Impacts beyond the brain: Unraveling molecular mechanisms linking psychiatric, metabolic, and inflammatory conditions.

By establishing semaphorin 6D expression in the amygdala as a central coordinator of brain, metabolic, and immunologic function, the Neuron publication by Nakanishi et al.1 provides new insight to how primary brain deficiency impacts physiological systems beyond the brain.

Motor innervation directs the correct development of the mouse sympathetic nervous system.

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the "fight-or-flight response". Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

Enhanced methamphetamine sensitisation in a rat model of the brain-derived neurotrophic factor Val66Met variant: Sex differences and dopamine receptor gene expression.

Brain-derived neurotrophic factor (BDNF) and the Val66Met polymorphism may play a role in the development of psychosis and schizophrenia. The aim of this study was to investigate long-term effects of methamphetamine (Meth) on psychosis-like behaviour and dopamine receptor and dopamine transporter gene expression in a novel rat model of the BDNF Val66Met polymorphism. At the end of a 7-day subchronic Meth treatment, female rats with the Met/Met genotype selectively showed locomotor hyperactivity sensitisation to the acute effect of Meth. Male rats showed tolerance to Meth irrespective of Val66Met genotype. Two weeks later, female Met/Met rats showed increased locomotor activity following both saline treatment or a low dose of Meth, a hyperactivity which was not observed in other genotypes or in males. Baseline PPI did not differ between the groups but the disruption of PPI by acute treatment with apomorphine was absent in Meth-pretreated Met/Met rats. Female Met/Met rats selectively showed down-regulation of dopamine D2 receptor gene expression in striatum. Behavioural effects of MK-801 or its locomotor sensitisation by prior Meth pretreatment were not influenced by genotype. These data suggest a selective vulnerability of female Met/Met rats to short-term and long-term effects of Meth, which could model increased vulnerability to psychosis development associated with the BDNF Val66Met polymorphism.

Interaction of Brain-Derived Neurotrophic Factor with the Effects of Chronic Methamphetamine on Prepulse Inhibition in Mice Is Independent of Dopamine D3 Receptors.

The aim of the present study was to gain a better understanding of the role of brain-derived neurotrophic factor (BDNF) and dopamine D3 receptors in the effects of chronic methamphetamine (METH) on prepulse inhibition (PPI), an endophenotype of psychosis. We compared the effect of a three-week adolescent METH treatment protocol on the regulation of PPI in wildtype mice, BDNF heterozygous mice (HET), D3 receptor knockout mice (D3KO), and double-mutant mice (DM) with both BDNF heterozygosity and D3 receptor knockout. Chronic METH induced disruption of PPI regulation in male mice with BDNF haploinsufficiency (HET and DM), independent of D3 receptor knockout. Specifically, these mice showed reduced baseline PPI, as well as attenuated disruption of PPI induced by acute treatment with the dopamine receptor agonist, apomorphine (APO), or the glutamate NMDA receptor antagonist, MK-801. In contrast, there were no effects of BDNF heterozygosity or D3 knockout on PPI regulation in female mice. Chronic METH pretreatment induced the expected locomotor hyperactivity sensitisation, where female HET and DM mice also showed endogenous sensitisation. Differential sex-specific effects of genotype and METH pretreatment were observed on dopamine receptor and dopamine transporter gene expression in the striatum and frontal cortex. Taken together, these results show a significant involvement of BDNF in the long-term effects of METH on PPI, particularly in male mice, but these effects appear independent of D3 receptors. The role of this receptor in psychosis endophenotypes therefore remains unclear.

Endothelial-derived angiocrine factors as instructors of embryonic development.

Blood vessels are well-known to play roles in organ development and repair, primarily owing to their fundamental function in delivering oxygen and nutrients to tissues to promote their growth and homeostasis. Endothelial cells however are not merely passive conduits for carrying blood. There is now evidence that endothelial cells of the vasculature actively regulate tissue-specific development, morphogenesis and organ function, as well as playing roles in disease and cancer. Angiocrine factors are growth factors, cytokines, signaling molecules or other regulators produced directly from endothelial cells to instruct a diverse range of signaling outcomes in the cellular microenvironment, and are critical mediators of the vascular control of organ function. The roles of angiocrine signaling are only beginning to be uncovered in diverse fields such as homeostasis, regeneration, organogenesis, stem-cell maintenance, cell differentiation and tumour growth. While in some cases the specific angiocrine factor involved in these processes has been identified, in many cases the molecular identity of the angiocrine factor(s) remain to be discovered, even though the importance of angiocrine signaling has been implicated. In this review, we will specifically focus on roles for endothelial-derived angiocrine signaling in instructing tissue morphogenesis and organogenesis during embryonic and perinatal development.

IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ.

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

Vascular cells improve functionality of human cardiac organoids.

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor ß (PDGFRß) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

PI(4,5)P2-dependent regulation of endothelial tip cell specification contributes to angiogenesis.

Dynamic positioning of endothelial tip and stalk cells, via the interplay between VEGFR2 and NOTCH signaling, is essential for angiogenesis. VEGFR2 activates PI3K, which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, activating AKT; however, PI3K/AKT does not direct tip cell specification. We report that PI(4,5)P2 hydrolysis by the phosphoinositide-5-phosphatase, INPP5K, contributes to angiogenesis. INPP5K ablation disrupted tip cell specification and impaired embryonic angiogenesis associated with enhanced DLL4/NOTCH signaling. INPP5K degraded a pool of PI(4,5)P2 generated by PIP5K1C phosphorylation of PI(4)P in endothelial cells. INPP5K ablation increased PI(4,5)P2, thereby releasing ß-catenin from the plasma membrane, and concurrently increased PI(3,4,5)P3-dependent AKT activation, conditions that licensed DLL4/NOTCH transcription. Suppression of PI(4,5)P2 in INPP5K-siRNA cells by PIP5K1C-siRNA, restored ß-catenin membrane localization and normalized AKT signaling. Pharmacological NOTCH or AKT inhibition in vivo or genetic ß-catenin attenuation rescued angiogenesis defects in INPP5K-null mice. Therefore, PI(4,5)P2 is critical for ß-catenin/DLL4/NOTCH signaling, which governs tip cell specification during angiogenesis.

Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells.

The ubiquitin ligase NEDD4 promotes neural crest cell (NCC) survival and stem-cell like properties to regulate craniofacial and peripheral nervous system development. However, how ubiquitination and NEDD4 control NCC development remains unknown. Here we combine quantitative analysis of the proteome, transcriptome and ubiquitinome to identify key developmental signalling pathways that are regulated by NEDD4. We report 276 NEDD4 targets in NCCs and show that loss of NEDD4 leads to a pronounced global reduction in specific ubiquitin lysine linkages. We further show that NEDD4 contributes to the regulation of the NCC actin cytoskeleton by controlling ubiquitination and turnover of Profilin 1 to modulate filamentous actin polymerization. Taken together, our data provide insights into how NEDD4-mediated ubiquitination coordinates key regulatory processes during NCC development.

Loss of NEDD4 causes complete XY gonadal sex reversal in mice.

Gonadogenesis is the process wherein two morphologically distinct organs, the testis and the ovary, arise from a common precursor. In mammals, maleness is driven by the expression of Sry. SRY subsequently upregulates the related family member Sox9 which is responsible for initiating testis differentiation while repressing factors critical to ovarian development such as FOXL2 and ß-catenin. Here, we report a hitherto uncharacterised role for the ubiquitin-protein ligase NEDD4 in this process. XY Nedd4-deficient mice exhibit complete male-to-female gonadal sex reversal shown by the ectopic upregulation of Foxl2 expression at the time of gonadal sex determination as well as insufficient upregulation of Sox9. This sex reversal extends to germ cells with ectopic expression of SYCP3 in XY Nedd4-/- germ cells and significantly higher Sycp3 transcripts in XY and XX Nedd4-deficient mice when compared to both XY and XX controls. Further, Nedd4-/- mice exhibit reduced gonadal precursor cell formation and gonadal size as a result of reduced proliferation within the developing gonad as well as reduced Nr5a1 expression. Together, these results establish an essential role for NEDD4 in XY gonadal sex determination and development and suggest a potential role for NEDD4 in orchestrating these cell fate decisions through the suppression of the female pathway to ensure proper testis differentiation.

Mandible Explant Assay for the Analysis of Meckel's Cartilage Development.

Ex vivo explant models are a valuable tool for analyzing organ and tissue morphogenesis, providing the opportunity to manipulate and interrogate specific cellular and/or molecular pathways that may not be possible using conventional methods in vivo. The mandible primordia is a remarkably self-organizing structure that has the ability to develop cartilage, bone, teeth, epithelial tissue, and the tongue when grown in culture ex vivo and closely mimics the development of these structures in vivo. Here we describe a robust protocol for the culture of mandibular explants using serum-free, chemically defined culture media. We also describe methods for manipulating mandible and/or Meckel's cartilage development by implantation of agarose beads soaked in various molecular factors to augment mandible development, as well as methods for Alcian blue staining of Meckel's cartilage and immunohistochemistry. This culture method can also be adapted for other molecular analyses, including addition of small-molecule inhibitors and/or growth factors to the culture media, as well as culturing explants from genetically modified mice.

Notch signaling is a critical initiator of roof plate formation as revealed by the use of RNA profiling of the dorsal neural tube.

BACKGROUND: The dorsal domain of the neural tube is an excellent model to investigate the generation of complexity during embryonic development. It is a highly dynamic and multifaceted region being first transiently populated by prospective neural crest (NC) cells that sequentially emigrate to generate most of the peripheral nervous system. Subsequently, it becomes the definitive roof plate (RP) of the central nervous system. The RP, in turn, constitutes a patterning center for dorsal interneuron development. The factors underlying establishment of the definitive RP and its segregation from NC and dorsal interneurons are currently unknown. RESULTS: We performed a transcriptome analysis at trunk levels of quail embryos comparing the dorsal neural tube at premigratory NC and RP stages. This unraveled molecular heterogeneity between NC and RP stages, and within the RP itself. By implementing these genes, we asked whether Notch signaling is involved in RP development. First, we observed that Notch is active at the RP-interneuron interface. Furthermore, gain and loss of Notch function in quail and mouse embryos, respectively, revealed no effect on early NC behavior. Constitutive Notch activation caused a local downregulation of RP markers with a concomitant development of dI1 interneurons, as well as an ectopic upregulation of RP markers in the interneuron domain. Reciprocally, in mice lacking Notch activity, both the RP and dI1 interneurons failed to form and this was associated with expansion of the dI2 population. CONCLUSIONS: Collectively, our results offer a new resource for defining specific cell types, and provide evidence that Notch is required to establish the definitive RP, and to determine the choice between RP and interneuron fates, but not the segregation of RP from NC.

Compound heterozygous variants in LAMC3 in association with posterior periventricular nodular heterotopia.

BACKGROUND: Periventricular nodular heterotopia (PNH) is a malformation of cortical development characterized by nodules of abnormally migrated neurons. The cause of posteriorly placed PNH is not well characterised and we present a case that provides insights into the cause of posterior PNH. CASE PRESENTATION: We report a fetus with extensive posterior PNH in association with biallelic variants in LAMC3. LAMC3 mutations have previously been shown to cause polymicrogyria and pachygyria in the occipital cortex, but not PNH. The occipital location of PNH in our case and the proposed function of LAMC3 in cortical development suggest that the identified LAMC3 variants may be causal of PNH in this fetus. CONCLUSION: We hypothesise that this finding extends the cortical phenotype associated with LAMC3 and provides valuable insight into genetic cause of posterior PNH.

Exploring the Intracrine Functions of VEGF-A.

Vascular endothelial growth factor A (VEGF-A or VEGF) is a highly conserved secreted signalling protein best known for its roles in vascular development and angiogenesis. Many non-endothelial roles for VEGF are now established, with the discovery that VEGF and its receptors VEGFR1 and VEGFR2 are expressed in many non-vascular cell-types, as well as various cancers. In addition to secreted VEGF binding to its receptors in the extracellular space at the cell membrane (i.e., in a paracrine or autocrine mode), intracellularly localised VEGF is emerging as an important signalling molecule regulating cell growth, survival, and metabolism. This intracellular mode of signalling has been termed "intracrine", and refers to the direct action of a signalling molecule within the cell without being secreted. In this review, we describe examples of intracrine VEGF signalling in regulating cell growth, differentiation and survival, both in normal cell homeostasis and development, as well as in cancer. We further discuss emerging evidence for the molecular mechanisms underpinning VEGF intracrine function, as well as the implications this intracellular mode of VEGF signalling may have for use and design of anti-VEGF cancer therapeutics.

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages.

Vessel-derived angiocrine IGF1 promotes Meckel's cartilage proliferation to drive jaw growth during embryogenesis.

Craniofacial development is a complex morphogenic process that requires highly orchestrated interactions between multiple cell types. Blood vessel-derived angiocrine factors are known to promote proliferation of chondrocytes in Meckel's cartilage to drive jaw outgrowth, however the specific factors controlling this process remain unknown. Here, we use in vitro and ex vivo cell and tissue culture, as well as genetic mouse models, to identify IGF1 as a novel angiocrine factor directing Meckel's cartilage growth during embryonic development. We show that IGF1 is secreted by blood vessels and that deficient IGF1 signalling underlies mandibular hypoplasia in Wnt1-Cre; Vegfafl/fl mice that exhibit vascular and associated jaw defects. Furthermore, conditional removal of IGF1 from blood vessels causes craniofacial defects including a shortened mandible, and reduced proliferation of Meckel's cartilage chondrocytes. This demonstrates a crucial angiocrine role for IGF1 during craniofacial cartilage growth, and identifies IGF1 as a putative therapeutic for jaw and/or cartilage growth disorders.

Notch signalling defines dorsal root ganglia neuroglial fate choice during early neural crest cell migration.

BACKGROUND: The dorsal root ganglia (DRG) are a critical component of the peripheral nervous system, and function to relay somatosensory information from the body's periphery to sensory perception centres within the brain. The DRG are primarily comprised of two cell types, sensory neurons and glia, both of which are neural crest-derived. Notch signalling is known to play an essential role in defining the neuronal or glial fate of bipotent neural crest progenitors that migrate from the dorsal ridge of the neural tube to the sites of the DRG. However, the involvement of Notch ligands in this process and the timing at which neuronal versus glial fate is acquired has remained uncertain. RESULTS: We have used tissue specific knockout of the E3 ubiquitin ligase mindbomb1 (Mib1) to remove the function of all Notch ligands in neural crest cells. Wnt1-Cre; Mib1fl/fl mice exhibit severe DRG defects, including a reduction in glial cells, and neuronal cell death later in development. By comparing formation of sensory neurons and glia with the expression and activation of Notch signalling in these mice, we define a critical period during embryonic development in which early migrating neural crest cells become biased toward neuronal and glial phenotypes. CONCLUSIONS: We demonstrate active Notch signalling between neural crest progenitors as soon as trunk neural crest cells delaminate from the neural tube and during their early migration toward the site of the DRG. This data brings into question the timing of neuroglial fate specification in the DRG and suggest that it may occur much earlier than originally considered.

Proteomics in Neural Crest Cell Research.

Detailed investigation of the neural crest cell proteome has lingered behind that of the transcriptome due to the challenge of obtaining sufficient starting material for subsequent proteomic investigation. Compounded by the complexity of protein abundance, large number of posttranslational modifications, and the stochastic nature of proteomics approaches, little data so far exists describing the true neural crest cell proteome. However, recent advances in instrument sensitivity and recovery of material during sample preparation have alleviated many of these problems and make proteomics analysis an underutilized tool to study neural crest cell biology. Here we present a quantitative proteomics protocol for deep analysis of both whole proteome and posttranslational modifications and a separate protocol for ultrasensitive proteomic profiling from submicrogram amounts of protein.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.