Tallo: A global tree allometry and crown architecture database.

Data capturing multiple axes of tree size and shape, such as a tree's stem diameter, height and crown size, underpin a wide range of ecological research-from developing and testing theory on forest structure and dynamics, to estimating forest carbon stocks and their uncertainties, and integrating remote sensing imagery into forest monitoring programmes. However, these data can be surprisingly hard to come by, particularly for certain regions of the world and for specific taxonomic groups, posing a real barrier to progress in these fields. To overcome this challenge, we developed the Tallo database, a collection of 498,838 georeferenced and taxonomically standardized records of individual trees for which stem diameter, height and/or crown radius have been measured. These data were collected at 61,856 globally distributed sites, spanning all major forested and non-forested biomes. The majority of trees in the database are identified to species (88%), and collectively Tallo includes data for 5163 species distributed across 1453 genera and 187 plant families. The database is publicly archived under a CC-BY 4.0 licence and can be access from: https://doi.org/10.5281/zenodo.6637599. To demonstrate its value, here we present three case studies that highlight how the Tallo database can be used to address a range of theoretical and applied questions in ecology-from testing the predictions of metabolic scaling theory, to exploring the limits of tree allometric plasticity along environmental gradients and modelling global variation in maximum attainable tree height. In doing so, we provide a key resource for field ecologists, remote sensing researchers and the modelling community working together to better understand the role that trees play in regulating the terrestrial carbon cycle.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Retinoic Acid Activity in Undifferentiated Neural Progenitors Is Sufficient to Fulfill Its Role in Restricting Fgf8 Expression for Somitogenesis.

Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.

Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM.

Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.

Embryonic heart progenitors and cardiogenesis.

The mammalian heart is a highly specialized organ, comprised of many different cell types arising from distinct embryonic progenitor populations during cardiogenesis. Three precursor populations have been identified to contribute to different myocytic and nonmyocytic cell lineages of the heart: cardiogenic mesoderm cells (CMC), the proepicardium (PE), and cardiac neural crest cells (CNCCs). This review will focus on molecular cues necessary for proper induction, expansion, and lineage-specific differentiation of these progenitor populations during cardiac development in vivo. Moreover, we will briefly discuss how the knowledge gained on embryonic heart progenitor biology can be used to develop novel therapeutic strategies for the management of congenital heart disease as well as for improvement of cardiac function in ischemic heart disease.

Retinoic acid functions as a key GABAergic differentiation signal in the basal ganglia.

Although retinoic acid (RA) has been implicated as an extrinsic signal regulating forebrain neurogenesis, the processes regulated by RA signaling remain unclear. Here, analysis of retinaldehyde dehydrogenase mutant mouse embryos lacking RA synthesis demonstrates that RA generated by Raldh3 in the subventricular zone of the basal ganglia is required for GABAergic differentiation, whereas RA generated by Raldh2 in the meninges is unnecessary for development of the adjacent cortex. Neurospheres generated from the lateral ganglionic eminence (LGE), where Raldh3 is highly expressed, produce endogenous RA, which is required for differentiation to GABAergic neurons. In Raldh3⁻/⁻ embryos, LGE progenitors fail to differentiate into either GABAergic striatal projection neurons or GABAergic interneurons migrating to the olfactory bulb and cortex. We describe conditions for RA treatment of human embryonic stem cells that result in efficient differentiation to a heterogeneous population of GABAergic interneurons without the appearance of GABAergic striatal projection neurons, thus providing an in vitro method for generation of GABAergic interneurons for further study. Our observation that endogenous RA is required for generation of LGE-derived GABAergic neurons in the basal ganglia establishes a key role for RA signaling in development of the forebrain.

Sex-specific timing of meiotic initiation is regulated by Cyp26b1 independent of retinoic acid signalling.

Sex-specific initiation of meiosis in the fetal ovary has been suggested to require retinoic acid (RA) for induction of Stra8, with expression of the RA-degrading enzyme Cyp26b1 in fetal testis delaying meiosis until postnatal development. In this study, we investigate Raldh2(-/-) mice lacking RA synthesis and signalling in mesonephros and adjacent gonad and reveal that Stra8 expression in the fetal ovary does not require RA signalling. In contrast to previous observations, we find that Stra8 is expressed in the absence of physiologically detectable levels of RA. Ketoconazole inhibition of Cyp26b1 in Raldh2(-/-) testis allows RA-independent induction of Stra8, but only when the mesonephros remains attached, pointing to a non-RA signal from the mesonephros that induces Stra8 in the adjacent gonad. These findings demonstrate that Cyp26b1 prevents the onset of meiosis by metabolizing a substrate other than RA that controls Stra8 expression, thus changing the paradigm for how studies on Cyp26 function are conducted.

Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2.

Epicardial signaling and Rxra are required for expansion of the ventricular myocardial compact zone. Here, we examine Raldh2(-/-) and Rxra(-/-) mouse embryos to investigate the role of retinoic acid (RA) signaling in this developmental process. The heart phenotypes of Raldh2 and Rxra mutants are very similar and are characterized by a prominent defect in ventricular compact zone growth. Although RA activity is completely lost in Raldh2(-/-) epicardium and the adjacent myocardium, RA activity is not lost in Rxra(-/-) hearts, suggesting that RA signaling in the epicardium/myocardium is not required for myocardial compact zone formation. We explored the possibility that RA-mediated target gene transcription in non-cardiac tissues is required for this process. We found that hepatic expression of erythropoietin (EPO), a secreted factor implicated in myocardial expansion, is dependent on both Raldh2 and Rxra. Chromatin immunoprecipitation studies support Epo as a direct target of RA signaling in embryonic liver. Treatment of an epicardial cell line with EPO, but not RA, upregulates Igf2. Furthermore, both Raldh2(-/-) and Rxra(-/-) hearts exhibit downregulation of Igf2 mRNA in the epicardium. EPO treatment of cultured Raldh2(-/-) hearts restores epicardial Igf2 expression and rescues ventricular cardiomyocyte proliferation. We propose a new model for the mechanism of RA-mediated myocardial expansion in which RA directly induces hepatic Epo resulting in activation of epicardial Igf2 that stimulates compact zone growth. This RA-EPO-IGF2 signaling axis coordinates liver hematopoiesis with heart development.

Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit-interdigit junction.

Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA-generating enzyme retinaldehyde dehydrogenase-2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2-/- mouse embryos lose expression of the RARE-lacZ RA-reporter transgene and matrix metalloproteinase-11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit-interdigit junction. Raldh2-/- autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down-regulated in Raldh2-/- autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit-interdigit junction suggests RA controls tissue remodeling as well as apoptosis.

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells.

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.

The amphibian second heart field: Xenopus islet-1 is required for cardiovascular development.

Islet-1 is a LIM-homeodomain transcription factor that has been defined to label cardiac progenitor cells of the second heart field. Here we provide the first analysis of the expression pattern of Xenopus islet-1 (Xisl-1) in the context of cardiovascular development. During early stages of heart development Xisl-1 is co-expressed with Nkx2.5 in the cardiac crescent in Xenopus supporting the notion of an initially single heart field. At subsequent stages of cardiogenesis the expression domains of Xisl-1 and Nkx2.5 become more distinct with Xisl-1 being detected more anterior to Nkx2.5, however both factors continue to be co-expressed in the dorsal mesocardium and pericardial roof of the linear heart tube. The presence of a cardiac Xisl-1 progenitor pool in an amphibian whose heart lacks an anatomically separated right ventricle is intriguing. Functional analyses show that Xisl-1 is required for normal heart development. Inhibition of Xisl-1 results in defects in heart morphogenesis and in the downregulation of early cardiac markers implicating a role for Xisl-1 in cardiac specification. Additionally, Xisl-1 loss-of-function affects the expression of several vascular markers demonstrating the involvement of Xisl-1 in vasculogenesis.

The role of Wnt signalling in cardiac development and tissue remodelling in the mature heart.

The heart is one of the first organs to function in the developing embryo. Vertebrate heart development can be subdivided into different phases, e.g. specification of myo- and endocardial progenitor cells during the establishment of heart-forming fields within the anterior lateral plate mesoderm, formation of the linear heart tube by merging of the paired heart-forming fields in front of the foregut, looping of the heart tube and transformation of the tubular embryonic heart into the four-chambered heart. The molecular mechanisms underlying these processes are phylogenetically remarkably conserved and involve the activation of specific transcriptional programs by different extracellular growth factors. In this review, we will focus on the functions of the Wnt family of growth factors in normal cardiac development and in tissue remodelling in the mature heart under pathological conditions.