Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic ...plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.
The Reprimo (
) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (
) embryogenesis, and primarily ...locates in the nervous system. Its most characterized member,
, which duplicated to give rise
and
in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three
genes (
, and
l) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish,
mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE),
is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas
is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of
has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of
gene products during zebrafish and mouse development as a first step to approach the physiological role of the
gene family.
Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the ...accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.
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•CHAPS forms smaller micelles allowing full permeabilization of aged human organs•SHANEL enables centimeters deep molecular labeling and clearing of whole human organs•SHANEL renders intact adult human brain and kidney transparent•Deep learning and light-sheet microscopy with SHANEL allows human organ mapping
Zhao et al. present an approach for intact human organ mapping that uses a new tissue permeabilization method to clear and deeply label whole organs followed by light-sheet microscopy imaging and a deep learning-based pipeline for 3D reconstruction and data analysis.
Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through ...commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.
The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show ...that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.
Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of ...the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.
The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative ...p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.
The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF) and surrounded by neuroepithelial cells. Several lines of evidence ...suggest that the eCSF contains diffusible factors regulating the survival, proliferation, and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. In addition, SCO-spondin is required for the correct morphogenesis of the posterior commissure and pineal gland. Because SCO-spondin is secreted by the diencephalon, we sought to corroborate the long-range function of this protein in vitro by performing gain and loss of function experiments on mesencephalic explants. We find that culture medium enriched in SCO-spondin causes an increased neurodifferentiation of explanted mesencephalic region. Conversely, inhibitory antibodies against SCO-spondin cause a reduction in neurodifferentiation and an increase of mitosis when such explants are cultured in eCSF. Our results suggest that SCO-spondin is a crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.
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