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Mouse Nr2f1haploinsufficiencyunveils newpathologicalmechanisms of a humanoptic atrophysyndrome Michele BertacchiAgnès GruartPolynikis Kaimakis2019 год

Mouse Nr2f1 haploinsufficiency unveils new pathological mechanisms of a human optic atrophy syndrome
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Аннотация: Article18 July 2019Open Access Transparent process Mouse Nr2f1 haploinsufficiency unveils new pathological mechanisms of a human optic atrophy syndrome Michele Bertacchi Corresponding Author [email protected] orcid.org/0000-0002-4402-4974 CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Search for more papers by this author Agnès Gruart Division of Neurosciences, Pablo de Olavide University, Seville, Spain Search for more papers by this author Polynikis Kaimakis Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Campus de la Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Cécile Allet Jean-Pierre Aubert Research Center (JPArc), Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Inserm, Lille, France University of Lille, FHU 1,000 Days for Health, Lille, France Search for more papers by this author Linda Serra CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Department of Biotechnology and Biological Sciences, University of Milano-Bicocca, Milano, Italy Search for more papers by this author Paolo Giacobini orcid.org/0000-0002-3075-1441 Jean-Pierre Aubert Research Center (JPArc), Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Inserm, Lille, France University of Lille, FHU 1,000 Days for Health, Lille, France Search for more papers by this author José M Delgado-García Division of Neurosciences, Pablo de Olavide University, Seville, Spain Search for more papers by this author Paola Bovolenta Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Campus de la Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Michèle Studer Corresponding Author [email protected] orcid.org/0000-0001-7105-2957 CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Search for more papers by this author Michele Bertacchi Corresponding Author [email protected] orcid.org/0000-0002-4402-4974 CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Search for more papers by this author Agnès Gruart Division of Neurosciences, Pablo de Olavide University, Seville, Spain Search for more papers by this author Polynikis Kaimakis Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Campus de la Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Cécile Allet Jean-Pierre Aubert Research Center (JPArc), Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Inserm, Lille, France University of Lille, FHU 1,000 Days for Health, Lille, France Search for more papers by this author Linda Serra CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Department of Biotechnology and Biological Sciences, University of Milano-Bicocca, Milano, Italy Search for more papers by this author Paolo Giacobini orcid.org/0000-0002-3075-1441 Jean-Pierre Aubert Research Center (JPArc), Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Inserm, Lille, France University of Lille, FHU 1,000 Days for Health, Lille, France Search for more papers by this author José M Delgado-García Division of Neurosciences, Pablo de Olavide University, Seville, Spain Search for more papers by this author Paola Bovolenta Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain CIBER de Enfermedades Raras (CIBERER), Campus de la Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Michèle Studer Corresponding Author [email protected] orcid.org/0000-0001-7105-2957 CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France Search for more papers by this author Author Information Michele Bertacchi *,1,2, Agnès Gruart3, Polynikis Kaimakis4,5, Cécile Allet6,7, Linda Serra1,8, Paolo Giacobini6,7, José M Delgado-García3, Paola Bovolenta4,5 and Michèle Studer *,1 1CNRS, Inserm, iBV, Université Côte d'Azur, Nice, France 2Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy 3Division of Neurosciences, Pablo de Olavide University, Seville, Spain 4Centro de Biología Molecular "Severo Ochoa", CSIC-UAM, Madrid, Spain 5CIBER de Enfermedades Raras (CIBERER), Campus de la Universidad Autónoma de Madrid, Madrid, Spain 6Jean-Pierre Aubert Research Center (JPArc), Laboratory of Development and Plasticity of the Neuroendocrine Brain, UMR-S 1172, Inserm, Lille, France 7University of Lille, FHU 1,000 Days for Health, Lille, France 8Department of Biotechnology and Biological Sciences, University of Milano-Bicocca, Milano, Italy *Corresponding author. Tel: +33 489150723; E-mail: [email protected] *Corresponding author. Tel: +33 489150720; E-mail: [email protected] EMBO Mol Med (2019)11:e10291https://doi.org/10.15252/emmm.201910291 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Optic nerve atrophy represents the most common form of hereditary optic neuropathies leading to vision impairment. The recently described Bosch-Boonstra-Schaaf optic atrophy (BBSOA) syndrome denotes an autosomal dominant genetic form of neuropathy caused by mutations or deletions in the NR2F1 gene. Herein, we describe a mouse model recapitulating key features of BBSOA patients—optic nerve atrophy, optic disc anomalies, and visual deficits—thus representing the only available mouse model for this syndrome. Notably, Nr2f1-deficient optic nerves develop an imbalance between oligodendrocytes and astrocytes leading to postnatal hypomyelination and astrogliosis. Adult heterozygous mice display a slower optic axonal conduction velocity from the retina to high-order visual centers together with associative visual learning deficits. Importantly, some of these clinical features, such the optic nerve hypomyelination, could be rescued by chemical drug treatment in early postnatal life. Overall, our data shed new insights into the cellular mechanisms of optic nerve atrophy in BBSOA patients and open a promising avenue for future therapeutic approaches. Synopsis This study proposes Nr2f1 heterozygous mice as a model of BBSOA syndrome, an optic nerve (ON) atrophy associated with intellectual disability. Nr2f1 mutants reveal ON hypomyelination, astrogliosis and reduced axonal conductance velocity. Treatment with Miconazole rescues ON myelination defects. Mouse Nr2f1 and human NR2F1 expression profiles are highly conserved in the developing retina and ON. Nr2f1 heterozygous and homozygous mutant mice recapitulate the human disease, displaying optic disc abnormalities, cerebral visual impairment and ON atrophy. Nr2f1 regulates retinal ganglion cell differentiation and accurate balance between ON oligodendrocytes and astrocytes. Nr2f1 modulates ON axonal conductance velocity and is involved in associative visual learning. Miconazole treatment at early postnatal ages rescues ON myelination level, hence representing a promising therapeutic approach for optic neuropathies. Introduction Optic atrophy denotes the loss of part or all the nerve fibers in the optic nerve (ON), often leading to widening of the optic cup, and represents an important sign of advanced ON disease frequently associated with gradual vision loss or reduced visual acuity. Optic neuropathies may range from non-syndromic genetic diseases to rare syndromic multisystemic disorders. The most common forms of inherited optic neuropathies, described so far, are the Leber's optic neuropathy (LHON), and the dominant optic atrophy (DOA) caused by mutations in the nuclear gene OPA1 (Carelli et al, 2017; Chun & Rizzo, 2017). Recently, patients carrying deletions or missense point mutations in the NR2F1 locus have also been diagnosed with optic atrophy associated with developmental delay and intellectual disability (Al-Kateb et al, 2013; Bosch et al, 2014; Chen et al, 2016; Kaiwar et al, 2017). This autosomal dominant disorder resulting from NR2F1 haploinsufficiency is currently named as Bosch-Boonstra-Schaaf optic atrophy (BBSOA) syndrome (OMIM: 615722). BBSOA patients display a variable array of clinical deficits, both visual and cognitive, where malformed optic disc (OD), ON atrophy, decreased visual acuity, developmental delay, epilepsy, and mild-to-moderate intellectual disability are among the most common deficiencies (reviewed in Bertacchi et al, 2018). The clinical features of BBSOA syndrome are still evolving, as the number of reported cases is continuously increasing since 2014, when the first patients with missense mutations were reported (Bosch et al, 2014; Chen et al, 2016; Kaiwar et al, 2017; Martin-Hernandez et al, 2018). This suggests that the prevalence of BBSOA syndrome might be still underestimated, which prompted us to further understand the mechanisms of this newly identified neurodevelopmental disease. NR2F1, also known as COUP-TFI, is an orphan nuclear receptor belonging to the superfamily of steroid/thyroid hormone receptors and acting as a strong transcriptional regulator (Alfano et al, 2013; Bertacchi et al, 2018). Two major homologs of this family have been identified in vertebrates: NR2F1 and NR2F2 (also named COUP-TFII; Ritchie et al, 1990; Wang et al, 1991; Alfano et al, 2013). Their molecular structure encompasses two highly conserved domains, the DNA-binding domain (DBD) and the ligand-binding domain (LBD). Most of the pathogenic mutations causing BBSOA syndrome are located in the NR2F1 DBD, hence disrupting its activity as a transcription factor, but some have been reported also in the LBD or in the start codon (ATG; Bosch et al, 2014; Chen et al, 2016; Bertacchi et al, 2018). The mouse Nr2f1 and human NR2F1 proteins are very well conserved during evolution and share 98–100% of sequence homology in both DBDs and LBDs (Bertacchi et al, 2018). Nr2f1 is widely and dynamically expressed in several mouse brain regions (Wang et al, 1991; Qiu et al, 1995; Tripodi et al, 2004; Armentano et al, 2006; Lodato et al, 2011b; Alfano et al, 2013; Flore et al, 2017; Parisot et al, 2017; Bertacchi et al, 2018), and its expression pattern seems to be well conserved in human embryos and fetuses, as recently shown (Alzu'bi et al, 2017a,b). Thus, structural and expression similarities between mouse and humans strongly suggest a conserved role of NR2F1 during development of the central nervous system (CNS). Multiple data in mice have highlighted the multi-faceted functions of Nr2f1 in the development of several mouse brain structures (Alfano et al, 2013; Bertacchi et al, 2018), but its exact role during eye development is still vague. Indeed, there is still inconsistency between the ocular phenotype obtained in mouse and the clinical features described in BSSOA patients, as the ON atrophy and cerebral visual deficits identified in several patients have not been reproduced in mice lacking solely Nr2f1 (Tang et al, 2015; Bertacchi et al, 2018). Only the combined inactivation of both homologs, Nr2f1 and Nr2f2, produced severe early ocular defects, such as coloboma and microphthalmia, suggesting a genetic compensation in the mouse (Tang et al, 2010, 2015). This is surprising since BBSOA patients are haploinsufficient for NR2F1, i.e., lack only one copy of the gene, but develop ocular impairments with high prevalence (Chen et al, 2016; Bertacchi et al, 2018). This discrepancy could depend either on species-specific functional differences or on the conditional mouse model used by Tang and colleagues (Swindell et al, 2006; Tang et al, 2010). Since genetically modified mice still offer a unique opportunity to decipher the mechanisms underlying eye development and assembly of the visual pathway, we investigated the role of Nr2f1 during visual development from the retina to the visual cortex, using heterozygotes (HET) and homozygotes (KO or null) of the constitutive mouse knock-out (KO) model (Armentano et al, 2006). We reasoned that constitutive loss of one Nr2f1 allele would better reproduce the human disease condition, in which Nr2f1 dosage is decreased in all cells and from the earliest stages of development. In this study, we report that Nr2f1/NR2F1 is expressed in the peripheral visual system in both mice and humans, particularly in cell types involved in the development and maturation of the ON, such as neural retina cells, ON astrocytes, and oligodendrocytes. Furthermore, we show that Nr2f1 HET and KO mice have clear ocular abnormalities, from OD malformations, delayed retinal ganglion cell (RGC) differentiation and apoptosis to decreased ON myelination and increased astrogliosis, resulting in reduced axonal conduction velocity from the retina to higher order centers. At adult stages, Nr2f1 HET mice have visual and associative learning deficits, reproducing in some ways the cerebral visual impairment described in patients (Bosch et al, 2014; Chen et al, 2016). Notably, Miconazole treatment in early postnatal pups rescues the ON demyelination defect, by restoring appropriate levels of oligodendrocytes, but has little effect on astrogliosis, indicating that the two events are independently controlled in this optic atrophy syndrome. Overall, we show that Nr2f1 mutant mice can be used as a model to reproduce the BBSOA syndrome and, more broadly, could serve as a tool to test possible therapeutic approaches aimed at counteracting ON neuropathies. Results NR2F1 is dynamically expressed in the mouse and human neural retina (NR) and optic nerve As most BBSOA patients develop ON atrophy, we carefully assessed NR2F1 expression in both the mouse and human developing eye and ON. As previously reported (Tang et al, 2010), the murine Nr2f1 protein is strongly expressed in both the presumptive dorsal and ventral optic stalk (OS), the precursor of the ON at embryonic day (E) 10.5, and shows a ventral-high to dorsal-low gradient in NR progenitors (Fig 1A). At E12.5, when the optic vesicle invaginates forming a bi-layered optic cup, Nr2f1 graded expression is maintained in the ventral retina, in the OS, and in the optic disc (OD; Fig 1B–B"). Nr2f1 co-localizes with virtually all Sox2+ retinal progenitors at E13.5 (Fig 1C–C") and with Brn3a+ early differentiating RGCs (Fig 1D–D"), even if at lower levels when compared to progenitors (Fig 1C'–D"). At E18.5 and postnatal (P) stages, Nr2f1 is still expressed in both Brn3a+ RGC neurons forming long-distance projections constituting the ON (Fig 1E–F"), and in residual progenitors (Sox2+ cells; Fig 1G and G'). Beside retinal neurons, Nr2f1 is also localized in astrocytes and oligodendrocytes of the ON. Between E13.5 and E18.5, Nr2f1 is expressed in virtually all Glast+/NF1A+ astrocytes of the OS/ON (Figs 1H–I" and EV1A–A"), then maintained in 70% of Glast+/NF1A+ astrocytes at P7 and P28 (Fig EV1B), and in around 80% of Sox10+ oligodendrocytes at postnatal stages (Figs 1J–K" and EV1C). In summary, Nr2f1 is highly expressed in both retinal and optic nerve components of the peripheral visual system in mouse. Figure 1. Nr2f1/NR2F1 expression during neural retina and optic nerve development in mice and humans A–B''. Nr2f1 (red) and Pax6 (green, retinal progenitors) immunofluorescences (IF) on sagittal sections of E10.5 and E12.5 mouse optic vesicles. Note the high-ventral to low-dorsal Nr2f1 gradient in the presumptive neural retina (pNR), and high Nr2f1 expression in both the presumptive dorsal and ventral optic stalk (pvOS and pdOS). Cr, crystal lens; Di, distal; Pr, proximal; presumptive retinal pigmented epithelium (pRPE). C–D". IF on E13.5 mouse eye sagittal sections with Nr2f1 (red) and Sox2 (green, progenitors in C, C') or Brn3a (green, RGCs in D, D') showing Nr2f1 expression in NR progenitors and post-mitotic RGCs (insets in C'–D"). E–F". Nr2f1 (red) and Brn3a (green) IF on E18.5 and P7 mouse eyes depicting Nr2f1 expression in virtually all RGCs in the ganglion cell layer (GCL). Arrowheads point to double-labeled cells. G, G'. Nr2f1 (red) and Sox2 (green) IF on P7 mouse retina illustrating low Nr2f1 expression in the inner nuclear layer (INL) and progenitors (NPs), and high levels in the GCL and ciliary marginal zone (CMZ). No expression in the outer nuclear layer (ONL). H–K". IF on cross-sections of E13.5 optic stalks (OS), and E18.5 and P7 optic nerves (ONs) with Nr2f1 (red) and Glast (green, astrocytic progenitors in H–H''), NF1A (green, astrocytes in I–I''), or Sox10 (green, oligodendrocyte precursors in J–K'') showing Nr2f1 expression in both astrocytic and oligodendrocytic lineages. Arrowheads point to double-labeled cells. See Fig EV1B and C for quantification. L–L". NR2F1 (red) and PAX6 (green; NR domain) IF on sagittal sections of human eye primordia at gestational week (GW) 11 illustrating high NR2F1 expression in both ON and NR cells. M–N". NR2F1 (red) and S100β (green, astrocytes) IF on cross-sections of human GW11 and GW14 ONs. Higher magnifications (M'–N") show that most of S100β+ astrocytes co-express NR2F1. Lower magnification views are shown in Fig EV1E–E". O–Q"'. NR2F1 (red) and SOX2 (green in O–P') or BRN3a (green in Q–Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''', P, P'), differentiating RGCs (Q–Q'''), and in the majority of ON astrocytic progenitors (O'). Arrowheads point to double-labeled cells. Data information: Nuclei counterstaining (blue) was obtained with DAPI. Scale bars: 50 or 100 μm for mouse and human sections, respectively. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Nr2f1/NR2F1 expression in mouse and human retina and optic nerve A–A''. Nr2f1 (red) and Glast (green, astrocyte precursors) immunofluorescences (IF) in a wild-type (WT) P0 mouse optic nerve (ON) showing that almost all Glast+ astrocytes are co-labeled with Nr2f1 (insets A', A''). B. Graph illustrating the percentage of double NF1A+/Nr2f1+ astrocytes (yellow) over the total NF1A+ population in P0, P7 and P28 mouse ONs. C. Graph showing the percentage of double Sox10+/Nr2f1+ oligodendrocytes (yellow) over the total Sox10+ population, in P0, P7 and P28 mouse ONs. D–D''. NR2F1 (red) and SOX2 (green, NR progenitors) IF of gestational week (GW) 11 human eye, showing high NR2F1 expression in all NR progenitors. GCL, ganglion cell layer; NP, neural progenitor layer; NR, neural retina. E–E''. NR2F1 (red) and S100β (green) IF in a sagittal GW14 eye section showing NR2F1 expression in almost all astrocytes of the ON (E'), in the NR and ciliary marginal zone (CMZ; E''). High-magnification views of the ON are shown in Fig 1N–N". Cr, lens crystal. F–F'''. NR2F1 (red) and SOX10 (green, oligodendrocyte precursors) IF in a sagittal GW11 human brain section (proximal ON and basal forebrain region) illustrating the presence of SOX10+ oligodendrocyte progenitors at the ventricular zone and in proximity of the ON (arrows in F'–F"'). Data information: Nuclei (blue) were stained with DAPI. In (B, C) data were normalized to the total NF1A+ or Sox10+ cells and represented as mean ± SEM; N = 3. Scale bars: 50 μm in (A), 100 μm in (D–F). Download figure Download PowerPoint Staining of NR2F1 on human embryonic sections of gestational week (GW) 11 confirmed expression conservation in both the developing neural retina (identified by the retinal marker PAX6) and the ON (Fig 1L–L"). Notably, NR2F1 is highly expressed in progenitors (SOX2+) as well as in post-mitotic differentiated retinal cells (SOX2−; Fig EV1D–D"). Along the ON, from the optic disc (distal) to the chiasm (proximal) regions, almost all cells are positive for NR2F1 and 93% of them co-express the astrocytic marker S100β (Fig 1M–N"). High co-expression of NR2F1 with SOX2 and S100β (Figs 1O–P' and EV1E–E"), as well as with BRN3a in the ganglion cell layer (GCL) of the neural retina (Fig 1Q–Q"'), is maintained at GW14, indicating that NR2F1 expression follows cell differentiation in both the NR (from progenitor to post-mitotic RGCs) and the ON. Moreover, oligodendrocytes have not reached the ON at these early stages, but can be distinguished in the preoptic area, where they are generated and co-express NR2F1 (93% double-positive cells; Fig EV1F–F"'). The high expression of NR2F1 in the human and mouse NR, OD and ON suggests a conserved role for this gene in the development of the peripheral visual system. We observed that along the human ON, NR2F1 expression levels seem even higher than in mouse (compare Fig 1I–I" with Fig 1L–L"), suggesting an important role for NR2F1 in human ON development. Optic disc malformations in Nr2f1 mutant mice In light of the early Nr2f1 expression gradient in the developing optic vesicle, key markers of proximo-distal (P-D) eye patterning, such as Pax6 and Pax2 (Schwarz et al, 2000), were first analyzed in WT, HET, and KO embryos. We found ectopic Pax6 expression in the presumptive ventral OS of HET and KO E10.5 embryos (Fig EV2A–F), as well as Pax2 downregulation and reciprocal Pax6 upregulation in invaginating E11.5 mutant optic cups (Fig EV2G–M"). Thus, differently from previous reports (Tang et al, 2010, 2015), loss of Nr2f1 alone is sufficient to affect early patterning of the developing optic vesicle, indicating that Nr2f1 can control key molecular regulators of early identity acquisition during eye development. Click here to expand this figure. Figure EV2. Nr2f1-dependent control of early OS/NR molecular domains A–C". Nr2f1 (red) and Pax6 (green, NR) IF on sagittal sections of E10.5 optic vesicles in wild-type (WT), heterozygous (HET), and full knock-out (KO) embryos. (A'–C') Note the progressive reduction of Nr2f1 protein levels from WT to KO. (A"–C") Full and empty arrowheads point to increased Pax6 expression in the ventral and dorsal regions, respectively, of the optic vesicle. D, E. Pixel intensity quantification of Pax6 and Nr2f1 IF in presumptive neural retina (pNR; D) and ventral optic stalk (vOS; E) of E10.5 embryos confirming an inverse relationship between Nr2f1 and Pax6 protein levels. Data represent the mean ± SEM; N = 4–5. F. Scheme illustrating the expansion of Pax6+ pNR domain (green) toward the pvOS domain (red) in Nr2f1 KO mutants. pdOS, presumptive dorsal optic stalk; pRPE, presumptive retinal pigmented epithelium. G. Scheme depicting an E11.5 mouse eye primordium and the structures visible after cross-section at different levels, from distal (1) to middle (2) to proximal (3). H–J''. Nr2f1 (red) and Pax6 (green, NR) IF on E11.5 WT, HET, and KO optic cups, at the levels indicated above showing the inverse correlation between Nr2f1 and Pax6 expression levels from WT to KO (arrowheads in I, J, I', J'). Note the presence of Pax6+ cells in the proximal region of the pvOS of KO animals (J''), suggesting that pNR cells substitute pvOS cells along the OS axis. K–M''. Nr2f1 (red) and Pax2 (green) IF on E11.5 WT, HET, and KO optic cups at the levels indicated above and showing reduced Pax2 expression and reciprocal Pax6 is upregulation in the ventral region of the pNR of HET and KO animals (arrowheads in L, M, M'). Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50 μm. Download figure Download PowerPoint The refinement of P-D marker expression is essential for the establishment of a structural border between OS and neural retina (NR), called the optic disc (OD), which forms at the most proximal region of the optic cup. In light of the ventral shift of the presumptive OS/NR border in Nr2f1 mutants, and of BBSOA patients displaying OD abnormalities (Bosch et al, 2014; Chen et al, 2016), we decided to closely follow the morphological and molecular development of the OD in Nr2f1 HET and KO eyes. Abnormal positioning and aberrant morphology of the presumptive OD was found in E12.5 Nr2f1 mutants (Fig 2A–B"), particularly in Nr2f1 KO optic vesicles, in which Pax2 expression is lost in the proximal OS region and ectopic Pax6+ cells appear to differentiate into Tuj1+ neurons in loco (arrowheads and inset in Fig 2C–D"). Cells positive for Pax2 remain abnormally low in the OD domain of Nr2f1 HET and KO mutants until E18.5 (Fig 2E and F) and fail to properly surround and constrain the extension of Tuj1+ axons already at E15.5 (Fig 2G–G'"). Early tissue patterning defects ultimately impinge on the final morphology and cell organization of the OD at later stages, in which Tuj1+ fibers and Brn3a+ RGC bodies remain severely misplaced in E18.5 KO fetuses (Fig 2H–I'). Together, our data show that reduced Nr2f1 expression levels affect P-D molecular patterning and lead to an abnormal OD organization from which RGC axons exit and form the ON. Interestingly, these defects are reminiscent of various OD abnormalities described in BBSOA patients, including small discs, pale discs, and disc excavations (Bosch et al, 2014; Chen et al, 2016). Figure 2. Optic disc (OD) malformations and optic nerve (ON) atrophy in Nr2f1-deficient mice A–A''. Pax2 (red, OD) and TujI (green, axons) IF on E12.5 optic cup cross-sections in wild-type (WT), heterozygous (HET), and knock-out (KO) embryos showing abnormal ODs (white dotted lines and arrowhead). B–B''. Pax6 (red, retinal progenitors) and Tuj1 (green) IF indicating ectopic Pax6+ retinal tissue in HET (B') and KO embryos (B", arrowhead). C–D". High-magnification views highlight a morphological displacement of Pax2− (C–C") and Pax6+ (D–D") cells. Arrowheads in (D', D") point to ectopic Tuj1/Pax6+ neurons. E, E'. Pax2 (red) and Tuj1 (green) IF on tangential sections of E13.5 optic cups revealing strong OD reduction in KO embryos (dotted line and arrowhead in E'), compared to WT (surrounded by a white dotted line in E). F. Histogram quantifying the gradual reduction of Pax2+ OD cells in HET and KO at different ages. G–G'''. Low (G) and high (G'–G''') magnifications of E15.5 optic cups stained for Pax2 (red) and Tuj1 (green) confirming Pax2+ reduction in HET and KO ODs and severe morphological malformations in KO (arrowheads in G'''). H–I'. Tangential (H, H') and sagittal (I, I') sections of the OD in E18.5 WT and KO retinae stained for Brn3a (red; RGCs) and Tuj1 (green; axons) highlighting the malformed OD and disorganized arrangement of RGCs in KO. In (H, H'), white dotted line delineates the borders of the OD and in (I, I') the RGC layer; red dotted line surrounds mesodermal cells forming the hyaloid vessel. Arrowheads in (I') point to ectopically placed RGCs. J–K'. IF on E13.5 optic cup cross-sections in WT and KO embryos showing increased Pax6 (green, retinal progenitors) and decreased Tuj1+ (red, differentiating cells) in the ventral retina of HET and KO embryos. Arrowhead in (J') points to increased thickness and bending of the ventral retina, while arrowheads in (K, K') indicate Tuj1+ axons exiting the OD. L. Histogram confirming the decreased number of Tuj1+ neurons in the ventral, but not dorsal, retina of Nr2f1 mutants. M–N'. Double Tuj1/Brn3a IF on E13.5 WT and KO optic cup sections revealing decreased numbers of differentiating RGCs in bending ventral retinae of KO embryos (N'). O, O'. Details of P28 WT and HET retinae indicating decreased number of Brn3a+ neurons (arrowheads) in the GCL of HET animals. P. Graph illustrating the dynamics of RGC differentiation from E13.5 to P28, normalized to WT animals. Q–R. Cross-sections of (Q–Q'') DiI-labeled P0 ONs and (R, R') Tuj1+ P8 ON fibers in WT and KO pups showing a progressive volume loss from P0 to P8. S. Quantification of the surface occupied by Tuj1+ or DiI+ fibers in different genotypes and ages, as indicated. T–U'. Cleaved Caspase3 (green, apoptotic cells) and Brn3a (red, RGCs) IF on transverse sections of E18.5 retinae in WT and KO fetuses. High magnifications (T'–T'") highlight an example of a double-labeled RGC undergoing apoptosis (arrowheads) in the ganglion cell layer (GCL). Apoptotic RGCs are increased in KO animals (arrowheads in U'), compared to WT (U). V. Histogram quantifying the number of RGC apoptotic cells in WT, HET, and KO from E18.5 to P5. Note the significant increase of dying cells at E18.5. Data information: Nuclei (blue) were stained with DAPI. In (F, L, P, S, V), data are represented as mean ± SEM; N = 3–4 for (F, L, P, V); N = 4–5 for (S). Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 50 μm. Download figure Download PowerPoint Pre- and early postnatal optic nerve defects in Nr2f1 mutants Since Nr2f1 promotes cell differentiation in different regions of the CNS (Faedo et al, 2008; Lodato et al, 2011a; Parisot et al, 2017; Bertacchi et al, 2018), we asked whether partial or complete loss of Nr2f1 function in the NR would affect cell proliferation and/or RGC differentiation (Fig 2J–V). Retinal neurons start to be generated around E11.5 in the mouse eye and express the neuron-specific β-tubulin marker Tuj1 and the RGC marker Brn3a (Heavner & Pevny, 2012). A reduction of Tuj1+ differentiating cells in the ventral but not dorsal retina was detected in E13.5 HET and KO optic cups (Fig 2J–L), and consequently, fewer axons entered the OS (arrowheads in Fig 2K and K'). Reduced rates of differentiation were most probably due to an increased number of proliferative EdU+/Ki67+ cells resulting in tissue expansion and bending (arrowhead in Fig 2J' and Appendix Fig S1A–B'). Even if these morphological deformations might resemble a colobom
Год издания: 2019
Авторы: Michele Bertacchi, Agnès Gruart, Polynikis Kaimakis, Cécile Allet, Linda Serra, Paolo Giacobini, J.M. Delgado-García, Paola Bovolenta, Michèle Studer
Издательство: Springer Nature
Источник: EMBO Molecular Medicine
Ключевые слова: Retinal Development and Disorders, Telomeres, Telomerase, and Senescence, Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Другие ссылки: EMBO Molecular Medicine (PDF)
EMBO Molecular Medicine (HTML)
HAL (Le Centre pour la Communication Scientifique Directe) (HTML)
DOAJ (DOAJ: Directory of Open Access Journals) (HTML)
Europe PMC (PubMed Central) (PDF)
Europe PMC (PubMed Central) (HTML)
DIGITAL.CSIC (Spanish National Research Council (CSIC)) (PDF)
DIGITAL.CSIC (Spanish National Research Council (CSIC)) (HTML)
PubMed Central (HTML)
PubMed (HTML)