The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner.

Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.

Capillary contact points determine beta cell polarity, control secretion and are disrupted in the db/db mouse model of diabetes.

AIMS/HYPOTHESIS: Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. METHODS: We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. RESULTS: We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. CONCLUSIONS/INTERPRETATION: Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.

Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Cell and chloroplast anatomical features are poorly estimated from 2D cross-sections.

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function.

No evidence of homeostatic regulation of leaf temperature in Eucalyptus parramattensis trees: integration of CO2 flux and oxygen isotope methodologies.

Thermoregulation of leaf temperature (Tleaf ) may foster metabolic homeostasis in plants, but the degree to which Tleaf is moderated, and under what environmental contexts, is a topic of debate. Isotopic studies inferred the temperature of photosynthetic carbon assimilation to be a constant value of c. 20°C; by contrast, leaf biophysical theory suggests a strong dependence of Tleaf on environmental drivers. Can this apparent disparity be reconciled? We continuously measured Tleaf and whole-crown net CO2 uptake for Eucalyptus parramattensis trees growing in field conditions in whole-tree chambers under ambient and +3°C warming conditions, and calculated assimilation-weighted leaf temperature (TL-AW ) across 265 d, varying in air temperature (Tair ) from -1 to 45°C. We compared these data to TL-AW derived from wood cellulose δ18 O. Tleaf exhibited substantial variation driven by Tair , light intensity, and vapor pressure deficit, and Tleaf was strongly linearly correlated with Tair with a slope of c. 1.0. TL-AW values calculated from cellulose δ18 O vs crown fluxes were remarkably consistent; both varied seasonally and in response to the warming treatment, tracking variation in Tair . The leaves studied here were nearly poikilothermic, with no evidence of thermoregulation of Tleaf towards a homeostatic value. Importantly, this work supports the use of cellulose δ18 O to infer TL-AW , but does not support the concept of strong homeothermic regulation of Tleaf.

Isotopic steady state or non-steady state transpiration? Insights from whole tree chambers.

Unravelling the complexities of transpiration can be assisted by understanding the oxygen isotope composition of transpired water vapour (δE). It is often assumed that δE is at steady state, thereby mirroring the oxygen isotope composition of source water (δsource), but this assumption has never been tested at the whole-tree scale. This study utilised the unique infrastructure of 12 whole-tree chambers (WTC) enclosing Eucalyptus parramattensis trees to measure δE along with concurrent temperature and gas exchange data. Six chambers tracked ambient air temperature and six were exposed to an ambient +3 °C warming treatment. Day-time means for δE were within 1.2‰ of δsource (-3.3‰) but varied considerably throughout the day. Our observations show that Eucalyptus parramattensis trees are seldom transpiring at isotopic steady state over a diel period, but transpiration approaches source water isotopic composition over longer time periods.

Creating a virtual leaf.

When microscopy meets modelling the exciting concept of a 'virtual leaf' is born. The goal of a 'virtual leaf' is to capture complex physiology in a virtual environment, resulting in the capacity to run experiments computationally. One example of a 'virtual leaf' application is capturing 3D anatomy from volume microscopy data and estimating where water evaporates in the leaf and the proportions of apoplastic, symplastic and gas phase water transport. The same 3D anatomy could then be used to improve established 3D reaction-diffusion models, providing a better understanding of the transport of CO2 across the stomata, through the airspace and across the mesophyll cell wall. This viewpoint discusses recent progress that has been made in transitioning from a bulk leaf approach to a 3D understanding of leaf physiology, in particular, the movement of CO2 and H2O within the leaf.

Embracing 3D Complexity in Leaf Carbon-Water Exchange.

Leaves are a nexus for the exchange of water, carbon, and energy between terrestrial plants and the atmosphere. Research in recent decades has highlighted the critical importance of the underlying biophysical and anatomical determinants of CO2 and H2O transport, but a quantitative understanding of how detailed 3D leaf anatomy mediates within-leaf transport has been hindered by the lack of a consensus framework for analyzing or simulating transport and its spatial and temporal dynamics realistically, and by the difficulty of measuring within-leaf transport at the appropriate scales. We discuss how recent technological advancements now make a spatially explicit 3D leaf analysis possible, through new imaging and modeling tools that will allow us to address long-standing questions related to plant carbon-water exchange.