrane, co-localizing with hemidesmosomes as determined by staining with bullous pemphigoid antiserum. We also examined the distribution of three known ligands for keratinocyte integrins: laminin and collagen type IV were present in the basement membrane zone at all stages of development, whereas fibronectin was only evident there until about 13 weeks estimated gestational age. Finally, we found that the changes in integrin expression that occur on initiation of stratification in vivo could be reproduced in organ cultures of developing skin; such cultures therefore provided a useful experimental model for further studies of the role of integrins in epidermal stratification.

Overexpression of Krüppel-like factor 5 in esophageal epithelia in vivo leads to increased proliferation in basal but not suprabasal cells

2007 ◽  
Vol 292 (6) ◽  
pp. G1784-G1792 ◽  
Author(s):  
Bree G. Goldstein ◽  
Hann-Hsiang Chao ◽  
Yizeng Yang ◽  
Yuliya A. Yermolina ◽  
John W. Tobias ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Ectopic Expression ◽  
Basal Layer ◽  
Keratinocyte Differentiation ◽  
Basal Cells ◽  
Proliferating Cells ◽  
Barr Virus

Krüppel-like factor 5 ( Klf5; also called IKLF or BTEB2), a zinc-finger transcription factor with proproliferative and transforming properties in vitro, is expressed in proliferating cells of gastrointestinal tract epithelia, including in basal cells of the esophagus. Thus, Klf5 is an excellent candidate to regulate esophageal epithelial proliferation in vivo. Nonetheless, the function of Klf5 in esophageal epithelial homeostasis and tumorigenesis in vivo has not previously been determined. Here, we used the ED- L2 promoter of the Epstein-Barr virus to express Klf5 throughout esophageal epithelia. ED-L2/ Klf5 transgenic mice were born at the appropriate Mendelian ratio, survived to at least 1 yr of age, and showed no evidence of esophageal dysplasia or cancer. Staining for bromodeoxyuridine (BrdU) demonstrated increased proliferation in the basal layer of ED-L2/ Klf5 mice, but no proliferation was seen in suprabasal cells, despite ectopic expression of Klf5 in these cells. Notably, expression of the KLF family member Klf4, which binds the same DNA sequences as Klf5 and which inhibits proliferation and promotes differentiation, was not altered in ED-L2/ Klf5 transgenic mice. In primary esophageal keratinocytes that overexpressed Klf5, expression of Klf4 still inhibited proliferation and promoted differentiation, providing a possible mechanism for the persistence of keratinocyte differentiation in ED-L2/ Klf5 mice. To identify additional targets for Klf5 in esophageal epithelia, we performed functional genomic analyses and identified a total of 15 differentially expressed genes. In summary, while Klf5 positively regulates proliferation in basal cells, it is not sufficient to maintain proliferation in the esophageal epithelium.

In vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations

2004 ◽  
Vol 286 (4) ◽  
pp. L643-L649 ◽  
Author(s):  
Kyung U. Hong ◽  
Susan D. Reynolds ◽  
Simon Watkins ◽  
Elaine Fuchs ◽  
Barry R. Stripp
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Tracheal Epithelium ◽  
Secretory Cells ◽  
Basal Cells ◽  
Cell Type ◽  
Cell Hyperplasia ◽  
Differentiation Potential ◽  
Airway Injury

The composition of the conducting airway epithelium varies significantly along the proximal to distal axis, with that of the tracheal epithelium exhibiting the greatest complexity. A number of progenitor cells have been proposed to contribute to the maintenance of this cellular diversity both in the steady state and in response to injury. However, individual roles for each progenitor cell type are poorly defined in vivo. The present study was undertaken to investigate the hypothesis that basal cells represent a multipotent progenitor cell type for renewal of the injured tracheal epithelium. To understand their contribution to epithelial repair, mice were exposed to naphthalene to induce airway injury and depletion of the secretory cell progenitor pool. Injury resulted in a rapid induction of cytokeratin 14 (K14) expression among the majority of GSI-B4-reactive cells and associated hyperplasia of basal cells. Restoration of depleted secretory cells occurred after 6 days of recovery and was associated with regression of the basal cell hyperplasia, suggesting a progenitor-progeny relationship. Multipotent differentiation of basal cells was confirmed using a bitransgenic ligand-regulated Cre-loxP reporter approach in which expression of a ubiquitously expressed LacZ reporter was activated within K14-expressing progenitor cells during airway repair. With the use of this approach, it was determined that K14-expressing cells include subsets capable of either multipotent or unipotent differentiation in vivo. We conclude that basal cells have the capacity for restoration of a fully differentiated epithelium.

scholarly journals AP-2α: a regulator of EGF receptor signaling and proliferation in skin epidermis

2006 ◽  
Vol 172 (3) ◽  
pp. 409-421 ◽  
Author(s):  
Xuan Wang ◽  
Diana Bolotin ◽  
David H. Chu ◽  
Lisa Polak ◽  
Trevor Williams ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Basal Layer ◽  
Growth Factor Receptor ◽  
Conditional Knockout ◽  
Direct Target Gene ◽  
Elevated Expression ◽  
Epidermal Growth ◽  
Skin Epidermis ◽  
Knockout Technology

AP-2 transcription factors have been implicated in epidermal biology, but their functional significance has remained elusive. Using conditional knockout technology, we show that AP-2α is essential for governing the balance between growth and differentiation in epidermis. In vivo, epidermis lacking AP-2α exhibits elevated expression of the epidermal growth factor receptor (EGFR) in the differentiating layers, resulting in hyperproliferation when the receptors are activated. Chromatin immunoprecipitation and promoter activity assays identify EGFR as a direct target gene for AP-2α repression, and, in the absence of AP-2α, this is manifested primarily in excessive EGF-dependent phosphoinositol-3 kinase/Akt activity. Together, our findings unveil a hitherto unrecognized repressive role for AP-2α in governing EGFR gene transcription as cells exit the basal layer and withdraw from the cell cycle. These results provide insights into why elevated AP-2α levels are often associated with terminal differentiation and why tumor cells often display reduced AP-2α and elevated EGFR proteins.

scholarly journals RAB6 GTPase regulates mammary secretory function by controlling the activation of STAT5

Development ◽  
2020 ◽  
Vol 147 (19) ◽  
pp. dev190744 ◽  
Author(s):  
Surya Cayre ◽  
Marisa M. Faraldo ◽  
Sabine Bardin ◽  
Stéphanie Miserey-Lenkei ◽  
Marie-Ange Deugnier ◽  
...  
Keyword(s):  
Prolactin Receptor ◽  
Retrograde Transport ◽  
Mammary Epithelial ◽  
Epithelial Cell Line ◽  
Rab Gtpases ◽  
Cellular Functions ◽  
Membrane Expression ◽  
Transport Pathways

ABSTRACTThe Golgi-associated RAB GTPases, RAB6A and RAB6A′, regulate anterograde and retrograde transport pathways from and to the Golgi. In vitro, RAB6A/A′ control several cellular functions including cell division, migration, adhesion and polarity. However, their role remains poorly described in vivo. Here, we generated BlgCre; Rab6aF/F mice presenting a specific deletion of Rab6a in the mammary luminal secretory lineage during gestation and lactation. Rab6a loss severely impaired the differentiation, maturation and maintenance of the secretory tissue, compromising lactation. The mutant epithelium displayed a decreased activation of STAT5, a key regulator of the lactogenic process primarily governed by prolactin. Data obtained with a mammary epithelial cell line suggested that defective STAT5 activation might originate from a perturbed transport of the prolactin receptor, altering its membrane expression and signaling cascade. Despite the major functional defects observed upon Rab6a deletion, the polarized organization of the mammary epithelial bilayer was preserved. Altogether, our data reveal a crucial role for RAB6A/A′ in the lactogenic function of the mammary gland and suggest that the trafficking pathways controlled by RAB6A/A′ depend on cell-type specialization and tissue context.

Characteristics of magnetically separated rat tracheal epithelial cell populations

1992 ◽  
Vol 263 (5) ◽  
pp. L568-L574 ◽  
Author(s):  
J. R. Ford ◽  
M. Terzaghi-Howe
Keyword(s):  
Basal Cell ◽  
Significant Proportion ◽  
Cell Types ◽  
Cell Fraction ◽  
Secretory Cells ◽  
Basal Cells ◽  
Proliferative Capacity ◽  
Tracheal Mucosa ◽  
Tracheal Epithelial

A simple magnetic separation technique has been developed using lectins specific for two of the cell types found in the tracheal mucosa. The resulting populations of basal and secretory cells were examined for proliferative capacity in culture and in vivo. The basal cell fraction contains the cells that proliferate in culture and respond to 12-O-tetradecanoylphorbol-13-acetate. In addition, the basal cell fraction exhibited the highest proliferative capacity in vivo during the first few days after transplantation. Repopulation of inverted intestinal segments showed that only with suspensions containing a significant proportion of basal cells could a mucociliary lining be established. Segments receiving the same number of unsorted or predominantly mucous secreting cells did not repopulate in vivo. These data support the hypothesis that the basal cell is most likely the stem cell of the tracheal epithelium.

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