J Regularization Improves Imbalanced Multiclass Segmentation

We propose a new loss formulation to further advance the multiclass segmentation of cluttered cells under weakly supervised conditions. When adding a Youden's J statistic regularization term to the cross entropy loss we improve the separation of touching and immediate cells, obtaining sharp segmentation boundaries with high adequacy. This regularization intrinsically supports class imbalance thus eliminating the necessity of explicitly using weights to balance training. Simulations demonstrate this capability and show how the regularization leads to correct results by helping advancing the optimization when cross entropy stagnates. We build upon our previous work on multiclass segmentation by adding yet another training class representing gaps between adjacent cells. This addition helps the classifier identify narrow gaps as background and no longer as touching regions. We present results of our methods for 2D and 3D images, from bright field images to confocal stacks containing different types of cells, and we show that they accurately segment individual cells after training with a limited number of images, some of which are poorly annotated

Plant Stem Cell Signaling Involves Ligand-Dependent Trafficking of the CLAVATA1 Receptor Kinase

Background: Cell numbers in above-ground meristems of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex [1]. CLV3 produced in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 and WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. Overexpression of CLV3 eliminates the stem cells, causing meristem termination [2], and loss of CLV3 function allows meristem overproliferation [3]. There are many questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype [4]. Results: Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model [5]. Conclusions: This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants

Modulation of Asymmetric Division Diversity through Cytokinin and SPEECHLESS Regulatory Interactions in the Arabidopsis Stomatal Lineage

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types—pavement cells and guard cells—in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the prime regulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomatal lineage divisions and forms a regulatory circuit with SPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition

A Robust and Sensitive Synthetic Sensor to Monitor the Transcriptional Output of the Cytokinin Signaling Network in Planta

Cytokinins are classic plant hormones that orchestrate plant growth, development, and physiology. They affect gene expression in target cells by activating a multistep phosphorelay network. Type-B response regulators, acting as transcriptional activators, mediate the final step in the signaling cascade. Previously, we have introduced a synthetic reporter, Two Component signaling Sensor (TCS)::green fluorescent protein (GFP), which reflects the transcriptional activity of type-B response regulators. TCS::GFP was instrumental in uncovering roles of cytokinin and deepening our understanding of existing functions. However, TCS-mediated expression of reporters is weak in some developmental contexts where cytokinin signaling has a documented role, such as in the shoot apical meristem or in the vasculature of Arabidopsis (Arabidopsis thaliana). We also observed that GFP expression becomes rapidly silenced in TCS::GFP transgenic plants. Here, we present an improved version of the reporter, TCS new (TCSn), which, compared with TCS, is more sensitive to phosphorelay signaling in Arabidopsis and maize (Zea mays) cellular assays while retaining its specificity. Transgenic Arabidopsis TCSn::GFP plants exhibit strong and dynamic GFP expression patterns consistent with known cytokinin functions. In addition, GFP expression has been stable over generations, allowing for crosses with different genetic backgrounds. Thus, TCSn represents a significant improvement to report the transcriptional output profile of phosphorelay signaling networks in Arabidopsis, maize, and likely other plants that display common response regulator DNA-binding specificities

Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases

The CLAVATA3 (CLV3)-CLAVATA1 (CLV1) ligand-receptor kinase pair negatively regulates shoot stem cell proliferation in plants. clv1 null mutants are weaker in phenotype than clv3 mutants, but the clv1 null phenotype is enhanced by mutations in the related receptor kinases BARELY ANY MERISTEM 1, 2 and 3 (BAM1, 2 and 3). The basis of this genetic redundancy is unknown. Here, we demonstrate that the apparent redundancy in the CLV1 clade is in fact due to the transcriptional repression of BAM genes by CLV1 signaling. CLV1 signaling in the rib meristem (RM) of the shoot apical meristem is necessary and sufficient for stem cell regulation. CLV3-CLV1 signaling in the RM represses BAM expression in wild-type Arabidopsis plants. In clv1 mutants, ectopic BAM expression in the RM partially complements the loss of CLV1. BAM regulation by CLV1 is distinct from CLV1 regulation of WUSCHEL, a proposed CLV1 target gene. In addition, quadruple receptor mutants are stronger in phenotype than clv3, pointing to the existence of additional CLV1/BAM ligands. These data provide an explanation for the genetic redundancy seen in the CLV1 clade and reveal a novel feedback operating in the control of plant stem cells

Computational morphodynamics of plants: integrating development over space and time

The emerging field of computational morphodynamics aims to understand the changes that occur in space and time during development by combining three technical strategies: live imaging to observe development as it happens; image processing and analysis to extract quantitative information; and computational modelling to express and test time-dependent hypotheses. The strength of the field comes from the iterative and combined use of these techniques, which has provided important insights into plant development

J Regularization Improves Imbalanced Multiclass Segmentation

We propose a new loss formulation to further advance the multiclass segmentation of cluttered cells under weakly supervised conditions. When adding a Youden's J statistic regularization term to the cross entropy loss we improve the separation of touching and immediate cells, obtaining sharp segmentation boundaries with high adequacy. This regularization intrinsically supports class imbalance thus eliminating the necessity of explicitly using weights to balance training. Simulations demonstrate this capability and show how the regularization leads to correct results by helping advancing the optimization when cross entropy stagnates. We build upon our previous work on multiclass segmentation by adding yet another training class representing gaps between adjacent cells. This addition helps the classifier identify narrow gaps as background and no longer as touching regions. We present results of our methods for 2D and 3D images, from bright field images to confocal stacks containing different types of cells, and we show that they accurately segment individual cells after training with a limited number of images, some of which are poorly annotated

ETR1 Integrates Response to Ethylene and Cytokinins into a Single Multistep Phosphorelay Pathway to Control Root Growth

Cytokinins and ethylene control plant development via sensors from the histidine kinase (HK) family. However, downstream signaling pathways for the key phytohormones are distinct. Here we report not only cytokinin but also ethylene is able to control root apical meristem (RAM) size through activation of the multistep phosphorelay (MSP) pathway. We find both cytokinin and ethylene-dependent RAM shortening requires ethylene binding to ETR1 and its HK activity. The receiver domain of ETR1 interacts with MSP signaling intermediates acting downstream of cytokinin receptors, further substantiating the role of ETR1 in MSP signaling. Our studies find both cytokinin and ethylene induce the MSP in similar and distinct cell types with ETR1-mediated ethylene signaling controlling MSP output specifically in the root transition zone. We identified members of the MSP pathway specific and common to both hormones and show that ETR1-regulated ARR3 controls RAM size. ETR1-mediated MSP spatially differs from canonical CTR1/EIN2/EIN3 ethylene signaling and is independent of EIN2, indicating that both pathways can be spatially and functionally separated. Furthermore, we demonstrate that canonical ethylene signaling controls MSP responsiveness to cytokinin specifically in the root transition zone, presumably via regulation of ARR10, one of the positive regulators of MSP signaling in Arabidopsis

Calcium signals are necessary to establish auxin transporter polarity in a plant stem cell niche

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca^(2+)) and that transient Ca^(2+) response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca^(2+) occur during development of the SAM and this Ca^(2+) response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca^(2+) is not necessary for the response of MTs to mechanical perturbations revealing that Ca^(2+) specifically acts downstream of mechanics to regulate PIN1 polarity response