Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex.

The role of WNT/ß-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/ß-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.

Arrest of WNT/ß-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries.

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic ß-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in ß-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of ß-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/ß-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that ß-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.

Adrenal glands are zonated endocrine organs that are essential in controlling body homeostasis. How zonation is induced and maintained and how renewal of the adrenal cortex is ensured remain a mystery. Here we show that capsular RSPO3 signals to the underlying steroidogenic compartment to induce ß-catenin signaling and imprint glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. Importantly, Rspo3 function remains essential in adult life to ensure replenishment of lost cells and maintain the properties of the zona glomerulosa. Thus, the adrenal capsule acts as a central signaling center that ensures replacement of damaged cells and is required to maintain zonation throughout life.

Arabidopsis Transcriptomics Reveals the Role of Lipoxygenase2 (AtLOX2) in Wound-Induced Responses.

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit.

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.

A mosaic of induced and non-induced branches promotes variation in leaf traits, predation and insect herbivore assemblages in canopy trees.

Forest canopies are complex and highly diverse environments. Their diversity is affected by pronounced gradients in abiotic and biotic conditions, including variation in leaf chemistry. We hypothesised that branch-localised defence induction and vertical stratification in mature oaks constitute sources of chemical variation that extend across trophic levels. To test this, we combined manipulation of plant defences, predation monitoring, food-choice trials with herbivores and sampling of herbivore assemblages. Both induction and vertical stratification affected branch chemistry, but the effect of induction was stronger. Induction increased predation in the canopy and reduced herbivory in bioassays. The effects of increased predation affected herbivore assemblages by decreasing their abundance, and indirectly, their richness. In turn, we show that there are multiple factors contributing to variation across canopies. Branch-localised induction, variation between tree individuals and predation may be the ones with particularly strong effects on diverse assemblages of insects in temperate forests.

The Sexually Dimorphic Adrenal Cortex: Implications for Adrenal Disease.

Many adrenocortical diseases are more prevalent in women than in men, but the reasons underlying this sex bias are still unknown. Recent studies involving gonadectomy and sex hormone replacement experiments in mice have shed some light onto the molecular basis of sexual dimorphism in the adrenal cortex. Indeed, it has been shown that gonadal hormones influence many aspects of adrenal physiology, ranging from stem cell-dependent tissue turnover to steroidogenesis and X-zone dynamics. This article reviews current knowledge on adrenal cortex sexual dimorphism and the potential mechanisms underlying sex hormone influence of adrenal homeostasis. Both topics are expected to contribute to personalized and novel therapeutic approaches in the future.

Gradient Photonic Materials Based on One-Dimensional Polymer Photonic Crystals.

In nature, animals such as chameleons are well-known for the complex color patterns of their skin and the ability to adapt and change the color by manipulating sophisticated photonic crystal systems. Artificial gradient photonic materials are inspired by these color patterns. A concept for the preparation of such materials and their function as tunable mechanochromic materials is presented in this work. The system consists of a 1D polymer photonic crystal on a centimeter scale on top of an elastic poly(dimethylsiloxane) substrate with a gradient in stiffness. In the unstrained state, this system reveals a uniform red reflectance over the entire sample. Upon deformation, a gradient in local strain of the substrate is formed and transferred to the photonic crystal. Depending on the magnitude of this local strain, the thickness of the photonic crystal decreases continuously, resulting in a position-dependent blue shift of the reflectance peak and hence the color in a rainbow-like fashion. Using more sophisticated hard-soft-hard-soft-hard gradient elastomers enables the realization of stripe-like reflectance patterns. Thus, this approach allows for the tunable formation of reflectance gradients and complex reflectance patterns. Envisioned applications are in the field of mechanochromic sensors, telemedicine, smart materials, and metamaterials.

Myocardial-specific R-spondin3 drives proliferation of the coronary stems primarily through the Leucine Rich Repeat G Protein coupled receptor LGR4.

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal R-spondin 3 receptor in the heart.

Confinement templates for hierarchical nanoparticle alignment prepared by azobenzene-based surface relief gratings.

Alignment of nanoparticles to hierarchical periodic structures is an emerging field in the development of patterned surfaces. Common alignment methods are based on templates that guide particle self-assembly. These can be formed using lithographic methods offering an almost free choice of the motif, while being expensive and time-consuming for large-scale production. Alternatively, template formation by controlled wrinkling offers a low-cost formation, but often suffers from the formation of defect structures like line-defects and cracks. Here, we show a preparation technique for nanoparticle alignment substrates that is based on the inscription of holographic surface relief gratings with a periodic sinusoidal wave pattern on the surface of azobenzene films. As interference patterns are employed for structure formation, very uniform and defect-free gratings with tunable grating height and grating period can be prepared. These substrates were successfully replicated to poly(dimethyl siloxane) and the replicas used for the alignment of polystyrene latex particles. Accordingly produced substrates exhibiting gratings with a variation in grating height allow for efficient screening of nanoparticle alignment in a geometrical confinement in one single experiment. We anticipate our studies as a promising tool for the development of sensors, tunable gratings and metamaterials.

Awakening the Bowman: inhibition of CXCL12 signaling activates parietal epithelial cells.

Glomerular podocytes are terminally differentiated cells, and podocyte loss is a common feature of progressive renal pathologies. In this issue, Romoli et al. focus on podocyte progenitors that were proposed to reside in the Bowman's capsule. They demonstrate that suppression of CXCL12/CXCR4 signaling activates parietal epithelial cells that integrate into glomeruli, express podocyte specific markers, and interdigitate with existing cells. The data may open new therapeutic avenues for the treatment of glomerular diseases.

Sox11 gene disruption causes congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital abnormalities of the kidney and the urinary tract (CAKUT) belong to the most common birth defects in human, but the molecular basis for the majority of CAKUT patients remains unknown. Here we show that the transcription factor SOX11 is a crucial regulator of kidney development. SOX11 is expressed in both mesenchymal and epithelial components of the early kidney anlagen. Deletion of Sox11 in mice causes an extension of the domain expressing Gdnf within rostral regions of the nephrogenic cord and results in duplex kidney formation. On the molecular level SOX11 directly binds and regulates a locus control region of the protocadherin B cluster. At later stages of kidney development, SOX11 becomes restricted to the intermediate segment of the developing nephron where it is required for the elongation of Henle's loop. Finally, mutation analysis in a cohort of patients suffering from CAKUT identified a series of rare SOX11 variants, one of which interferes with the transactivation capacity of the SOX11 protein. Taken together these data demonstrate a key role for SOX11 in normal kidney development and may suggest that variants in this gene predispose to CAKUT in humans.

Controlled Wrinkling of Gradient Metal Films.

Controlled wrinkling is a rather simple method of fabricating surface topographies. The production process is based on the spontaneous formation of wrinkles upon compression of a hard film attached to a soft elastic substrate. Controlled wrinkling typically features large-scale wrinkled samples with a discrete wavelength and amplitude. In this report, we employ an approach utilizing linear metal layer thickness gradients for the controlled formation of gradient wrinkle patterns. The observed wavelength modulation was experimentally achieved by preparing layer thickness gradients of gold, chromium, and indium by physical vapor deposition in combination with a poly(dimethyl siloxane) elastomer substrate. In case of chromium and indium, a thin SiO x surface layer was sufficient to ensure adhesion. However, in case of gold, an additional thin chromium adhesion layer was required. For the wrinkled gradient gold film, it was possible to tune the wavelength from 3.4 to 12.2 µm on a single substrate. The experimental data correspond well to the theoretical bilayer model from Stafford et al. Chromium has a significant higher Young's modulus and melting temperature than gold. However, chromium was successfully evaporated and gradient wrinkle patterns with wavelengths from 1.0 to 3.5 µm were realized. In contrast, indium has a considerable lower Young's modulus than gold and chromium, respectively. Consequently, lower wavelengths (0.6-1.0 µm) of the wrinkled gradient indium film were observed. These tunable wrinkled gradient metal films can be envisioned as components in sensors and optical and electro-optical devices.

A knock-in mouse line conditionally expressing the tumor suppressor WTX/AMER1.

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele.

Repression of CMIP transcription by WT1 is relevant to podocyte health.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

Detection of Cellulose-Derived Chromophores by Ambient Ionization-MS.

The detection of individual chromophores that contribute to the overall discoloration of paper ("yellowing") is a challenge because those substances are only present in very small amounts. In this research, two analytical approaches based on ambient ionization techniques, namely, desorption electrospray ionization and paper spray, both coupled to mass spectrometry, are compared to each other with regard to their suitability for detecting acetylated cellulosic key chromophores. The paper spray approach proved to be the more sensitive and versatile method. Subsequently, paper spray (PS)-mass spectrometry was applied to model papers and historical papers in which the acetylated chromophores were detected successfully. Independent accurate mass measurements confirmed the results obtained from reference compounds, model samples, and real-world specimens.

Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.

The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad.

The gonad arises from the thickening of the coelomic epithelium and then commits into the sex determination process. Testis differentiation is activated by the expression of the Y-linked gene Sry, which promotes cell proliferation and differentiation of Sertoli cells, the supporting cells of the testis. In absence of Sry (XX individuals), activation of WNT/CTNNB1 signalling, via the upregulation of Rspo1 and Wnt4, promotes ovarian differentiation. However, Rspo1 and Wnt4 are expressed in the early undifferentiated gonad of both sexes, and Axin2-lacZ, a reporter of canonical WNT/CTNNB1 signalling, is expressed in the coelomic region of the E11.5 gonadal primordium, suggesting a role of these factors in early gonadal development. Here, we show that simultaneous ablation of Rspo1 and Wnt4 impairs proliferation of the cells of the coelomic epithelium, reducing the number of progenitors of Sertoli cells in XY mutant gonads. As a consequence, in XY Wnt4(-/-); Rspo1(-/-) foetuses, this leads to the differentiation of a reduced number of Sertoli cells and the formation of a hypoplastic testis exhibiting few seminiferous tubules. Hence, this study identifies Rspo1 and Wnt4 as two new regulators of cell proliferation in the early gonad regardless of its sex, in addition to the specific role of these genes in ovarian differentiation.

Hierarchical line-defect patterns in wrinkled surfaces.

We demonstrate a novel approach for controlling the formation of line-defects in wrinkling patterns by introducing step-like changes in the Young's modulus of elastomeric substrates supporting thin, stiff layers. Wrinkles are formed upon treating the poly(dimethylsiloxane) (PDMS) substrates by UV/Ozone (UVO) exposure in a uniaxially stretched state and subsequent relaxation. Line defects such as minutiae known from fingerprints are a typical feature in wrinkling patterns. The position where these defects occur is random for homogenous substrate elasticity and film thickness. However, we show that they can be predetermined by using PDMS substrates consisting of areas with different cross-linking densities. While changing the cross-linking density is well known to influence the wrinkling wavelength, we use this parameter in this study to force defect formation. The defect formation is monitored in situ using light microscopy and the mechanical parameters/film thicknesses are determined using imaging AFM indentation measurements. Thus the observed wrinkle-wavelengths can be compared to theoretical predictions. We study the density and morphology of defects for different changes in elasticity and compare our findings with theoretical considerations based on a generalized Swift-Hohenberg-equation to simply emulate the observed pattern-formation process, finding good agreement. The fact that for suitable changes in elasticity, well-ordered defect patterns are observed is discussed with respect to formation of hierarchical structures for applications in optics and nanotechnology.