Neutrophil immune profile guides spinal cord regeneration in zebrafish.

Spinal cord injury triggers a strong innate inflammatory response in both non-regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet, their role in the repair process, particularly in a regenerative context, remains largely unknown. Here, we show that, following rapid recruitment to the injured spinal cord, neutrophils mostly reverse migrate throughout the zebrafish body. In addition, promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil-specific RNA-seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf-α expression in macrophage/microglia populations. Conversely, blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether, these findings provide new insights into the role of neutrophils in spinal cord regeneration, emphasizing the significant impact of their immune profile on the outcome of the repair process.

Mouse Spinal Cord Vascular Transcriptome Analysis Identifies CD9 and MYLIP as Injury-Induced Players.

Traumatic spinal cord injury (SCI) initiates a cascade of cellular events, culminating in irreversible tissue loss and neuroinflammation. After the trauma, the blood vessels are destroyed. The blood-spinal cord barrier (BSCB), a physical barrier between the blood and spinal cord parenchyma, is disrupted, facilitating the infiltration of immune cells, and contributing to a toxic spinal microenvironment, affecting axonal regeneration. Understanding how the vascular constituents of the BSCB respond to injury is crucial to prevent BSCB impairment and to improve spinal cord repair. Here, we focus our attention on the vascular transcriptome at 3- and 7-days post-injury (dpi), during which BSCB is abnormally leaky, to identify potential molecular players that are injury-specific. Using the mouse contusion model, we identified Cd9 and Mylip genes as differentially expressed at 3 and 7 dpi. CD9 and MYLIP expression were injury-induced on vascular cells, endothelial cells and pericytes, at the injury epicentre at 7 dpi, with a spatial expression predominantly at the caudal region of the lesion. These results establish CD9 and MYLIP as two new potential players after SCI, and future studies targeting their expression might bring promising results for spinal cord repair.

Identification of Dmrt2a downstream genes during zebrafish early development using a timely controlled approach.

BACKGROUND: Dmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: left-right asymmetry, synchronisation of the somite clock genes and fast muscle differentiation. Despite the described functions, Dmrt2a mechanism of action is unknown. Therefore, with this work, we propose to identify Dmrt2a downstream genes during zebrafish early development. RESULTS: We generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines. We characterised dmrt2a overexpression phenotype and verified that it was very similar to the one described after knockdown of this gene, with left-right asymmetry defects and desynchronisation of somite clock genes. Additionally, we identified a new phenotype of somite border malformation. We generated several dmrt2a mutant lines, but we only detected a weak to negligible phenotype. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. We found that dmrt2b does not seem to compensate the lack of dmrt2a. Furthermore, we took advantage of one of our mutant lines to confirm dmrt2a morpholino specificity, which was previously shown to be a robust knockdown tool in two independent studies. Using the described genetic tools to perform and validate a microarray, we were able to identify six genes downstream of Dmrt2a: foxj1b, pxdc1b, cxcl12b, etv2, foxc1b and cyp1a. CONCLUSIONS: In this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. The identified genes will be crucial to the future understanding of Dmrt2a mechanism of action in zebrafish.

The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1.

Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

A Comparative Analysis of the Venom System between Two Morphotypes of the Sea Anemone Actinia equina.

The current study investigates the venom-delivery system of green and red morphotypes of the sea anemone Actinia equina to disclose its potential as a source of bioactive compounds. We compared the two morphotypes using electron and optical microscopy, proteomics, and toxicity assessment on zebrafish embryos. Specialized venom-injecting cells (nematocysts) are equally distributed and found in the tentacles of both varieties. Proteomics revealed proteins of interest in both red and green Actinia, yielding the three most abundant Gene Ontology (GO) terms related to the biological processes "proteolysis", "hemolysis in another organism" and "lipid catabolic process". Neurotoxins and cytolytic toxins similar to known cnidarian toxins like PsTX-60A and AvTX-60A, for instance, were identified in both types. Extracts from green and red anemones were toxic to zebrafish embryos, with green anemone venom appearing to be more potent. The findings highlight the presence of proteinaceous toxins in A. equina and the potential for different varieties to possess distinct bioactive compounds. Notably, pore-forming toxins are suggested for molecular probes and immunotoxins, making them valuable assets for potential biotechnological and biomedical purposes.

Notch signalling regulates left-right asymmetry through ciliary length control.

The importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer's vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer's vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury.

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Development and repair of blood vessels in the zebrafish spinal cord.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

An amputation resets positional information to a proximal identity in the regenerating zebrafish caudal fin.

BACKGROUND: Zebrafish has emerged as a powerful model organism to study the process of regeneration. This teleost fish has the ability to regenerate various tissues and organs like the heart, spinal cord, retina and fins. In this study, we took advantage of the existence of an excellent morphological reference in the zebrafish caudal fin, the bony ray bifurcations, as a model to study positional information upon amputation. We investigated the existence of positional information for bifurcation formation by performing repeated amputations at different proximal-distal places along the fin. RESULTS: We show that, while amputations performed at a long distance from the bifurcation do not change its final proximal-distal position in the regenerated fin, consecutive amputations done at 1 segment proximal to the bifurcation (near the bifurcation) induce a positional reset and progressively shift its position distally. Furthermore, we investigated the potential role of Shh and Fgf signalling pathways in the determination of the bifurcation position and observed that they do not seem to be involved in this process. CONCLUSIONS: Our results reveal that, an amputation near the bifurcation inhibits the formation of the regenerated bifurcation in the pre-amputation position, inducing a distalization of this structure. This shows that the positional memory for bony ray bifurcations depends on the proximal-distal level of the amputation.

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock.

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.

Activation of Nkx2.5 transcriptional program is required for adult myocardial repair.

The cardiac developmental network has been associated with myocardial regenerative potential. However, the embryonic signals triggered following injury have yet to be fully elucidated. Nkx2.5 is a key causative transcription factor associated with human congenital heart disease and one of the earliest markers of cardiac progenitors, thus it serves as a promising candidate. Here, we show that cardiac-specific RNA-sequencing studies reveal a disrupted embryonic transcriptional profile in the adult Nkx2.5 loss-of-function myocardium. nkx2.5-/- fish exhibit an impaired ability to recover following ventricular apex amputation with diminished dedifferentiation and proliferation. Complex network analyses illuminate that Nkx2.5 is required to provoke proteolytic pathways necessary for sarcomere disassembly and to mount a proliferative response for cardiomyocyte renewal. Moreover, Nkx2.5 targets embedded in these distinct gene regulatory modules coordinate appropriate, multi-faceted injury responses. Altogether, our findings support a previously unrecognized, Nkx2.5-dependent regenerative circuit that invokes myocardial cell cycle re-entry, proteolysis, and mitochondrial metabolism to ensure effective regeneration in the teleost heart.

DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential.

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential.

The right time for senescence.

Cellular senescence is a highly complex and programmed cellular state with diverse and, at times, conflicting physiological and pathological roles across the lifespan of an organism. Initially considered a cell culture artifact, senescence evolved from an age-related circumstance to an intricate cellular defense mechanism in response to stress, implicated in a wide spectrum of biological processes like tissue remodelling, injury and cancer. The development of new tools to study senescence in vivo paved the way to uncover its functional roles in various frameworks, which are sometimes hard to reconcile. Here, we review the functional impact of senescent cells on different organismal contexts. We provide updated insights on the role of senescent cells in tissue repair and regeneration, in which they essentially modulate the levels of fibrosis and inflammation, discussing how "time" seems to be the key maestro of their effects. Finally, we overview the current clinical research landscape to target senescent cells and contemplate its repercussions on this fast-evolving field.

Targeting senescent cells improves functional recovery after spinal cord injury.

Persistent senescent cells (SCs) are known to underlie aging-related chronic disorders, but it is now recognized that SCs may be at the center of tissue remodeling events, namely during development or organ repair. In this study, we show that two distinct senescence profiles are induced in the context of a spinal cord injury between the regenerative zebrafish and the scarring mouse. Whereas induced SCs in zebrafish are progressively cleared out, they accumulate over time in mice. Depletion of SCs in spinal-cord-injured mice, with different senolytic drugs, improves locomotor, sensory, and bladder functions. This functional recovery is associated with improved myelin sparing, reduced fibrotic scar, and attenuated inflammation, which correlate with a decreased secretion of pro-fibrotic and pro-inflammatory factors. Targeting SCs is a promising therapeutic strategy not only for spinal cord injuries but potentially for other organs that lack regenerative competence.

Induced pluripotent stem cell-derived vascular networks to screen nano-bio interactions.

The vascular bioactivity/safety of nanomaterials is typically evaluated by animal testing, which is of low throughput and does not account for biological differences between animals and humans such as ageing, metabolism and disease profiles. The development of personalized human in vitro platforms to evaluate the interaction of nanomaterials with the vascular system would be important for both therapeutic and regenerative medicine. A library of 30 nanoparticle (NP) formulations, in use in imaging, antimicrobial and pharmaceutical applications, was evaluated in a reporter zebrafish model of vasculogenesis and then tested in personalized humanized models composed of human-induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) with "young" and "aged" phenotypes in 3 vascular network formats: 2D (in polystyrene dish), 3D (in Matrigel) and in a blood vessel on a chip. As a proof of concept, vascular toxicity was used as the main readout. The results show that the toxicity profile of NPs to hiPSC-ECs was dependent on the "age" of the endothelial cells and vascular network format. hiPSC-ECs were less susceptible to the cytotoxicity effect of NPs when cultured in flow than in static conditions, the protective effect being mediated, at least in part, by glycocalyx. Overall, the results presented here highlight the relevance of in vitro hiPSC-derived vascular systems to screen vascular nanomaterial interactions.

Author Correction: A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Low doses of ionizing radiation enhance angiogenesis and consequently accelerate post-embryonic development but not regeneration in zebrafish.

Low doses of ionizing radiation (LDIR) activate endothelial cells inducing angiogenesis. In zebrafish, LDIR induce vessel formation in the sub-intestinal vessels during post-embryonic development and enhance the inter-ray vessel density in adult fin regeneration. Since angiogenesis is a crucial process involved in both post-embryonic development and regeneration, herein we aimed to understand whether LDIR accelerate these physiological conditions. Our data show that LDIR upregulate the gene expression of several pro-angiogenic molecules, such as flt1, kdr, angpt2a, tgfb2, fgf2 and cyr61in sorted endothelial cells from zebrafish larvae and this effect was abrogated by using a vascular endothelial growth factor receptor (VEGFR)-2 tyrosine kinase inhibitor. Irradiated zebrafish present normal indicators of developmental progress but, importantly LDIR accelerate post-embryonic development in a VEGFR-2 dependent signaling. Furthermore, our data show that LDIR do not accelerate regeneration after caudal fin amputation and the gene expression of the early stages markers of regeneration are not modulated by LDIR. Even though regeneration is considered as a recapitulation of embryonic development and LDIR induce angiogenesis in both conditions, our findings show that LDIR accelerate post-embryonic development but not regeneration. This highlights the importance of the physiological context for a specific phenotype promoted by LDIR.

Differential requirements for COPI transport during vertebrate early development.

The coatomer vesicular coat complex is essential for normal Golgi and secretory activities in eukaryotic cells. Through positional cloning of genes controlling zebrafish notochord development, we found that the sneezy, happy, and dopey loci encode the alpha, beta, and beta' subunits of the coatomer complex. Export from mutant endoplasmic reticulum is blocked, Golgi structure is disrupted, and mutant embryos eventually degenerate due to widespread apoptosis. The early embryonic phenotype, however, demonstrates that despite its "housekeeping" functions, coatomer activity is specifically and cell autonomously required for normal chordamesoderm differentiation, perinotochordal basement membrane formation, and melanophore pigmentation. Hence, differential requirements for coatomer activity among embryonic tissues lead to tissue-specific developmental defects. Moreover, we note that the mRNA encoding alpha coatomer is strikingly upregulated in notochord progenitors, and we present data suggesting that alpha coatomer transcription is tuned to activity- and cell type-specific secretory loads.

A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

Spinal cord injury (SCI) is a complex condition, with limited therapeutic options, that results in sensory and motor disabilities. To boost discovery of novel therapeutics, we designed a simple and efficient drug screening platform. This innovative approach allows to determine locomotor rescue properties of small molecules in a zebrafish (Danio rerio) larval spinal cord transection model. We validated our screening platform by showing that Riluzole and Minocycline, two molecules that are in clinical trials for SCI, promote rescue of the locomotor function of the transected larvae. Further validation of the platform was obtained through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Importantly, we identified Tranexamic acid and further showed that this molecule maintains its locomotor recovery properties in a rodent female contusion model. Our screening platform, combined with drug repurposing, promises to propel the rapid translation of novel therapeutics to improve SCI recovery in humans.

Fine-tuning of fgf8a expression through alternative polyadenylation has a selective impact on Fgf-associated developmental processes.

The formation of distinct 3'UTRs through alternative polyadenylation is a mechanism of gene expression regulation that has been implicated in many physiological and pathological processes. However, its functions in the context of vertebrate embryonic development have been largely unaddressed, in particular with a gene-specific focus. Here we show that the most abundant 3'UTR for the zebrafish fgf8a gene in the developing embryo mediates a strong translational repression, when compared to a more sparsely used alternative 3'UTR, which supports a higher translational efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we show a temporally and spatially specific impact of fgf8a 3'UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. These results reveal a critical functional importance of gene-specific alternative 3'UTRs in vertebrate embryonic development.