Pancreatic Ductal Morphogenesis and the Pdx1 Homeodomain Transcription Factor

Embryonic development of the pancreas is marked by an early phase of dramatic morphogenesis, in which pluripotent progenitor cells of the developing pancreatic epithelium give rise to the full array of mature exocrine and endocrine cell types. The genetic determinants of acinar and islet cell lineages are somewhat well defined; however, the molecular mechanisms directing ductal formation and differentiation remain to be elucidated. The complex ductal architecture of the pancreas is established by a reiterative program of progenitor cell expansion and migration known as branching morphogenesis, or tubulogenesis, which proceeds in mouse development concomitantly with peak Pdx1 transcription factor expression. We therefore evaluated Pdx1 expression with respect to lineage-specific markers in embryonic sections of the pancreas spanning this critical period of duct formation and discovered an unexpected population of nonislet Pdx1-positive cells displaying physical traits of branching. We then established a 3D cell culture model of branching morphogenesis using primary pancreatic duct cells and identified a transient surge of Pdx1 expression exclusive to branching cells. From these observations we propose that Pdx1 might be involved temporally in a program of gene expression sufficient to facilitate the biochemical and morphological changes necessary for branching morphogenesis.

Download Full-text

The basic-helix-loop-helix protein pod1 is critically important for kidney and lung organogenesis

Development ◽

10.1242/dev.126.24.5771 ◽

1999 ◽

Vol 126 (24) ◽

pp. 5771-5783 ◽

Cited By ~ 19

Author(s):

S.E. Quaggin ◽

L. Schwartz ◽

S. Cui ◽

P. Igarashi ◽

J. Deimling ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cell Types ◽

Perinatal Period ◽

Branching Morphogenesis ◽

Bhlh Transcription Factor ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Kidney Morphogenesis

Epithelial-mesenchymal interactions are required for the development of all solid organs but few molecular mechanisms that underlie these interactions have been identified. Pod1 is a basic-helix-loop-helix (bHLH) transcription factor that is highly expressed in the mesenchyme of developing organs that include the lung, kidney, gut and heart and in glomerular visceral epithelial cells (podocytes). To determine the function of Pod1 in vivo, we have generated a lacZ-expressing null Pod1 allele. Null mutant mice are born but die in the perinatal period with severely hypoplastic lungs and kidneys that lack alveoli and mature glomeruli. Although Pod1 is exclusively expressed in the mesenchyme and podocytes, major defects are observed in the adjacent epithelia and include abnormalities in epithelial differentiation and branching morphogenesis. Pod1 therefore appears to be essential for regulating properties of the mesenchyme that are critically important for lung and kidney morphogenesis. Defects specific to later specialized cell types where Pod1 is expressed, such as the podocytes, were also observed, suggesting that this transcription factor may play multiple roles in kidney morphogenesis.

Download Full-text

Balancing dynamic tradeoffs drives cellular reprogramming

10.1101/393934 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kimberley N. Babos ◽

Kate E. Galloway ◽

Kassandra Kisler ◽

Madison Zitting ◽

Yichen Li ◽

...

Keyword(s):

Transcription Factor ◽

Cell Division ◽

Motor Neuron ◽

Molecular Mechanisms ◽

Cell Types ◽

Cellular Reprogramming ◽

Cellular Event ◽

Chemical Factors ◽

Number Of Cells ◽

Transcription And Replication

AbstractAlthough cellular reprogramming continues to generate new cell types, reprogramming remains a rare cellular event. The molecular mechanisms that limit reprogramming, particularly to somatic lineages, remain unclear. By examining fibroblast-to-motor neuron conversion, we identify a previously unappreciated dynamic between transcription and replication that determines reprogramming competency. Transcription factor overexpression forces most cells into states that are refractory to reprogramming and are characterized by either hypertranscription with little cell division, or hyperproliferation with low transcription. We identify genetic and chemical factors that dramatically increase the number of cells capable of both hypertranscription and hyperproliferation. Hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. We demonstrate that elevated topoisomerase expression endows cells with privileged reprogramming capacity, suggesting that biophysical constraints limit cellular reprogramming to rare events.

Download Full-text

Hepatobiliary Differentiation: Principles from Embryonic Liver Development

Seminars in Liver Disease ◽

10.1055/s-0040-1709679 ◽

2020 ◽

Vol 40 (04) ◽

pp. 365-372

Author(s):

Scott H. Freeburg ◽

Wolfram Goessling

Keyword(s):

Molecular Mechanisms ◽

Alagille Syndrome ◽

Cell Types ◽

Extrinsic Cues ◽

Hepatic Portal Vein ◽

Hepatic Portal ◽

Transcription Factor Expression ◽

In Vitro Data

AbstractHepatocytes and biliary epithelial cells (BECs), the two endodermal cell types of the liver, originate from progenitor cells called hepatoblasts. Based principally on in vitro data, hepatoblasts are thought to be bipotent stem cells with the potential to produce both hepatocytes and BECs. However, robust in vivo evidence for this model has only recently emerged. We examine the molecular mechanisms that stimulate hepatoblast differentiation into hepatocytes or BECs. In the absence of extrinsic cues, the default fate of hepatoblasts is hepatocyte differentiation. Inductive cues from the hepatic portal vein, however, initiate transcription factor expression in hepatoblasts, driving biliary specification. Defining the mechanisms of hepatobiliary differentiation provides important insights into congenital disorders, such as Alagille syndrome, and may help to better characterize the poorly understood hepatic lineage relationships observed during regeneration from liver injury.

Download Full-text

Proliferation and Migration of Human Osteoblasts on Porous Three Dimensional Scaffolds

Materials Science Forum ◽

10.4028/www.scientific.net/msf.638-642.506 ◽

2010 ◽

Vol 638-642 ◽

pp. 506-511 ◽

Cited By ~ 5

Author(s):

Claudia Bergemann ◽

Ernst Dieter Klinkenberg ◽

Frank Lüthen ◽

Arne Weidmann ◽

Regina Lange ◽

...

Keyword(s):

Cell Culture ◽

Morphological Changes ◽

Three Dimensional ◽

3D Cell Culture ◽

Porous Tantalum ◽

Human Osteoblasts ◽

A Cell ◽

And Migration ◽

Migration Of Cells

Porous tantalum (Ta) biomaterial is designed to function as a scaffold for osseous ingrowths and has found applications in orthopedics. Integration of this Ta foam into the neighboring bone requires that osteoprogenitor cells attach to the implant, grow into the scaffold, proliferate and differentiate to osteoblasts. The aim of the present study was to create an in vitro 3D model system to investigate the interaction of human osteoblasts with porous Ta in the depth of the corpus. To explore active migration of osteoblasts into the Ta scaffold two porous Ta discs (Zimmer, Poland) were horizontally fixed within a clamping ring. Thereby a 3D Ta module with 4 levels is generated, which is placed into a cell culture well with the appropriate medium. Osteoblast-like cells were seeded apical onto the Ta module and cultured for 7 days in humidified atmosphere. Active migration of cells into the scaffold was monitored by field emission scanning electron microscopy (FESEM) imaging of the apical, medial and basal layers. A problem in 3D cell culture is the nutrition of cells inside of the scaffold. Therefore morphological changes and differentiation of the cells in distinct layers were analyzed.

Download Full-text

OR16-05 Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.655 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Leonard Cheung ◽

Alexandre Daly ◽

Michelle Brinkmeier ◽

Sally Ann Camper

Keyword(s):

Transcription Factor ◽

Single Cell ◽

Molecular Mechanisms ◽

Thyroid Stimulating Hormone ◽

Pars Tuberalis ◽

Pituitary Cells ◽

Pars Distalis ◽

Mouse Development ◽

Single Cell Sequencing ◽

Stimulating Hormone

Abstract We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear. Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

Download Full-text

PSVII-36 Examining the effect of a physiological dose of the polyamine; spermine, on myogenic regulatory transcription factor expression

Journal of Animal Science ◽

10.1093/jas/skz258.717 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 359-359

Author(s):

Avani gouru ◽

Gordon Murdoch

Keyword(s):

Transcription Factor ◽

Biological Effects ◽

Muscle Growth ◽

Cell Types ◽

Myogenic Regulatory Factors ◽

Regulatory Factors ◽

Factor Expression ◽

C2c12 Muscle Cells ◽

Physiological Dose ◽

Transcription Factor Expression

Abstract Examining the effect of a physiological dose of the polyamine; spermine, on myogenic regulatory transcription factor expression. Spermine is a micronutrient derived from amino acids. It affects cell growth, proliferation, differentiation and gene regulation in many cell types. Polyamines have long been overlooked with respect to their biological effects on muscle growth. Myogenic regulatory factors (myoD, myf5) initiate, promote and regulate myogenesis. Supporting myogenic transcription factors transcription and by such enhancing muscle production in livestock, through nutrition may be possible with dietary spermine supplementation. We examined the effect of spermine (0.5mM) in undifferentiated c2c12 muscle cells at two time points (8hr and 16hr). Using TaqMan-MGB qRT-PCR we quantified mRNA for key myogenic regulatory factors in a minimum of three experiments each containing 3 technical replicates. We report a significant increase in myoD (P = 0.02) and myf5 (P = 0.05) mRNA at 8hr following spermine treatment as compared to controls (no spermine). After 16 hr exposure to spermine (0.5mM) treatment myoD (P = 0.01) remained significantly different from controls. Our results indicate that spermine supports myogenesis through expression of increased myogenic regulatory factors at early stages of myogenesis. These findings support the need to further test the hypothesis that spermine promotes increased muscle growth and ultimately may represent a dietary means to maximize muscle growth in livestock species.

Download Full-text

209 Rho signaling-directed regulation of YAP/TAZ in parthenogenetic stem cells

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab209 ◽

2020 ◽

Vol 32 (2) ◽

pp. 233

Author(s):

G. Pennarossa ◽

S. Arcuri ◽

F. Gandolfi ◽

T. Brevini

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Rho Gtpase ◽

Hippo Pathway ◽

Genetic Material ◽

Embryonic Stem ◽

Cell Types ◽

Embryonic Cell ◽

And Migration ◽

The Hippo Pathway

Mammalian oocyte maturation is characterised by asymmetric meiotic division that is regulated by specific cytoskeleton organisation. Similarly, during early embryonic divisions, one of the most important steps is the establishment of polarity that allows cells to adopt distinct developmental fates. All of these events are driven by dynamic changes in actin filaments. It has been demonstrated recently that the Rho signalling pathway plays a key role in the organisation and rearrangement of actin-containing structures, regulating cell polarity and migration. In addition, beside its effect on cell cytoskeleton, Rho directly interacts with the Hippo pathway, influencing both embryonic cell proliferation and differentiation. Because both Rho and Hippo are expressed by the oocyte and maternally inherited (Zhang et al. 2014 Cell Cycle 13, 3390-3403, https://doi.org/10.4161/15384101.2014.952967; Menchero et al. 2017 Dev. Dyn. 246, 245-261, https://doi.org/10.1002/dvdy.24471), we investigated their regulation in parthenogenetic embryonic stem cells (ParthESC) that possess exclusively maternal genetic material, and compared the results with biparental ESCs. Previous results obtained by whole-transcriptome analysis revealed the presence of several differentially expressed genes involved in the Rho pathway and showed no differences for most of the Hippo signalling genes. To better elucidate the molecular mechanisms involved, in the present study, we dissected the expression pattern of the Rho and Hippo regulatory genes in human biparental ESCs and ParthESC. Experiments were performed on 4 biparental ESC and 4 ParthESC lines using cells between passages 5 to 25. The results showed significantly increased transcription of the Rho GTPase family genes (RHOA, RHOB, and RHOC) in ParthESC compared with biparental ESCs. Consistent with this, 12 of 17 Rho activators were significantly upregulated, whereas 8 of 11 Rho inhibitors were significantly decreased in ParthESC. Furthermore, monoparental cells displayed significantly higher expression levels of YAP and TAZ, whereas the upstream genes involved in the Hippo pathway (LATS1/2, MOB1, MST1/2, NF2) were comparable in the two cell types. Interestingly, a significantly higher total YAP protein content was detected in ParthESC, whereas the quantity of the phosphorylated form was comparable in the two cell types. This accounts for the observed upregulation of Rho genes, which stimulate the assembly of contractile actin stress fibres, inhibiting LATS1/2 phosphorylation and preventing subsequent phosphorylation of YAP/TAZ (Yu and Guan 2013 Genes Dev. 27, 355-371; https://doi.org/10.1101/gad.210773.112). Altogether, our results suggest that the Rho pathway may regulate YAP/TAZ behaviour via a LATS/MST/NF2-independent process in ParthESC, similarly to a previous report in oocytes (Posfai and Rossant 2016 Cell Res. 26, 393-394; https://doi.org/10.1038/cr.2016). Although further clarifications are needed, we hypothesise that the regulatory mechanisms detected in ParthESC may be related to their strictly maternal origin, with a possible impact on their plasticity and potency. This study was supported by Carraresi Foundation. Authors are members of the COST Actions CA16119.

Download Full-text

SIRT1 Inhibition Affects Angiogenic Properties of Human MSCs

BioMed Research International ◽

10.1155/2014/783459 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Botti Chiara ◽

Caiafa Ilaria ◽

Coppola Antonietta ◽

Cuomo Francesca ◽

Miceli Marco ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Molecular Mechanisms ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Therapeutic Angiogenesis ◽

Induced Response ◽

Human Mscs ◽

Hypoxic Conditions ◽

And Migration

Human mesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewal and of differentiating into several cell types. Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesis in ischemic disorders. However, the molecular mechanisms underlying these effects remain unclear. A recent report has connected MSCs multipotency to sirtuin families, showing that SIRT1 can regulate MSCs function. Furthermore, SIRT1 is a critical modulator of endothelial angiogenic functions. Here, we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic and pharmacological level. We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA and protein level. Inhibition of SIRT1 by sirtinol, a cell-permeable inhibitor, or by specific sh-RNA resulted in an increase of premature-senescence phenotype, a reduction of proliferation rate with increased apoptosis. Furthermore, we observed a consistent reduction of tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1α protein and its transcriptional activity in hMSCs. Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCs and may contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cells in ischemic disorders through SIRT1 modulation.

Download Full-text

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover

Cancers ◽

10.3390/cancers13225673 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5673

Author(s):

Ha Hyung Moon ◽

Nina-Naomi Kreis ◽

Alexandra Friemel ◽

Susanne Roth ◽

Dorothea Schulte ◽

...

Keyword(s):

Cell Motility ◽

Molecular Mechanisms ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Focal Adhesions ◽

Protein Composition ◽

Cell Types ◽

Migration And Invasion ◽

And Migration ◽

Cytoskeleton Dynamics

The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

Download Full-text

Expression profiling reveals novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

10.1101/114363 ◽

2017 ◽

Author(s):

Montserrat Torres-Oliva ◽

Julia Schneider ◽

Gordon Wiegleb ◽

Felix Kaufholz ◽

Nico Posnien

Keyword(s):

Transcription Factor ◽

Blood Brain Barrier ◽

Molecular Mechanisms ◽

Cell Types ◽

Cell Development ◽

Brain Barrier ◽

Gene Clustering ◽

Glia Cell ◽

Blood Brain

AbstractThe development of different cell types must be tightly coordinated in different organs. The developing head of Drosophila melanogaster represents an excellent model to study the molecular mechanisms underlying this coordination because the eye-antennal imaginal discs contain the organ anlagen of nearly all adult head structures, such as the compound eyes or the antennae. We studied the genome wide gene expression dynamics during eye-antennal disc development in D. melanogaster to identify new central regulators of the underlying gene regulatory network. Expression based gene clustering and transcription factor motif enrichment analyses revealed a central regulatory role of the transcription factor Hunchback (Hb). We confirmed that hb is expressed in two polyploid retinal subperineurial glia cells (carpet cells). Our functional analysis shows that Hb is necessary for carpet cell development and loss of Hb function results in abnormal glia cell migration and photoreceptor axon guidance patterns. Additionally, we show for the first time that the carpet cells are an integral part of the blood-brain barrier.

Download Full-text