scholarly journals Lung branching morphogenesis is accompanied by temporal metabolic changes towards a glycolytic preference

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hugo Fernandes-Silva ◽  
Marco G. Alves ◽  
Henrique Araújo-Silva ◽  
Ana M. Silva ◽  
Jorge Correia-Pinto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Glucose Consumption ◽  
Branching Morphogenesis ◽  
Explant Culture ◽  
Metabolic Changes ◽  
Specific Expression ◽  
Expression Levels ◽  
Early Stages ◽  
Lung Branching

Abstract Background Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching. Methods In this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity. Results Glucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal–distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis. Conclusions This report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development.

Dual Sympathetic Input into Developing Salivary Glands

2019 ◽  
Vol 98 (10) ◽  
pp. 1122-1130 ◽  
Author(s):  
T.H.N. Teshima ◽  
A.S. Tucker ◽  
S.V. Lourenço
Keyword(s):  
Salivary Gland ◽  
Superior Cervical Ganglion ◽  
Time Course ◽  
Sympathetic Nerves ◽  
Branching Morphogenesis ◽  
Developmental Time ◽  
Explant Culture ◽  
Early Stages ◽  
Cervical Ganglion ◽  
Gland Development

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

scholarly journals Comparative transcriptomic analysis of maize ear heterosis during the inflorescence meristem differentiation stage

2021 ◽  
Author(s):  
Xia Shi ◽  
Weihua li ◽  
Zhanyong Guo ◽  
Mingbo Wu ◽  
Xiangge Zhang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Transcriptomic Analysis ◽  
Inflorescence Meristem ◽  
Specific Expression ◽  
Ear Development ◽  
Expression Levels ◽  
Transcriptional Variation ◽  
Heterotic Locus ◽  
Segment Substitution ◽  
Maize Ear

AbstractHeterosis is widely used in many crops; however, its genetic mechanisms are only partly understood. Here, we sampled inflorescence meristem (IM) ears from the single-segment substitution maize (Zea mays) line lx9801hlEW2b, containing a heterotic locus hlEW2b associated with ear width, the receptor parent lx9801, the test parent Zheng58, and their corresponding hybrids. After transcriptomic analysis, 1638 genes were identified in at least one hybrid with nonadditively expressed patterns and different expression levels between the two hybrids. In particular, 2263 (12.89%) and 2352 (14.65%) genes showed allele-specific expression (ASE) in Zheng58 × lx9801 and Zheng58 × lx9801hlEW2b, respectively. A functional analysis showed that these genes were enriched in development-related processes and biosynthesis and catabolism processes, which are potentially associated with heterosis. Additionally, nonadditive expression and ASE may fine-tune the expression levels of crucial genes (such as WUS and KNOX that control IM development) controlling auxin metabolism and ear development to optimal states, and transcriptional variation may play important roles in maize ear heterosis. The results provide new information that increases our understanding of the relationship between transcriptional variation and heterosis formation during maize ear development, which may be helpful in clarifying the genetic and molecular mechanisms of heterosis.

scholarly journals Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland

2020 ◽  
Author(s):  
Qiang Lan ◽  
Marja L. Mikkola
Keyword(s):  
Mammary Gland ◽  
Gene Transfer ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Transcriptional Profiling ◽  
Branching Morphogenesis ◽  
Culture Technique ◽  
Adeno Associated Virus ◽  
Profiling Techniques

Abstract Branching morphogenesis of the murine mammary gland starts during late embryogenesis. It is regulated by the signals emanating both from the epithelium and the mesenchyme, yet the molecular mechanisms regulating this process remain poorly understood. We have previously developed a unique whole organ culture technique for embryonic mammary glands, which provides a powerful tool to monitor and manipulate branching morphogenesis ex vivo. Nowadays, RNA sequencing and other transcriptional profiling techniques provide robust methods to identify components of gene regulatory networks driving branching morphogenesis. However, validation of the candidate genes still mainly depends on the use of the transgenic mouse models, especially in mammary gland studies. By comparing different serotypes of recombinant adeno-associated virus (rAAVs), we found out that rAAVs provide sufficient efficiency for gene transfer with different tissue preferences depending on the serotypes of the virus. AAV-2 and AAV-8 preferentially target epithelial and mesenchymal compartments, respectively, while AAV-9 infects both tissues. Here, we describe a protocol for AAV-mediated gene transfer in ex vivo cultured murine embryonic mammary gland facilitating gene function studies on mammary gland branching morphogenesis.

scholarly journals Expression of Plasmodium vivaxcrt-oIs Related to Parasite Stage but NotEx VivoChloroquine Susceptibility

2015 ◽  
Vol 60 (1) ◽  
pp. 361-367 ◽  
Author(s):  
Zuleima Pava ◽  
Irene Handayuni ◽  
Grennady Wirjanata ◽  
Sheren To ◽  
Leily Trianty ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Drug Susceptibility ◽  
Fold Change ◽  
Low Grade ◽  
Content Type ◽  
Expression Levels ◽  
Stage Of Development ◽  
Parasite Stage

ABSTRACTChloroquine (CQ)-resistantPlasmodium vivaxis present in most countries whereP. vivaxinfection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression ofP. vivaxcrt-o(pvcrt-o) has been correlated within vivoCQ resistance in an area with low-grade resistance. We assessedpvcrt-oexpression in isolates from Papua (Indonesia), whereP. vivaxis highly CQ resistant.Ex vivodrug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels ofpvcrt-owere measured using a novel real-time quantitative reverse transcription-PCR method. Large variations inpvcrt-oexpression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with theP. vivaxβ-tubulin gene and from 0.01 to 24 relative to that seen with theP. vivaxaldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5;P< 0.001). Twenty-nine isolates fulfilled the criteria forex vivodrug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found thatpvcrt-oexpression levels did not correlate with theex vivoresponse to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuringpvcrt-oexpression and suggest thatpvcrt-otranscription is not a primary determinant ofex vivodrug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels andP. vivaxdrug resistance.

scholarly journals Lung branching morphogenesis, in the chicken model, is accompanied by temporal metabolic changes

2017 ◽  
Vol 2 (5) ◽  
pp. 222-223
Author(s):  
H. Fernandes-Silva ◽  
M. G. Alves ◽  
J. Correia-Pinto ◽  
P. F. Oliveira ◽  
R. S. Moura
Keyword(s):  
Branching Morphogenesis ◽  
Metabolic Changes ◽  
Chicken Model ◽  
Lung Branching

Adenovirus-mediated decorin gene transfer prevents TGF-β-induced inhibition of lung morphogenesis

1999 ◽  
Vol 277 (2) ◽  
pp. L412-L422 ◽  
Author(s):  
Jingsong Zhao ◽  
Patricia J. Sime ◽  
Pablo Bringas ◽  
Jack Gauldie ◽  
David Warburton
Keyword(s):  
Transforming Growth Factor ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Surfactant Protein ◽  
Branching Morphogenesis ◽  
Lung Growth ◽  
Lung Morphogenesis ◽  
Ex Vivo Culture ◽  
Lung Branching ◽  
Embryonic Lungs

Excessive transforming growth factor (TGF)-β signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-β could be useful therapeutic agents. Because exogenous TGF-β ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-β activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-β. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-β efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-β-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-β-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-β could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-β-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-β during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-β signaling in the developing lung.

Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins

2004 ◽  
Vol 286 (2) ◽  
pp. L293-L300 ◽  
Author(s):  
Wei Shi ◽  
Hui Chen ◽  
Jianping Sun ◽  
Cheng Chen ◽  
Jingsong Zhao ◽  
...  
Keyword(s):  
Lung Development ◽  
Molecular Mechanisms ◽  
Branching Morphogenesis ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Cell Protein ◽  
Mouse Lung ◽  
Mrna Levels ◽  
Embryonic Lung ◽  
Lung Branching

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.

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