Differences in neural stem cell identity and differentiation capacity drive divergent regenerative outcomes in lizards and salamanders

While lizards and salamanders both exhibit the ability to regenerate amputated tails, the outcomes achieved by each are markedly different. Salamanders, such as Ambystoma mexicanum, regenerate nearly identical copies of original tails. Regenerated lizard tails, however, exhibit important morphological differences compared with originals. Some of these differences concern dorsoventral patterning of regenerated skeletal and spinal cord tissues; regenerated salamander tail tissues exhibit dorsoventral patterning, while regrown lizard tissues do not. Additionally, regenerated lizard tails lack characteristically roof plate-associated structures, such as dorsal root ganglia. We hypothesized that differences in neural stem cells (NSCs) found in the ependyma of regenerated spinal cords account for these divergent regenerative outcomes. Through a combination of immunofluorescent staining, RT-PCR, hedgehog regulation, and transcriptome analysis, we analyzed NSC-dependent tail regeneration. Both salamander and lizard Sox2+ NSCs form neurospheres in culture. While salamander neurospheres exhibit default roof plate identity, lizard neurospheres exhibit default floor plate. Hedgehog signaling regulates dorsalization/ventralization of salamander, but not lizard, NSCs. Examination of NSC differentiation potential in vitro showed that salamander NSCs are capable of neural differentiation into multiple lineages, whereas lizard NSCs are not, which was confirmed by in vivo spinal cord transplantations. Finally, salamander NSCs xenogeneically transplanted into regenerating lizard tail spinal cords were influenced by native lizard NSC hedgehog signals, which favored salamander NSC floor plate differentiation. These findings suggest that NSCs in regenerated lizard and salamander spinal cords are distinct cell populations, and these differences contribute to the vastly different outcomes observed in tail regeneration.

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Control of dorsoventral pattern in vertebrate neural development: induction and polarizing properties of the floor plate

Development ◽

10.1242/dev.113.supplement_2.105 ◽

1991 ◽

Vol 113 (Supplement_2) ◽

pp. 105-122 ◽

Cited By ~ 18

Author(s):

Marysia Placzek ◽

Toshiya Yamada ◽

Marc Tessier-Lavigne ◽

Thomas Jessell ◽

Jane Dodd

Keyword(s):

Neural Tube ◽

Neural Development ◽

Cell Types ◽

Floor Plate ◽

Neural Cells ◽

Dorsoventral Patterning ◽

Cellular Mechanisms ◽

Cell Position

Distinct classes of neural cells differentiate at specific locations within the embryonic vertebrate nervous system. To define the cellular mechanisms that control the identity and pattern of neural cells we have used a combination of functional assays and antigenic markers to examine the differentiation of cells in the developing spinal cord and hindbrain in vivo and in vitro. Our results suggest that a critical step in the dorsoventral patterning of the embryonic CNS is the differentiation of a specialized group of midline neural cells, termed the floor plate, in response to local inductive signals from the underlying notochord. The floor plate and notochord appear to control the pattern of cell types that appear along the dorsoventral axis of the neural tube. The fate of neuroepithelial cells in the ventral neural tube may be defined by cell position with respect to the ventral midline and controlled by polarizing signals that originate from the floor plate and notochord.

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Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord

Development ◽

10.1242/dev.126.16.3649 ◽

1999 ◽

Vol 126 (16) ◽

pp. 3649-3659

Author(s):

M.P. Matise ◽

M. Lustig ◽

T. Sakurai ◽

M. Grumet ◽

A.L. Joyner

Keyword(s):

Spinal Cord ◽

Critical Role ◽

Floor Plate ◽

Posterior Commissure ◽

Ventral Midline ◽

Mouse Spinal Cord ◽

Commissural Axons ◽

Midline Cells

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(−/−) embryos. Netrin1 mRNA expression was detected in Gli2(−/−) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(−/−) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(−/−) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(−/−) embryos, although they are less well organized in Gli2(−/−)mutants.

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Perfecting the Imperfect: Introducing dorsoventral patterning into adult regenerating lizard tails with gene-edited embryonic neural stem cells.

10.21203/rs.3.rs-142271/v1 ◽

2021 ◽

Author(s):

Thomas Lozito ◽

Ricardo Londono ◽

Aaron Sun ◽

Megan Hudnall

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Hedgehog Signaling ◽

Regenerative Process ◽

Dorsoventral Patterning ◽

Tail Regeneration ◽

Neural Tubes ◽

Roof Plate ◽

Lepidodactylus Lugubris

Abstract Lizards are able to regrow amputated tails, but the lizard tail regenerative process fails to recapitulate the dorsoventral patterning achieved during embryonic tail development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. Embryonic NTs are, themselves, dorsoventrally patterned, with Pax7+ Shh- dorsal roof plate domains that restrict cartilage skeletal formation induced by Pax7- Shh+ floor domains to ventral tail regions. However, adult regenerated tail ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tube skeletons. Both NTs and ETs contain populations of neural stem cells (NSCs), but only embryonic NSC populations are able to differentiate into roof plate identities and neurons. Embryonic NSCs transplanted into regenerated tail ETs retain the capacity to form roof domains but are ultimately ventralized by the unchecked hedgehog signaling of regenerated lizard tail environments. We hypothesized that only the simultaneous repression of hedgehog signaling and enhancement of NCS roof plate differentiation capacity would induce patterning in lizard ETs and, hence, regenerated cartilage. This was tested through the use of a novel genetic engineering process in which NSCs are isolated from embryos of the parthenogenetic lizard Lepidodactylus lugubris, gene-edited in vivo, and implanted back into clonally-identical adults to regulate tail regeneration. Embryonic lizard NSC lines unresponsive to hedgehog stimulation were generated through the use of CRISPR/Cas9 technologies to knockout (KO) the signaling regulator smoothened (Smo). Exogenous Smo KO NSCs were injected into adult tail spinal cords, where they engrafted to endogenous ependymal cell populations and contributed to dorsal domains in regenerated tail ETs. Embryonic Smo KO NSCs maintained roof plate identities in vivo, and lizards treated with edited NSCs regrew tails that lacked cartilage in dorsal regions. These studies represent an important milestone in the creation of the first regenerated lizard tails with dorsoventrally patterned ETs and skeletal tissues.

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Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing

Genes & Development ◽

10.1101/gad.268086.115 ◽

2015 ◽

Vol 29 (24) ◽

pp. 2617-2632

Author(s):

Berenice Hernandez-Enriquez ◽

Zhuhao Wu ◽

Edward Martinez ◽

Olav Olsen ◽

Zaven Kaprielian ◽

...

Keyword(s):

Spinal Cord ◽

Floor Plate ◽

Axon Pathfinding ◽

Developmentally Regulated ◽

Commissural Axon ◽

Commissural Axons ◽

Receptor Complexes ◽

Midline Crossing

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

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A Collagen-Based Scaffold for Promoting Neural Plasticity in a Rat Model of Spinal Cord Injury

Polymers ◽

10.3390/polym12102245 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2245

Author(s):

Jue-Zong Yeh ◽

Ding-Han Wang ◽

Juin-Hong Cherng ◽

Yi-Wen Wang ◽

Gang-Yi Fan ◽

...

Keyword(s):

Spinal Cord ◽

Spinal Cord Injury ◽

Rat Model ◽

Neural Plasticity ◽

Collagen Scaffold ◽

Glial Scar ◽

Beneficial Effects ◽

Cord Injury

In spinal cord injury (SCI) therapy, glial scarring formed by activated astrocytes is a primary problem that needs to be solved to enhance axonal regeneration. In this study, we developed and used a collagen scaffold for glial scar replacement to create an appropriate environment in an SCI rat model and determined whether neural plasticity can be manipulated using this approach. We used four experimental groups, as follows: SCI-collagen scaffold, SCI control, normal spinal cord-collagen scaffold, and normal control. The collagen scaffold showed excellent in vitro and in vivo biocompatibility. Immunofluorescence staining revealed increased expression of neurofilament and fibronectin and reduced expression of glial fibrillary acidic protein and anti-chondroitin sulfate in the collagen scaffold-treated SCI rats at 1 and 4 weeks post-implantation compared with that in untreated SCI control. This indicates that the collagen scaffold implantation promoted neuronal survival and axonal growth within the injured site and prevented glial scar formation by controlling astrocyte production for their normal functioning. Our study highlights the feasibility of using the collagen scaffold in SCI repair. The collagen scaffold was found to exert beneficial effects on neuronal activity and may help in manipulating synaptic plasticity, implying its great potential for clinical application in SCI.

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Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Cell Transplantation ◽

10.1177/0963689720978219 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097821

Author(s):

Andrea Tenorio-Mina ◽

Daniel Cortés ◽

Joel Esquivel-Estudillo ◽

Adolfo López-Ornelas ◽

Alejandro Cabrera-Wrooman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Neural Induction ◽

Human Keratinocytes ◽

Differentiation Potential ◽

Neuronal Proteins ◽

Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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Apoptosis in metanephric development.

The Journal of Cell Biology ◽

10.1083/jcb.119.5.1327 ◽

1992 ◽

Vol 119 (5) ◽

pp. 1327-1333 ◽

Cited By ~ 200

Author(s):

C Koseki ◽

D Herzlinger ◽

Q al-Awqati

Keyword(s):

Spinal Cord ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phorbol Esters ◽

Dna Degradation ◽

Morphological Evidence ◽

Metanephric Mesenchyme ◽

Embryonic Spinal Cord

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

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