Eph/Ephrin Signaling Controls Progenitor Identities In The Ventral Spinal Cord
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In the vertebrate spinal cord, motor neurons (MN) are generated in stereotypical numbers from a pool of dedicated progenitors (pMN) whose number depends on signals that control their specification but also their proliferation and differentiation rates. Although the initial steps of pMN specification have been extensively studied, how pMN numbers are regulated over time is less well characterized.
Here, we show that ephrinB2 and ephrinB3 are differentially expressed in progenitor domains in the ventral spinal cord with several Eph receptors more broadly expressed. Genetic loss-of-function analyses show that ephrinB2 and ephrinB3 inversely control pMN numbers and that these changes in progenitor numbers correlate with changes in motor neuron numbers. Detailed phenotypic analyses by immunostaining and genetic interaction studies between ephrinB2 and Shh indicate that changes in pMN numbers in ephrin mutants are due to alteration in progenitor identity at late stages of development.
Altogether our data reveal that Eph:ephrin signaling is required to control progenitor identities in the ventral spinal cord.
KeywordsEphrins Neural tube Progenitors Fate Motor neurons Sonic hedgehog Mouse
In vertebrates, motor neurons (MN) innervating skeletal muscles are born in the ventral neural tube, the future spinal cord, from a pool of progenitors located in the ventricular zone. As for other neuronal subtypes, the production of stereotyped numbers of MN requires the integration of different processes such as specification and proliferation of progenitors, followed by cell cycle exit and differentiation . While these processes are common to the genesis of all neuronal subtypes throughout the central nervous system, one distinguishing feature of MN development is the fact that progenitor specification is dependent on their spatial organization within the neural tube. Indeed, the vertebrate neural tube is organized along its dorsoventral axis in different progenitor domains which first give rise to distinct neuronal subtypes and later on to subtypes of glial cells. Combinatorial positional information provided by graded Sonic Hedgehog (Shh), Wnt, BMP and FGF signaling induces the regionalized expression of homeodomain and helix-loop-helix identity transcription factors (iTFs) in different progenitor domains . For instance, progenitors of motor neurons (pMNs) express the iTF Olig2 while adjacent progenitors (p3) which give rise to v3 interneurons express the iTF Nkx2.2. Ventral patterning of the spinal cord, including specification of pMN and p3 progenitors, is controlled by the morphogen Sonic Hedgehog (Shh) produced by the notochord and the floor plate . In addition to doses, different exposure times to Shh modulates the expression of these iTFs in progenitors, thus specifying the distinct progenitor identities [3, 4]. Specifically, the progressive emergence of a gene regulatory network (GRN) composed of three transcription factors- Pax6, Olig2 and Nkx2.2 whose expression is refined by cross-repressive interactions interprets graded Shh signaling, to control the size of the p3 and pMN progenitor domains [5, 6]. Although early steps of progenitor specification are fairly well characterized, mechanisms that ensure stereotypy in the size of progenitor domains as the tissue grows are less well understood. These mechanisms include maintenance of progenitor identities [4, 7, 8] as well as control of proliferation and differentiation rates that vary between progenitor types and over time . In addition, mechanisms such as cell sorting have been shown to participate in defining and/or maintaining domain boundaries thus indirectly contributing to pattern progenitor domains [10, 11, 12, 13].
Eph:ephrin signaling is a cell-to-cell communication pathway that has been implicated in numerous developmental processes [14, 15]. A distinctive feature of Eph:ephrin signaling is its ability to trigger forward signaling downstream of Eph receptors and reverse signaling downstream of ephrins. One of its major biological functions is to control cell adhesion and repulsion events in developing and adult tissues thus leading to the establishment and/or maintenance of axon tracts, tissue organization and patterning [16, 17]. In addition, Eph:ephrin signaling has been shown to control various aspects of neural progenitor development and homeostasis in the developing and adult mammalian cortex including self-renewal, proliferation, quiescence and differentiation . In the developing spinal cord, the role of Eph:ephrin signaling has been prominently studied in post-mitotic neurons, specifically in axon guidance and fasciculation of MNs [19, 20, 21], as a consequence, virtually nothing is known on the function of this pathway in spinal progenitors. Here, we show that two B-class ephrins, ephrinB2 and ephrinB3 are differentially expressed in pMN and p3 progenitors. Loss of function analyses indicate that expression of ephrinB2 and ephrinB3 is not required for initial specification of these progenitors. However, at later developmental stages, expression of ephrinB2 and ephrinB3 is essential to maintain appropriate numbers of pMN and p3 progenitors. Interestingly, ephrinB2 and ephrinB3 mutants exhibit opposite phenotypes, matching their opposite differential expression patterns. Detailed analyses of ephinB2 mutants indicate that the change in pMN number is not due to a change in proliferation or differentiation rates. Rather, our data shows that Efnb2 interacts with Shh to control the ratio between pMN and p3 progenitors. Lastly, loss of ephrinB3 -but not ephrinB2- leads to pMN and p3 progenitor intermingling. Altogether our data suggests that Eph:ephrin signaling plays a role in controlling progenitor identity.
Ephrin mutant mice were maintained in a mixed background and genotyped by PCR. The mouse lines Shh ko , Efnb3 ko , Efnb2 lox and Efnb2 GFP have been described previously [22, 23, 24]. The Olig2-Cre mouse line  was maintained in a pure C57Bl6/J genetic background. For Efnb2 cKO, control genotypes used in the study include Efnb2 lox/lox , Efnb2 lox/GFP , Efnb2 +/GFP and Efnb2 +/GFP ; Olig2-Cre. For Efnb3 KO, control genotypes are always Efnb3 +/- . E0.5 is defined as the day on which a vaginal plug was detected.
In Situ Hybridization
In situ hybridization was performed using standard protocols on 70μm vibratome sections at brachial level. Antisense RNA probes labeled with digoxigenin were used to detect in vivo gene expression with a 72 h incubation time.
All analyses for Efnb2 cKO were performed on control and mutant littermates collected from at least two different litters. On the other hand, control and Efnb3 mutant embryos were collected from independent litters. The number of embryo analyzed for each immunostaining and each developmental stage is indicated in the figure legends. To avoid bias in rostro-caudal axis, data was collected on thick vibratome sections covering the entire brachial region (600 μm). Antibody staining was performed following standard protocol on 70μm vibratome sections of mouse embryos at brachial level. For BrdU incorporation, pregnant dams were injected with BrdU (10mg/ml; 100mg/kg) with intraperitoneal injection. After 1 h, embryos were dissected in cold PBS and processed for subsequent immunostaining.
Antibodies used were: goat anti-Nkx2.2 (1/100, Santa Cruz Biotechnology); rabbit anti-Olig2 (1/1000, Sigma); mouse anti-Islet1/2, 39-4D5 (1/50, DSHB); rabbit anti-Foxp1 (1/200, Abcam), rabbit anti-P-H3 (1/1000, Millipore), rabbit anti-EphA4 (1/100, Santa Cruz Biotechnology), goat anti-EphB2 (1/50, R&D Systems), Tuj1 (1/1000, Covance). All secondary antibodies were from Jackson ImmunoResearch (1/1000).
Image processing and quantification
Images were collected on a Leica SP5 confocal microscope or Nikon eclipse 80i microscope for in situ hybridization data. Cell numbers were collected blindly on 5 vibratome sections (n=25 confocal Z-sections) per embryo and at least 2000 nuclei were recorded per embryo. The number of embryo analyzed for each immunostaining and each developmental stage is indicated in the figure legends. Acquisitions of nuclei 2D positions and semi quantitative analyses of fluorescence intensity were performed using Fiji . Spatial distribution of progenitor subtypes was quantified using the R Project (http://www.r-project.org/), see Additional file 1: (Sup Code) for details on the code.
For all analyses sample size was estimated empirically. Sample sizes are indicated in Figure legends and further details are provided in Additional file 2: Table S1. Statistical analyses were performed with GraphPad, using Mann-Whitney-Wilcoxon test or ANOVA, depending on the data set. P<0.05 was considered statistically significant.
EphrinB2 and ephrinB3 exhibit restricted expression in progenitors of the ventral spinal cord.
EphrinB2 controls the number of pMN and their progeny
Efnb2 interacts with Shh to control the ratio between pMN and p3 progenitors
EphrinB3 inversely controls the ratio between pMN and p3 identities
While early steps of ventral pMN specification have been extensively studied, highlighting the critical role of Shh, mechanisms that control the number of ventral progenitors over time are less well characterized. Here we show that Eph:ephrin signaling is required to precisely control the number of pMN (and p3) progenitors at late stages of development (after E9.5). It has been proposed previously that the modulation of p3 and pMN numbers after E9.5 is driven mainly by differentiation and/or proliferation, which vary over time and according to progenitor types . Despite the fact that a number of studies in the developing and adult cortex have shown a role for Eph:ephrinB signaling in controling the balance between proliferation and differentiation of neural progenitors in the cerebral cortex , our data unexpectedly show that changes in pMN progenitor numbers in ephrin mutants are not due to alterations of proliferation or differentiation rates.
As a consequence of these changes in progenitor identities, Efnb2 and Efnb3 mutants exhibit opposite alterations in MN numbers at E12.5. It would be interesting to assess whether these changes are still present postnataly, however, postnatal changes in MN number may be due to distinct mechanisms, for instance a decrease in MN number associated with cell death at later stages than those analyzed here has been described in EphA4 -/- mutants .
Traditionally, the role of Eph:ephrin signaling in specification processes has been linked to its function in boundary maintenance. For instance, a recent study has shown that loss of ephrinB2 in the developing cochlea leads to a switch in cell identity from supporting cell to hair cell fate and this was attributed to the mis-positioning of supporting cells into the hair cell layer . No mis-positioning of p3 and pMN progenitors was observed in the Efnb2 cKO mutants analyzed here. Whether excision of Efnb2 in all neural tube progenitors would lead to a similar phenotype remains an open question. Here, we observed mis-positioning of progenitors only in ephrinB3 mutants although both ephrinB2 and ephrinB3 mutants exhibited changes in p3 and pMN progenitor ratio, indicating that resolution of identity is independent of mis-positioning. This is consistent with a growing number of published studies reporting a role for Eph:ephrin signaling in lineage commitment or cell fate maintenance via the modulation of intracellular signal transduction pathways and gene expression, independently of cell sorting at boundaries [31, 32, 33, 34, 35, 36]. Another possibility, consistent with the genetic interaction between Efnb2 and Shh, could be that ephrins impact on Shh signal transduction cascade as was recently described for Notch [37, 38]. In fact, genetic interaction between Shh and cell surface proteins has been reported previously and such studies identified Gas1, Cdo and Boc as components of the Shh signaling pathway [7, 39, 40]. In this context, it would be interesting to test for a genetic interaction between Efnb3 and Shh in the control of p3 and pMN progenitor identity and positioning.
In conclusion, our study shows that ephrinB2 and ephrinB3 are required to control progenitor identities in the ventral spinal cord and suggests a role for Eph:ephrin signaling in refining morphogen-dependent tissue patterning.
The Islet 1/2 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Iowa City, IA 52242. We thank Dr Novitch for sharing the Olig2-Cre mice. Dr Kania and Dr Henkemeyer provided some molecular reagents used in this study. We are grateful to Brice Ronsin for his help with confocal microscopy (TRI Imaging Core Facility) and to Marion Aguirrebengoa for her help with statistical analyses. We thank the ABC facility and ANEXPLO for housing mice. We are grateful for Sylvain Touret’s help for writing the R code. We thank Eric Agius, Serge Plaza and Alain Vincent for critical reading of the manuscript.
Research in the Davy team is financed by the CNRS, by the Fondation ARC and by ANR (ANR-15-CE13-0010-01). JL received support from the French Ministère de l’Enseignement Supérieur et de la Recherche and from the Fondation pour la Recherche Médicale (FDT20140931010). DJL is funded by the Miami Project to Cure Paralysis and PAN by NIH/NINDS (NS089325).
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.
JL planned, performed and analyzed experiments, and he participated in writing the manuscript; AK, CA and NE performed experiments; PA and DL collected and provided mutant embryos and revised the manuscript; CS provided scientific input on the project and revised the manuscript; AD supervised the project, planned the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare no conflict of interest.
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All animal procedures were approved by the institution ethical committee (protocol number: APAFIS#1289-2015110609133558 v5).
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