Cell-cycle-linked growth reprogramming encodes developmental time into leaf morphogenesis.

How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.

The cellular basis for synergy between RCO and KNOX1 homeobox genes in leaf shape diversity.

Leaves of seed plants provide an attractive system to study the development and evolution of form. Leaves show varying degrees of margin complexity ranging from simple, as in Arabidopsis thaliana, to fully dissected into leaflets in the closely related species Cardamine hirsuta. Leaflet formation requires actions of Class I KNOTTED1-LIKE HOMEOBOX (KNOX1) and REDUCED COMPLEXITY (RCO) homeobox genes, which are expressed in the leaves of C. hirsuta but not A. thaliana. Evolutionary studies indicate that diversification of KNOX1 and RCO genes was repeatedly associated with increased leaf complexity. However, whether this gene combination represents a developmentally favored avenue for leaflet formation remains unknown, and the cell-level events through which the combined action of these genes drives leaflet formation are also poorly understood. Here we show, through a genetic screen, that when a C. hirsuta RCO transgene is expressed in A. thaliana, then ectopic KNOX1 expression in leaves represents a preferred developmental path for leaflet formation. Using time-lapse growth analysis, we demonstrate that KNOX1 expression in the basal domain of leaves leads to prolonged and anisotropic cell growth. This KNOX1 action, in synergy with local growth repression by RCO, is instrumental in generating rachises and petiolules, the linear geometrical elements, that bear leaflets in complex leaves. Our results show how the combination of cell-level growth analyses and genetics can help us understand how evolutionary modifications in expression of developmentally important genes are translated into diverse leaf shapes.

Accurate and versatile 3D segmentation of plant tissues at cellular resolution.

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

The detection of multiple DNA targets with a single probe using a conformation-sensitive acoustic sensor.

By using an acoustic wave methodology that allows direct sensing of biomolecular conformations, we achieved the detection of multiple target DNAs using a single probe, exploiting the fact that each bound target results in a hybridized product of a different shape.

Investigation of angiogenetic pathways in nasal polyposis.

Tissue angiogenesis is a complex phenomenon that results in the growth of new blood vessels from the microcirculation. This process has been known to play a crucial role in tumor growth as well as several benign diseases. The aim of this study was to assess mRNA expression of various angiogenic factors and chemokines in nasal polyps and compare the results to normal nasal mucosa. mRNA expression was measured using real-time RT-PCR for the following angiogenic factors and chemokines: VEGF, VEGFR-1, Ang-1, Ang-2, Tie-2A, Tie-2B, SDF-1α, SDF-1ß, CXCR4 and YY1. Biopsy specimens from nasal polyps in the polyposis group and middle turbinates in the control group were studied. A total of 18 nasal polyposis patients were studied and compared to 10 control subjects. Results showed VEGF, VEGFR-1, Ang-1, Ang-2, Tie-2A, Tie-2B, SDF-1α and SDF-1ß mRNA expression to be significantly higher in nasal polyposis patients compared to the control group (p<0.05). The findings of this study support the role of angiogenic growth factors in the pathogenesis of nasal polyposis. Further studies are required to confirm these results and evaluate potential clinical implications.

Investigation of Toll-like receptors in the pathogenesis of fibrotic and granulomatous disorders: a bronchoalveolar lavage study.

BACKGROUND AND AIM: Toll-like receptors (TLRs), a key component of innate immunity, have recently been implicated in the pathogenesis of interstitial lung diseases (ILDs). As the involvement of TLRs has not yet been fully elucidated, the aim of the current study was to examine the expression of various TLRs in the bronchoalveolar lavage fluid (BALF) of patients with ILDs. PATIENTS AND METHODS: We studied prospectively three groups of patients: (1) one group of 35 patients with fibrotic disorders, 16 with idiopathic pulmonary fibrosis (IPF) and 19 with fibrotic interstitial pneumonias associated with collagen tissue disorders (CTD-IPs); (2) one group of 14 patients with pulmonary sarcoidosis; and (3) 11 normal subjects. We evaluated TLR expression with flow cytometry and mRNA expression with real-time PCR. RESULTS: An overexpression of TLR-3 mRNA was found in fibrotic disorders (CTD-IPs/IPF) in comparison with sarcoidosis (mean ± SD, 1.104 ± 1.087 versus 0.038 ± 0.03; P = 0.04). Additionally, TLR-3 mRNA was increased in CTD-IPs in comparison with IPF (P = 0.001), sarcoidosis (P = 0.002) and controls (P = 0.05). An upregulation in TLR-7 and -9 mRNA expression was detected in IPF (P = 0.05) and sarcoidosis (P = 0.05), respectively, when compared to controls. A higher percentage of TLR-9-expressing cells was found in BALF of CTD-IPs when compared to IPF (mean ± SD, 36.7 ± 7.06 versus 14.85 ± 3.82; P = 0.025). CONCLUSION: We observed distinct profiles of TLR expression in fibrotic and granulomatous disorders. It is likely that they could play a key role in the pathogenesis of these diseases and represent future therapeutic targets.

Expression analysis of Akt and MAPK signaling pathways in lung tissue of patients with idiopathic pulmonary fibrosis (IPF).

PURPOSE OF THE STUDY: Several studies in patients with lung cancer have shown that epidermal growth factor receptor regulates various tumorigenic processes through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin and Ras/Raf/Mek/Erk (mitogen-activated protein kinase (MAPK)) signalling pathways. The aim of our study is to evaluate whether these pathways are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and to seek indirect evidence of a common pathogenetic pathway with lung cancer. m-RNA expression of oncogenes participating in these two signaling pathways, as well as the combined m-RNA expression of the suppressor genes R-kip and p53 in lung tissue of patients with IPF were evaluated. BASIC PROCEDURES: The study population was composed by two distinct groups. Patients with IPF (n = 25) and control subjects who underwent thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned oncogenes and suppressor genes was performed using real-time reverse transcription polymerase chain reaction. MAIN FINDINGS: We found no difference in the overall m- RNA expression between controls and IPF in both investigated pathways. However, Braf has been overexpressed in IPF samples (P = 0.01) in contrast with K-ras that has been found downregulated (P < 0.001) in comparison with controls. PRINCIPAL CONCLUSIONS: These findings cannot exclude the hypothesis of involvement of Akt and MAPK signalling pathways in pathogenesis of IPF. However, further investigation is needed in order to verify these data.

Role of VEGF-stromal cell-derived factor-1alpha/CXCL12 axis in pleural effusion of lung cancer.

CONTEXT AND OBJECTIVE: It has been suggested that stromal cell-derived factor-1alpha ((SDF-1alpha) or CXCL12, both transcripts, TR1 and TR2) and its cognate receptor CXCR4 may regulate cancer metastasis. We have investigated the role of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2) and the biological axis of CXCL12-CXCR4, in patients with malignant pleural effusions (PEs). MATERIAL AND METHODS: Twenty five patients, seven with transudative PEs due to heart failure and 18 with exudative malignant PEs (7 with small cell lung cancer (SCLC) and 11 with nonsmall cell lung cancer (NSCLC)) were included in the study. Expression analysis of the mediators was performed in pleural fluid pellet using real-time reverse transcription-PCR. Protein expression has been evaluated by western blot analysis. RESULTS: SDF-TR1 (P = 0.02) but not SDF-TR2 (P = 0.23) or CXCR4 levels (P = 0.23) were higher in malignant PEs than in transudates. SDF-TR1 (P = 0.04) and SDF- TR2 levels (P = 0.04) but not CXCR4 levels (P = 0.123) were higher in SCLC PEs than in heart failure PEs. SDF-TR1 (P = 0.03) but not SDF-TR2 levels (P = 0.6) and CXCR4 levels (P = 0.4) were higher in NSCLC PEs than in transudates. Ang-1 has not been expressed in PEs, whereas no significant difference has been detected in VEGF and Ang-2 expression between malignant PEs and transudates. However, protein expression showed increased VEGF and SDF expression in malignant PEs. CONCLUSIONS: These results suggest that elevated SDF-1alpha/CXCL12 levels would be suggestive of a link to metastasis and may participate in pleural trafficking in lung cancer.

Expression analysis of angiogenic growth factors and biological axis CXCL12/CXCR4 axis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is associated with aberrant repair, persistence of collagen deposition, and the development of vascular remodeling. However, the role of angiogenesis in the pathogenesis of IPF is still undetermined. The aim of this study was to evaluate the combined mRNA expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF1) epidermal growth factor (EGF), and its receptor (EGFR) in lung tissue obtained from IPF patients. We have also investigated the expression of chemokine CXCL12/stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, to identify alterations that maybe implicated in the pathogenesis of IPF. The subjects studied consisted of two distinct groups: patients with IPF (n = 25) and subjects (control) undergoing thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned growth factors and biological axis CXCL12/CXR4 analysis were performed using real-time RT-PCR. IGF-1, EGF, and FGF2 mRNA levels are significantly decreased in the patients compared to the controls (p = 0.028, p = 0.023 and p = 0.009, respectively). SDF1-TR1 and SDF1-TR2 transcript levels were significantly lower in patients compared to controls (p = 0.017 and p = 0.001). Significant coexpression of VEGF mRNA with IGF mRNA was observed in the group of the patients (p = 0.017). An additional coexpression of VEGF mRNA with SDF1-TR1 mRNA was demonstrated(p = 0.030). Our results show a downregulation in angiogenetic mechanisms in IPF. However, our results should be further verified by measuring other angiogenetic pathways in more samples.

Down-regulation of K-ras and H-ras in human brain gliomas.

Ras genes, a class of nucleotide-binding proteins that regulate normal and transformed cell growth, have been scarcely investigated in human brain tumours. We evaluated the mutational, mRNA and protein expression profile of the ras genes in 21 glioblastomas multiforme (grade IV), four fibrillary astrocytoma (grade II), four anaplastic astrocytoma (grade III) and 15 normal specimens. K-, H- and N-ras transcript levels were determined by real-time RT-PCR and mutational status by PCR-restriction fragment length polymorphism (RFLP) and direct sequencing. p21 protein was evaluated by Western blot analysis. Two K-ras mutations were found in codons 16 and 26 in one pathological and one normal sample, respectively. Glioblastoma multiforme cases exhibited significantly lower K- and H-ras mRNA levels compared to controls (P < 10(-4)). K- and H-ras mRNA down-regulation was not associated with patient outcome or survival. K-ras was positively correlated with H-ras in glioblastomas (P = 0.005), but not in normal specimens. p21 protein was absent in all samples. Our findings provide evidence of K- and H-ras involvement in brain malignant transformation through transcriptional down-regulation, while N-ras seems to contribute less to brain carcinogenesis.

Different activity of the biological axis VEGF-Flt-1 (fms-like tyrosine kinase 1) and CXC chemokines between pulmonary sarcoidosis and idiopathic pulmonary fibrosis: a bronchoalveolar lavage study.

BACKGROUND: We have previously shown a different local and systemic angiogenic profile of CXC chemokines in Idiopathic Pulmonary Fibrosis (IPF) patients compared to sarcoidosis. In particular, sarcoidosis showed an angiostatic microenvironment, as compared with the angiogenic cytokine milieu seen in IPF. Purpose of the Study. Our aim was to further investigate the aforementioned finding by measuring the expression of different chemokines in granulomatous and fibrotic diseases. We estimated the levels of vascular endothelial growth factor (VEGF) and its high-affinity receptor, Flt-1 (fms-like tyrosine kinase 1), in bronchoalveolar lavage fluid (BALF) of patients with IPF and pulmonary sarcoidosis. We have also investigated the mRNA expression of angiogenetic chemokines' receptors such as CXCR2 and CXCR3 and the biological axis of stromal derived factor-1 alpha (SDF-1 alpha or CXCL12 alpha/CXCL12 beta) and receptor, CXCR4. METHODS: We studied prospectively three groups of patients: (i) one group of 18 patients with IPF, (ii) one group of 16 patients with sarcoidosis, and (iii) 10 normal subjects. RESULTS: A statistically significant increase has been detected in VEGF mRNA expression in IPF in comparison with pulmonary sarcoidosis (P = .03). In addition, a significant increase has been measured in CXCL12 alpha in sarcoidosis in comparison to IPF (P = .02). Moreover, a statistically significant decrease has been found in Flt-1 protein levels in pulmonary sarcoidosis in comparison with IPF (P = .03). A significant increase in VEGF (P = .03) and CXCR4 (P = .03) mRNA levels has been also detected in sarcoidosis' patients when compared with healthy controls. CONCLUSIONS: Our data suggest that increased expression of Flt-1 and downregulation of CXCL12 alpha in IPF may further support the hypothesis of a different angiogenetic profile between fibrotic and granulomatous diseases. However, further studies are needed in order to better investigate these enigmatic diseases.