Neurogenesis is associated with changes in the concentration of metabolites

Decrease in the number of mitochondria is thought to result from environmental changes, like hypoxia, being used as a way to adapt the cellular metabolic status. We asked whether the strong decrease in the proportion of cells that contain MitoDsRed2-labelled mitochondria (Fig. 7D) was associated with metabolic changes. ¹H Magnetic Resonance Spectroscopy (MRS) allows investigating metabolite profiles in vivo with no disturbance of the embryo. Chicken and pigeon eggs were scanned at E6 and E8 in a horizontal 14.1T/26cm Varian magnet. The eyes were recognizable by imaging of the embryo in ovo (Fig. 8A, B). Lactate, citrate and glucose were reliably quantifiable even in spectra with low SNR (Fig. 8A, B).¹H-NMR spectroscopy data show an increase in lactate concentration (CRLB <

10%) in the chick vitreous body between E6 and E8. This concentration change was confirmed by high resolution ¹H-NMR spectroscopy (Fig. 8A, D). Increased lactate concentration was not accompanied by a reduction of the chick vitreous body’s pH as indicated by the citrate chemical shift displacement from 2.597±0.001 ppm to 2.592±0.003 ppm (mean±SEM) between E6 and E8 respectively (Fig. S4 A, B). Citrate contains three carboxylic acids; its four methylene proton chemical shifts are highly pH sensitive, which allows determining quantitatively whether the pH is lower than 7.00 or not (Kedir et al., 2014). The fact that no change in lactate concentration was detected both in the whole eye in vivo (CRLB < 20%) and in retina extracts in vitro (Fig. 8B, F) raises questions about the source of lactate that accumulates in the vitreous body. Interestingly, no increase in the concentration of lactate was detected in the pigeon vitreous body between E6 and E8 (Fig. 8A, D) suggesting a species-specific metabolic shift because of the delayed neurogenesis in pigeon retina. However, we have been unable to check whether lactate concentration increased in pigeon after E8, because in ovo embryo’s movements perturbed MRS recordings. Nonetheless, our data suggest that increased lactate concentration in the chick vitreous body could reflect ongoing cell differentiation in retina, that correlates with decreased mitochondrial activity and the upregulation of the glycolysis gene Pfkfb3 (Figs. 7A, D; 8E). The possibility that increased glycolysis in retina results in the accumulation of lactate

in the vitreous body is, however, challenged by the fact that higher concentration of lactate in the vitreous body than in retina at E8 (Fig. 8D, F) could prevent its transport along a concentration gradient through passive transporters like monocarboxylate transporters (MCT) (Hertz and Dienel, 2005). The fact that citrate concentration decreased in vitreous body and eye between E6 and E8 in chick, but not in pigeon, (Fig. S4 C, D, E) suggests an increase of its oxidation by mitochondria through the TCA cycle rather than a dilution of its preexisting pool upon growth of the eye since the eye grows at a similar pace in chick and pigeon (Rodrigues et al., 2016). Decrease of citrate concentration in the chick vitreous body correlates with the much increased number of RGC axons loaded with mitochondria and growing on the basal surface (Figs. 1A, 9), hence in direct contact with the vitreous body between E6 and E8.

Figure 8: in vivo ¹H-NMR for measuring metabolite concentrations in the chick and pigeon eyes

(A) Concentration of lactate increased in the vitreous body of chick, whereas no change is detected in pigeon. (A left) In vivo spectra of the vitreous body. The sum of all the spectra per group is shown. (A

middle) T2-weighted MRI image of a chick eye and the position of the voxel of interest (yellow rectangle) for localized ¹H-MRS. (A, right) Lactate quantification of the in vivo spectra (mean±SEM;

**p<0.005; unpaired t-test; n=5-6 per group). (B) No change in lactate concentration in the whole eye.

(B, left) In vivo spectra of the eye. The sum of all the spectra per group is shown. (B, middle) T2-weighted MRI image of a chick eye and the position of the voxel of interest (yellow rectangle) for localized ¹H-MRS. (B, right) Lactate quantification of the in vivo spectra (mean±SEM, n=5-6 per group).

(C) Typical high-resolution spectrum of retina extract. Identified metabolites include: lactate, glucose, alanine, pyruvate, citrate, creatine, N-acetyl aspartate (NAA), choline containing compounds (CCC), myo-inositol, scyllo-inositol, taurine, acetate, glutamate, aspartate, γ-aminobutyric acid (GABA). The NMR 0 ppm reference used was 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). (D) Lactate quantification obtained by high resolution ¹H-MRS of metabolites extracted from the vitreous bodies (mean±SEM; **p<0.005; unpaired t-test; n=5-7 per group). (E) Lactate increase in the chick vitreous body at E8 coincides with increased expression of the phosphofructokinase (Pfkfb3) expression in retina. Accumulation of transcripts measured in whole retinas by RT-qPCR analysis. At each developmental stage, data are from three biological replicates including 3-10 retinas and presented as mean±SD. (F) Lactate quantification obtained by high resolution ¹H-MRS of metabolites extracted from the retina. (mean±SEM, n=6-8 per group).

Figure S4: ¹H-NMR for measuring metabolite concentrations in the chick and pigeon eyes (A) The pH of the embryo vitreous body remains constant over time. (B) Vitreous body pH does not get more acidic upon lactate accumulation as revealed by the center of the citrate methylene protons chemical shift. (C) In vivo and in vitro citrate of the vitreous body (*p<0.05, **p<0.005; unpaired t-test;

n=5-6 per group). (D) Vitreous body metabolite quantifications from in vitro and in vivo ¹H-MRS

(*p<0.05, **p<0.005; unpaired t-test; n=5-7 per group). (E) On the left, metabolite quantifications from the in vivo spectra of the eye. Concentrations and Cramér-Rao lower bounds are given as mean±SEM, n=5-6 per group. N.A. refers to unquantifiable metabolites. On the right, typical ¹H-MRS spectrum fit for metabolite quantification. Spectrum of chick at embryonic stage E6. Typical basis set for the eye included alanine, aspartate, choline-containing compounds (phosphorylcholine, glycerophosphorylcholine and choline), citrate, creatine+phosphocreatine, glucose, glutamate, glutamine, lactate, myo-inositol, scyllo-inositol, N-acetylaspartate, taurine, as well as main lipid resonances. (F) On the left, metabolite quantifications from the high resolution ¹H-MRS spectra of the retina extracts. Concentrations and Cramér-Rao lower bounds are given as mean±SEM, n=6-8 per group. N.A. refers to unquantifiable metabolites. On the right, resonances used for quantification of the high resolution ¹H-MRS spectra of metabolite extracts.

Figure 9: model of mitochondria activity during retina development

Numerous active mitochondria are present in actively dividing uncommitted progenitors. Pre-committed progenitors expressing high level of Hes5.3 and low level of Atoh7 show strong decrease of mitochondria activity. While mitochondrial activity is regained in committed RGCs that up-regulate Atoh7, activity is thought to remain low in other retinal cell types, and mitochondria number is thought to decrease. Oxidative phosphorylation in RGCs could be responsible for decreased citrate concentration in vitreous body, while increased glycolysis in other cell types would increase vitreous body lactate concentration.

Discussion

Mitochondrial content and activity in the developing avian retina display several unexpected features.

While the number and activity of mitochondria peaks in uncommitted early retinal progenitors, there is a significant decrease in the content and activity of mitochondria, accompanied by an increase of glycolytic activity as differentiation proceeds. Comparative analysis between chick and pigeon retinas indicate that this metabolic shift is associated with neurogenesis, irrespectively of eye growth. Analysis with high spatiotemporal resolution of distinct subsets of retinal cells revealed sharp, but transient decrease in mitochondrial activity in RGC-biased pre-committed progenitors after the activation of Hes5.3 by ATOH7. A remarkable feature of the metabolic dynamics underlying the production of avian RGCs is reflected by the fact that, in contrast to other retinal cell types, cells committed to the RGC fate recover the high number of active mitochondria that characterizes uncommitted progenitors. We show how the switch from low to high Atoh7 expression levels could play a role in the mechanism that produces differences in metabolic profiles between RGCs and other retinal cell types (Fig. 9).

Different metabolic profiles underlay the production of RGCs in chick and mouse retinas In chick and pigeon retinas, the metabolism changes that occur along the pathway converting uncommitted into RGC-biased pre-committed progenitors and finally into RGCs appear to be quite different from what happens in mouse (Esteban-Martinez et al., 2017). The fact that newly generated mouse RGCs contain fewer mitochondria than retinal neuroblasts is one out of several striking differences in the production of RGCs between birds and rodents. Esteban-Martinez et al. (2017) reported that decreased mitochondrial number in newborn mouse RGCs results from increased mitophagy. This in turn increases glycolysis and promotes RGC differentiation. In chick, the overall number of mitochondria per cell remained fairly stable during the conversion of uncommitted progenitors into newborn RGCs despite a very significant, but transient decrease in the number of active mitochondria in the subset of RGC-biased pre-committed progenitors. Our data provide, however, no evidence of ongoing mitophagy in these cells. The overall decrease in mitochondria number and activity between E6 and E8, i.e. at the peak of cell differentiation in the chick retina, correlates with an increase in the glycolytic activity, as suggested by an increase in lactate concentration in the vitreous body and the robust up-regulation of the phosphofructokinase Pfkfb3 in the retina. However, the accumulation of active mitochondria in cells committed to the RGC fate, and then in RGCs, suggest that they mostly rely on oxidative phosphorylation for energy supply. That distinct metabolic profiles underlay the production of RGCs in mouse and chick can be explained by the fact that mouse and chicken RGCs differ in many aspects. First, there are significant differences between the production of RGCs in chick and mouse, which arise, at least in part, from variations in the regulation of Atoh7 (Skowronska-Krawczyk et al., 2009). Second, the avian gene Hes5.3, which facilitates the transition of Atoh7-expressing cells from progenitors to RGCs is absent in mammals. As

a consequence, while in chick and pigeon ~80% of Atoh7⁺ pre-committed progenitors develop into RGCs, the proportion drops to ~15% in mouse where the majority of Atoh7-expressing cells become photoreceptors (Brzezinski et al., 2012; Skowronska-Krawczyk et al., 2009; Yang et al., 2003). As a result, the density of RGCs and the ratio of RGCs to photoreceptors are much higher in chicken and pigeon than in mouse (Rodrigues et al., 2016). Mammals typically have two distinct cones, whereas birds have four types of cones. Moreover, the ratio of cones to rods is much higher in birds compared to mammals. One notable exception, however, is the foveal area in primates that resembles the bird retina in many respects. Consequently, the postsynaptic wiring is expected to be more complex in birds and the far more elaborate dendritic arbors of chicken RGCs compared with mouse RGCs might reflect such an increased complexity of signal processing (Kong et al., 2005; Naito and Chen, 2004;

Querubin et al., 2009). For those various reasons, the arrays of RGC subtypes (Masland, 2001) are expected to be different in bird and mammal retinas. It is therefore not surprising that the production of RGCs may have different energy requirement in chick and mouse. We show that ATOH7 level influences the accumulation of active mitochondria in the avian retina. The comparatively lower expression of Atoh7 in mouse (Skowronska-Krawczyk et al., 2009) could account, at least in part, for the development of RGCs with different energy requirements. Comparative analysis of the transcriptomes of RGCs from retinas dominated by rods (e.g., mouse) or by cones (e.g., pigeon) should help clarify this issue in future studies.

In both chick and pigeon retina, we found that mitochondria adopt very specific distribution during RGC differentiation, with an apical accumulation before the onset of axogenesis and RGC migration to the basal side. What could be the meaning of this apical accumulation? Mitosis occurs apically in neuroepithelia and apically positioned mitochondria could facilitate ATP supply for mitotic spindle formation and myosin ring contraction to complete cytokinesis (Lawrence and Mandato, 2013). The relocalization of mitochondria in the soma while cells migrate and extend their axons could be required for actomyosin activity and microtubule dynamics (Vaarmann et al., 2016). Moreover, accumulation of organelles at the position of cell division could ensure inheritance of mitochondria in both daughter cells (Mishra and Chan, 2014). Oxygen supply is another reason that could justify mitochondria accumulation on the apical surface. Bird retinas have higher metabolic rates than mammals, but have no blood vessels; hence they have developed a specialized structure called pecten that provides oxygen from inside the vitreous body (Pettigrew et al., 1990). However, early in development, when the pecten is not yet developed, oxygen comes from the choroid, making an apical oxygen gradient plausible. Cells committed to the RGC fate up-regulate Atoh7 and pause for ~20 h on the apical surface after their ultimate mitosis (Chiodini et al., 2013). We did not observe apical accumulation of mitochondria in E14 mouse retinas, i.e., at the peak of Atoh7 expression and RGC production (Fig. S1 G). Future studies in the insipient fovea of primate embryonic retinas should help

to determine whether accumulation of mitochondria on the apical side is a specific feature of RGCs produced in cone-dominated retinal areas.

Metabolic shift between uncommitted and pre-committed retinal progenitors

The quite dramatic change in metabolic pattern that occurs between uncommitted and pre-committed progenitors and the role that ATOH7 and HES5.3 could play emphasize the concept that metabolism is developmentally regulated and has the potential to be highly dynamic during retina ontogenesis. A remarkable feature of avian retinogenesis is the high content of active mitochondria and the low production of lactate in uncommitted progenitors suggesting that they predominantly utilize oxidative phosphorylation for energy supply. The fact that progenitors divide at a high rate with cell cycles that last 11-15 hours (Chiodini et al., 2013; Rodrigues et al., 2016) justifies high energetic requirement, though progenitors need a supply of glycolytic intermediates which are essential for anabolic reactions during cell division. It appears that the sharp decrease in the number of active mitochondria that occurs during the conversion of uncommitted progenitors into RGC-biased pre-committed progenitors could reflects a general trend toward fewer mitochondria when neurogenesis begins. The fact that the strong decrease in the number of active mitochondria is transient in progenitors that enter the RGC linage points to the need for metabolic changes at the onset of neurogenesis. We can argue that a shift in metabolic activity in pre-committed progenitors probably does not result from a downturn in oxygen and substrate supply, because a large subset of uncommitted progenitors at the same location maintain a high content of active mitochondria. In view of recent findings in the field of pluripotent stem cell (PSC) biology, it is tempting to suppose that a shift towards glycolysis could be required for providing metabolites necessary for chromatin remodeling and for changes in transcription that underlie the transition from uncommitted to committed cells (Wellen et al., 2009). Metabolic reprogramming from uncommitted to pre-committed progenitors that we describe in retina resembles that between naïve and primed PSCs. However, it is far from clear why naïve PSCs predominantly utilize oxidative phosphorylation while primed PSCs utilize glycolysis (reviewed in Ryall et al., 2015). Forced expression of Hes5.3 led to a sharp decrease in active mitochondria suggesting that it could directly participate in the metabolic reprogramming that precedes cell commitment. HES5.3-mediated lengthening of the cell cycle through the downregulation of Myl10 and the inhibitory effect of HES5.3 upon Hes1 create permissive conditions for Atoh7 to upregulate, and for cells to enter the RGC lineage (Chiodini et al., 2013; Rodrigues et al., in preparation). Da Silva and Cepko (2017) show how enzymes for retinoic acid (RA) degradation, e.g., CYP26A1, favor the production of RGCs and cones in the chicken area centralis. The inhibitory effect of RA on the production of RGCs (Da Silva and Cepko, 2017) is consistent with the fact that HES5.3 activates Cyp26A1 in RGC-biased pre-committed progenitors (Chiodini et al., 2013). In neuroblastoma, as well as in several other cell types, RA was shown to increase mitochondrial membrane potential and oxidative phosphorylation (see for instance: Schneider et al., 2011). Future experiments should

100

help to determine whether HES5.3 can decrease the mitochondrial membrane potential through the activation of Cyp26A1 in pre-committed retina progenitors.

Metabolic remodeling: mitochondrial mass vs. mitochondrial activity

Mitochondria are highly dynamic and many factors can influence cellular content including biogenesis, mitophagy as well as cytokinesis itself. The number of mitochondria per cell likely reflects the fine balance between these processes. The high mitochondrial mass in early retina keeps pace with the expansion of the pool of progenitors, suggesting a good coordination between rapid cell division of progenitors and efficient mitochondria biogenesis as evidenced by the high rates of NRF1 and TFAM expression in uncommitted retinal progenitors. Our live imaging data suggest that mitochondria apportioning in daughter cells of retinal progenitors is roughly equal. However, our experimental setup does not allow us to ascertain whether daughter cells receive exactly half the content of the parent cell. Both asymmetric segregation of mitochondria in daughter cells and mitophagy could lead to unbalance in mitochondria count in progenitors that transit from an uncommitted to pre-committed state. In mouse retina, mitophagy creates this unbalance in mitochondrial mass between neuroblasts and differentiating RGCs (Esteban-Martinez et al., 2017). However, in chick, TEM analysis revealed no significant decrease of mitochondria count in Hes5.3⁺ pre-committed progenitors, suggesting that the decrease in MitoDsRed2-labelling could rather reflect a transient loss of membrane potential. The MitoDsRed2 protein is a fusion between a sequence signal from the human CytC oxidase subunit VIII and DsRed2. A membrane potential is required to send the protein to the inner mitochondrial membrane (Hood et al., 2003; Rehling et al., 2001). Therefore, it is plausible that the quasi disappearance of fluorescent mitochondria in Hes5.3⁺ cells reflects the loss of mitochondrial activity. However, this loss is transient and coincides with the expression of Hes5.3 starting few hours before the penultimate mitosis and ending ~15 hours before the terminal mitosis (Chiodini et al., 2013). There is no detectable change of mitochondrial mass during this period of less than 24 hours.

As soon as Atoh7 is upregulated and Hes5.3 is turn off, cells recover mitochondrial activity. Indeed, MitoDsRed2-labelled mitochondria are abundant in newborn RGCs where they are rapidly translocated in growing axons. We surmise that the decrease in citrate concentration in the vitreous body when the majority of RGC differentiate could reflect the transfer of this metabolite into mitochondria of growing RGC axons (Fig. 9). In addition, citrate is a potent inhibitor of a glycolytic enzyme phosphofructokinase (Moreno-Sanchez et al., 2012; Pogson and Randle, 1966). A decrease of citrate in the environment could thus contribute to favor glycolysis in surrounding tissues. While differentiation of RGCs rely on oxidative phosphorylation for energy supply, there is an overall decrease of mitochondrial mass in the retina and an accumulation of lactate in the chick vitreous body between E6 and E8 (Fig. 9). We can argue that it reflects increased glycolysis in the retina concomitant to cell differentiation. This idea is strengthened by the fact that there is no change in lactate and citrate concentration in pigeon during the same period, presumably because of the delayed cell

101

differentiation (Rodrigues et al., 2016). Kanow et al. (2017) showed that photoreceptors (mostly rods) in adult mice retina have high rates of glycolysis and export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells, suggesting that lactate production could be a general feature of retina and demonstrating metabolic heterogeneity between cell types. However, it remains to be seen whether cone-dominated retinas display similar properties. Moreover, we do not yet understand how lactate produced in the chick retina could accumulate in the vitreous body against its concentration gradient. Even if our data do not provide functional metabolic fluxes quantification, they confirm the appearance of a metabolic switch leading to an unbalance between aerobic glycolysis and oxidative phosphorylation when retinal cells differentiate.

102

Materials and methods

Animals

Chick embryos from White Leghorn strain (UNIGE Animal Resources Centre) were staged according to Hamburger and Hamilton (1951). Fertilised pigeon eggs were supplied by Philippe Delaunay (Pigeonneau de la Suisse Normande, Croisilles, France). Experimental procedures were carried out in accordance with Federal Swiss Veterinary Regulations. Eyes were dissected in DPBS (ThermoFisher) and the surrounding retinal pigment epithelium (RPE) was removed.

Dans le document Atoh7 controls mitochondrial activity along the pathway converting pre-committed progenitors into retinal ganglion cells in retina adapted for high acuity vision (Page 91-116)