Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

Touch sensation requires the mechanically gated ion channel ELKIN1.

Touch perception is enabled by mechanically activated ion channels, the opening of which excites cutaneous sensory endings to initiate sensation. In this study, we identify ELKIN1 as an ion channel likely gated by mechanical force, necessary for normal touch sensitivity in mice. Touch insensitivity in Elkin1-/- mice was caused by a loss of mechanically activated currents (MA currents) in around half of all sensory neurons activated by light touch (low-threshold mechanoreceptors). Reintroduction of Elkin1 into sensory neurons from Elkin1-/- mice restored MA currents. Additionally, small interfering RNA-mediated knockdown of ELKIN1 from induced human sensory neurons substantially reduced indentation-induced MA currents, supporting a conserved role for ELKIN1 in human touch. Our data identify ELKIN1 as a core component of touch transduction in mice and potentially in humans.

Balancing WNT signalling in early forebrain development: The role of LRP4 as a modulator of LRP6 function.

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling.

Identification of disease-relevant modulators of the SHH pathway in the developing brain.

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

Neural tube closure requires the endocytic receptor Lrp2 and its functional interaction with intracellular scaffolds.

Pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we have combined analysis of neural tube closure in mouse and in the African Clawed Frog Xenopus laevis to elucidate the etiology of Lrp2-related NTDs. Lrp2 loss of function impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes, which are essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that, during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction, as well as PCP component trafficking in a temporospatial manner.

Neuromuscular performance of balance and posture control in childhood and adolescence.

This study examined a potential age-dependency of both posture and stability (balance) control in children and adolescents in a healthy population. Body posture with open and closed eyes was examined for a total of 456 test persons (age 6.7-17.6 years. Posture parameters (posture index, upper body tilt, trunk tilt) were assessed in the sagittal plane. Additionally, the oscillation of the center of pressure with open and closed eyes was additionally analyzed in a sub-sample of 318 subjects. Absolute values of stability control parameters changed significantly during childhood and adolescence for both boys (p = 0.005) and girls (p = 0.01). Relative changes of stability and posture parameters when closing the eyes did not change (p > 0.05) and were independent of age, gender or sports activity in healthy children and adolescents. The shifting of the body segments towards each other, as a result of the loss of visual information, does not seem to be primarily responsible for the increase in COP fluctuation. This is a further indication that stability control and posture control are complex interdependent mechanisms whose interaction is not yet fully understood.

Targeted Athletic Training Improves the Neuromuscular Performance in Terms of Body Posture From Adolescence to Adulthood - Long-Term Study Over 6 Years.

Poor posture in childhood and adolescence is held responsible for the occurrence of associated disorders in adult age. This study aimed to verify whether body posture in adolescence can be enhanced through the improvement of neuromuscular performance, attained by means of targeted strength, stretch, and body perception training, and whether any such improvement might also transition into adulthood. From a total of 84 volunteers, the posture development of 67 adolescents was checked annually between the age of 14 and 20 based on index values in three posture situations. 28 adolescents exercised twice a week for about 2 h up to the age of 18, 24 adolescents exercised continually up to the age of 20. Both groups practiced other additional sports for about 1.8 h/week. Fifteen persons served as a non-exercising control group, practicing optional sports of about 1.8 h/week until the age of 18, after that for 0.9 h/week. Group allocation was not random, but depended on the participants' choice. A linear mixed model was used to analyze the development of posture indexes among the groups and over time and the possible influence of anthropometric parameters (weight, size), of optional athletic activity and of sedentary behavior. The post hoc pairwise comparison was performed applying the Scheffé test. The significance level was set at 0.05. The group that exercised continually (TR20) exhibited a significant posture parameter improvement in all posture situations from the 2nd year of exercising on. The group that terminated their training when reaching adulthood (TR18) retained some improvements, such as conscious straightening of the body posture. In other posture situations (habitual, closed eyes), their posture results declined again from age 18. The effect sizes determined were between η2 = 0.12 and η2 = 0.19 and represent moderate to strong effects. The control group did not exhibit any differences. Anthropometric parameters, additional athletic activities and sedentary behavior did not influence the posture parameters significantly. An additional athletic training of 2 h per week including elements for improved body perception seems to have the potential to improve body posture in symptom free male adolescents and young adults.

Assessment of the posture of adolescents in everyday clinical practice: Intra-rater and inter-rater reliability and validity of a posture index.

OBJECTIVES: The assessment of the posture of children and adolescents using photometric methods has a long tradition in paediatrics, manual therapy and physiotherapy. It can be well integrated into the clinical routine and enables objective documentation. One-dimensional parameters such as angle sizes are mostly used in the diagnosis of postural defects in children and adolescents by means of photogrammetry. This study examined the posture index, a complex parameter, which evaluates the alignment of several trunk segments in the sagittal plane and is suitable for use as a screening parameter in everyday clinical practice. METHODS: For this postural photographs were taken in the sagittal plane of the habitual posture in a subgroup of 105 adolescents (12.9 ± 2.6 years) for analysing validity, and in a subgroup of 25 adolescents (12.1 ± 2.8 years) for analysing reliability and objectivity. Marker spheres (12 mm) were placed on five anatomical landmarks. The posture was also evaluated clinically by experienced investigators (PT, MD, DSc). The distances of the marker points to the plumb line through the malleolus lateralis were calculated and the posture index calculated from these. In order to determine the objectivity, reliability and validity of the posture index, statistical parameters were calculated. RESULTS: The posture index demonstrated very good objectivity (intraclass correlation coefficient ICC = 0.865), good reliability (Cronbach's alpha = 0.842) and good validity compared to the posture assessment done by the medical experts (Spearman's rho = 0.712). CONCLUSIONS: The posture index reflects a doctor's assessment of the posture of children and adolescents and is suitable as a clinical parameter for the assessment of postural defects.

Bimodal antagonism of PKA signalling by ARHGAP36.

Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease.

Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes.

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.

Age-dependency of posture parameters in children and adolescents.

[Purpose] Poor posture in children and adolescents is a well-known problem. Therefore, early detection of incorrect posture is important. Photometric posture analysis is a cost-efficient and easy method, but needs reliable reference values. As children's posture changes as they grow, the assessment needs to be age-specific. This study aimed to investigate the development of both one-dimensional posture parameter (body inclination angle) and complex parameter (posture index) in different age groups (childhood to adolescence). [Subjects and Methods] The participants were 372 symptom-free children and adolescents (140 girls and 232 boys aged 6-17). Images of their habitual posture were obtained in the sagittal plane. High-contrast marker points and marker spheres were placed on anatomical landmarks. Based on the marker points, the body inclination angle (INC) and posture index (PI) were calculated using the Corpus concepts software. [Results] The INC angle significantly increased with age. The PI did not change significantly among the age groups. No significant differences between the corresponding age groups were found for PI and INC for both sexes. [Conclusion] When evaluating posture using the body inclination angle, the age of the subject needs to be considered. Posture assessment with an age-independent parameter may be more suitable.

Changes in Habitual and Active Sagittal Posture in Children and Adolescents with and without Visual Input - Implications for Diagnostic Analysis of Posture.

INTRODUCTION: Poor posture in children and adolescents has a prevalence of 22-65% and is suggested to be responsible for back pain. To assess posture, photometric imaging of sagittal posture is widely used, but usually only habitual posture positions (resting position with minimal muscle activity) are analysed. AIM: The objective of this study was 1) to investigate possible changes in posture-describing parameters in the sagittal plane, when the subjects changed from a habitual passive posture to an actively corrected posture, and 2) to investigate the changes in posture parameters when an actively corrected posture was to be maintained with closed eyes. MATERIALS AND METHODS: In a group of 216 male children and adolescents (average 12.4 ± 2.5 years, range 7.0 - 17.6 years), six sagittal posture parameters (body tilt BT, trunk incline TI, posture index PI, horizontal distances between ear, shoulder and hip and the perpendicular to the ankle joint) were determined by means of photometric imaging in an habitual passive posture position, in an actively erect posture with eyes open, and in active stance with eyes closed. The change in these parameters during the transition between the posture positions was analysed statistically (dependent t-Test or Wilcoxon-Test) after Bonferroni correction (p<0.004). RESULTS: When moving from a habitual passive to an active posture BT, TI, PI, dEar, dShoulder, and dHip decreased significantly(p< 0.004). When the eyes were closed, only the perpendicular distances (dEar, dShoulder, and dHip) increased significantly. The parameters that describe the alignment of the trunk sections in relation to each other (BT, TI, PI), remained unchanged in both actively regulated posture positions. CONCLUSION: Changes in sagittal posture parameters that occur when a habitual passive posture switches into an active posture or when an active posture is to be maintained while the eyes are closed can be used for diagnostic purposes regarding poor posture and posture regulation.

LRP2 Acts as SHH Clearance Receptor to Protect the Retinal Margin from Mitogenic Stimuli.

During forebrain development, LRP2 promotes morphogen signaling as an auxiliary SHH receptor. However, in the developing retina, LRP2 assumes the opposing function, mediating endocytic clearance of SHH and antagonizing morphogen action. LRP2-mediated clearance prevents spread of SHH activity from the central retina into the retinal margin to protect quiescent progenitor cells in this niche from mitogenic stimuli. Loss of LRP2 in mice increases the sensitivity of the retinal margin for SHH, causing expansion of the retinal progenitor cell pool and hyperproliferation of this tissue. Our findings document the ability of LRP2 to act, in a context-dependent manner, as activator or inhibitor of the SHH pathway. Our current findings uncovered LRP2 activity as the molecular mechanism imposing quiescence of the retinal margin in the mammalian eye and suggest SHH-induced proliferation of the retinal margin as cause of the large eye phenotype observed in mouse models and patients with LRP2 defects.

LRP2 mediates folate uptake in the developing neural tube.

The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2) is a multifunctional cell-surface receptor expressed in the embryonic neuroepithelium. Loss of LRP2 in the developing murine central nervous system (CNS) causes impaired closure of the rostral neural tube at embryonic stage (E) 9.0. Similar neural tube defects (NTDs) have previously been attributed to impaired folate metabolism in mice. We therefore asked whether LRP2 might be required for the delivery of folate to neuroepithelial cells during neurulation. Uptake assays in whole-embryo cultures showed that LRP2-deficient neuroepithelial cells are unable to mediate the uptake of folate bound to soluble folate receptor 1 (sFOLR1). Consequently, folate concentrations are significantly reduced in Lrp2(-/-) embryos compared with control littermates. Moreover, the folic-acid-dependent gene Alx3 is significantly downregulated in Lrp2 mutants. In conclusion, we show that LRP2 is essential for cellular folate uptake in the developing neural tube, a crucial step for proper neural tube closure.

Endocytic receptor-mediated control of morphogen signaling.

Receptor-mediated endocytosis provides a mechanism by which cells take up signaling molecules from the extracellular space. Recent studies have shown that one class of endocytic receptors, the low-density lipoprotein receptor-related proteins (LRPs), is of particular relevance for embryonic development. In this Primer, we describe how LRPs constitute central pathways that modulate morphogen presentation to target tissues and cellular signal reception, and how LRP dysfunction leads to developmental disturbances in many species.

LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline for inductive signals.

Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM), a major forebrain organizer. We identified LRP2, a member of the LDL receptor gene family, as a component of the SHH signaling machinery in the RDVM. LRP2 acts as an apical SHH-binding protein that sequesters SHH in its target field and controls internalization and cellular trafficking of SHH/patched 1 complexes. Lack of LRP2 in mice and in cephalic explants results in failure to respond to SHH, despite functional expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increases SHH signaling capacity. Our data identify a critical role for LRP2 in SHH signaling and reveal the molecular mechanism underlying forebrain anomalies in mice and patients with Lrp2 defects.

Loss of Lrp2 in zebrafish disrupts pronephric tubular clearance but not forebrain development.

Low-density lipoprotein receptor-related protein 2 (LRP2) is a multifunctional cell surface receptor conserved from nematodes to humans. In mammals, it acts as regulator of sonic hedgehog and bone morphogenetic protein pathways in patterning of the embryonic forebrain and as a clearance receptor in the adult kidney. Little is known about activities of this LRP in other phyla. Here, we extend the functional elucidation of LRP2 to zebrafish as a model organism of receptor (dys)function. We demonstrate that expression of Lrp2 in embryonic and larval fish recapitulates the patterns seen in mammalian brain and kidney. Furthermore, we studied the consequence of receptor deficiencies in lrp2 and in lrp2b, a homologue unique to fish, using ENU mutagenesis or morpholino knockdown. While receptor-deficient zebrafish suffer from overt renal resorption deficiency, their brain development proceeds normally, suggesting evolutionary conservation of receptor functions in pronephric duct clearance but not in patterning of the teleost forebrain.

Mutation of megalin leads to urinary loss of selenoprotein P and selenium deficiency in serum, liver, kidneys and brain.

Distribution of selenium (Se) within the mammalian body is mediated by SePP (selenoprotein P), an Se-rich glycoprotein secreted by hepatocytes. Genetic and biochemical evidence indicate that the endocytic receptors ApoER2 (apolipoprotein E receptor 2) and megalin mediate tissue-specific SePP uptake. In the present study megalin-mutant mice were fed on diets containing adequate (0.15 p.p.m.) or low (0.08 p.p.m.) Se content and were analysed for tissue and plasma Se levels, cellular GPx (glutathione peroxidase) activities and protein expression patterns. Megalin-mutant mice displayed increased urinary Se loss, which correlated with SePP excretion in their urine. Accordingly, serum Se and SePP levels were significantly reduced in megalin-mutant mice, reaching marginal levels on the low-Se diet. Moreover, kidney Se content and expression of renal selenoproteins were accordingly reduced, as was SePP internalization along the proximal tubule epithelium. Although GPx4 expression was not altered in testis, Se and GPx activity in liver and brain were significantly reduced. When fed on a low-Se diet, megalin-mutant mice developed impaired movement co-ordination, but no astrogliosis. These findings suggest that megalin prevents urinary SePP loss and participates in brain Se/SePP uptake.

The soluble intracellular domain of megalin does not affect renal proximal tubular function in vivo.

Megalin-mediated endocytic uptake constitutes the main pathway for clearance of plasma proteins from the glomerular filtrate in proximal tubules. Little is known, however, about mechanisms that control megalin expression and activity in the kidney. A widely discussed hypothesis states that upon ligand binding a regulated intramembrane proteolysis releases the cytosolic domain of megalin and this fragment subsequently modulates megalin gene transcription. Here, we tested this by generating a mouse model that co-expressed both the soluble intracellular domain and full-length megalin. Despite pronounced synthesis in the proximal tubules, the soluble intracellular domain failed to exert distinct effects on renal proximal tubular function, including megalin expression, endocytic retrieval of proteins, or global renal gene transcription. Hence, our study argues that the soluble intracellular domain does not have a role in regulating the activity of megalin in the kidney.