Mitochondria in the RGC lineage - Atoh7 controls mitochondrial activity along the pathway conve

High concentration of mitochondria at the apical pole of retinal cells appears to be a general trend at the onset of neurogenesis. To determine whether this localization was related to cell cycle exit and cell differentiation, we monitored in real time the distribution of mitochondria in single Atoh7⁺ cells before their terminal mitosis (Fig. 3A cell 1; Movie S2). Apical accumulation of mitochondria roughly coincided with the upregulation of Atoh7 that occurs 8-15 h before the ultimate mitosis and marks the transition from pre-committed progenitors to cells committed to the RGC fate (Fig. 4A; Chiodini et al., 2013). During the initial phase of neurogenesis, ATOH7 accumulates at low levels in a fraction of progenitors and activates transcription of the gene encoding the bHLH transcription factor HES5.3.

HES5.3-mediated lengthening of the cell cycle is a prerequisite for cells to upregulate Atoh7 and to enter the RGC lineage. As progenitors enter the late phase, Hes5.3 is down-regulated and early RGC markers like the nicotinic acetylcholine receptor beta3 subunit (Chrnb3) are activated (Chiodini et al., 2013). To monitor mitochondria accumulation at the different stages along the pathway converting progenitors into newborn RGCs, E5 (HH25-26) retinas were electroporated with Atoh7-GFP, Hes5.3-GFP, Atoh7-RFP or Chrnb3-GFP in combination with CMV-MitoDsRed2 or CMV-MitoGFP reporter plasmid. Retinas were dissociated 8, 24 or 48 h later and selected by fluorescence-activated cell sorting (FACS). The subset of cells identified with GFP comprises pre-committed and committed Atoh7-expressing cells and only ~35% of cells in this subset contained MitoDsRed2-labelled mitochondria (Fig 4B, C). Interestingly, ~95% of cells expressing Atoh7 at high level and identified with Atoh7-RFP (Chiodini et al., 2013) contained fluorescent mitochondria (Fig. 4B, C). Consistent with the fact that these cells are committed and express early RGC markers, ~85% of cells identified with Chrnb3-GFP displayed mitochondrial labeling (Fig 4B, C). In contrast, in pre-committed progenitors identified with Hes5.3-GFP, fluorescent mitochondria were detected only in ~10% of cells 8 h after electroporation (Fig. 4B, C). This short incubation period was sufficient to label mitochondria and was selected on purpose, as it diminished the probability of counting cells that may have already entered the RGC lineage. To determine whether the ~9-fold increase in the proportion of cells with fluorescent mitochondria between pre-committed progenitors and cells committed to the RGC fate was reflecting higher mitochondria content, we measured the ratio of mitochondrial DNA (mt-DNA) to genomic DNA (g-DNA) (Fig. 4D) as follow: E5 retinas were co-electroporated with Atoh7-GFP and Atoh7-RFP

plasmids, retinas were dissociated 24 h later and two subsets of cells were selected by FACS. A subset of GFP⁺ cells that did not express RFP comprised Atoh7⁺ pre-committed progenitors, while the subset of RFP⁺ cells consisted of cells already committed to the RGC fate. Newborn RGCs and pre-committed progenitors were selected using, respectively, Chrnb3-GFP and Hes5.3-GFP. Quantitative PCR (qPCR) analysis with targets on mt-DNA and g-DNA revealed similar ratios of mt-DNA relative to g-DNA in progenitors and newborn RGCs, while this ratio was slightly higher in cells committed to the RGC fate (Fig. 4D). Lower ratio of mt-DNA to g-DNA in RGCs compared to committed cells could be explained by the fact that mitochondria, which were already located in RGC axons (Fig. 1B-D), were lost during cell dissociation and cell sorting. The small increase in the amounts of mt-DNA between pre-committed progenitors and pre-committed cells did not match the spectacular increase in the proportion of cells labelled with fluorescent mitochondria. Nonetheless, we wondered whether augmentation of mt-DNA in committed cells resulted from stimulation of mitochondria biogenesis. To test this idea, E5 retinas were co-electroporated with Atoh7-RFP and Atoh7-GFP and retinas were dissociated 24 h later. RFP⁺ cells and GFP⁺/RFP^- cells were separated by FACS and RNA was isolated and processed for RT-qPCR (Fig. 4E). Hes5.3 and the mitotic marker Kif11 were expressed at lower levels in RFP⁺ cells, while these cells displayed stronger expression of RGC markers such as Chrnb3 and stathmin 2 (Stmn2). This molecular signature confirmed that our assay accurately discriminated between pre-committed progenitors and cells pre-committed to the RGC fate. The upregulation of Atoh7 in pre-committed cells was not detected in this assay, because at the time of RNA isolation, a fraction of GFP⁺ cells have upregulated Atoh7, while on the other side, Atoh7 was already turned off in a fraction of RFP⁺ cells (Chiodini et al., 2013). Expression of PGC-1, Nrf1 and Tfam, i.e., the main regulators of mitochondria biogenesis, were down-regulated in RFP⁺ cells. Thus, it appears that the increase in mt-DNA content in committed cells (Fig. 4D) is related to the lengthening of the terminal cell cycle and cell cycle exit (Chiodini et al., 2013) rather than caused by mitochondria biogenesis (Fig. 4E).

Figure 4: mitochondria in pre-committed progenitors and in cells committed to the RGC fate (A) Schematic view of the time-course of Hes5.3 and Atoh7 expression during the penultimate and ultimate cell cycles. (B-D) E5 chick retinas were co-electroporated with reporter plasmids to identify different cell subsets. (B) Retinas were disaggregated 8 to 48 h later and subsets of cells were selected

by FACS before confocal imaging. (C) The proportions±SD of cells with MitoDsRed2- or MitoGFP-labelled mitochondria for each population. 12 to 16 retinas were pooled for each condition and processed as one biological replicate (Atoh7-GFP n=1879 cells, Hes5.3-GFP n=3531, Atoh7-RFP n=287, Chrnb3-GFP n=1571, CMV-GFP n=3837). All pairwise comparison show p<0.001 (Chi-square test). (D) The ratios±SD of mt-DNA to g-DNA from biological triplicate, each including 20-30 retinas, and with 4 targets on mt-DNA, (*p<0.05; unpaired t-test; n=3). (E) E5 retinas from biological triplicates, each including 30 retinas, were co-electroporated with Atoh7-RFP and Atoh7-GFP. Cells were dissociated 24 h later. RFP⁺ cells and GFP⁺, RFP^- cells were separated by FACS. RNA was isolated and processed for RT-qPCR. Histogram shows mean±SD (*p<0.05, **p<0.01; unpaired t-test; n=3).

5. HES5.3 decreases the number of active mitochondria in pre-committed progenitors The activation of Hes5.3 by ATOH7 marks the onset of neurogenesis and the transition from uncommitted to pre-committed progenitor cells. Hes5.3 is turn on at E4 in proliferating progenitors and turn off 8 to 15 hours before the ultimate mitosis (Fig. 5B inset; Chiodini et al., 2013). We monitored the proportion of Hes5. 3⁺ cells labelled with fluorescent mitochondria at E4, E5 and E6 (Fig. 5A, B). The fraction of double-labelled cells was the highest at E4, but it rapidly decreased between E4 and E5 and it reached a very low level at E6. In contrast, the proportion of uncommitted progenitors identified with Chrna7-GFP and containing fluorescent mitochondria was maintained at a high level during the same period (Fig. 5D). We wondered whether the ~10-fold decrease in the proportion of MitoDsRed2⁺ cells between E4 and E6 reflected a blockage in the import of MitoDsRed2 by mitochondria, or a decrease of mitochondria number. To address this issue, we did morphometric measurements on electron microscopy images of Hes5.3⁺ cells selected by FACS at E4 and E6 (Figs. 5C;

S3 C). Analysis at the single cell level did not reveal significant change in the total mitochondrial area per cell between E4 and E6 (Fig. 5C middle panel). A modest increase in the number of mitochondria (Fig. 5C left panel) and a decrease in the average area of individual mitochondria at E6 (Fig. 5C right panel) suggest that mitochondrial fragmentation could occur in Hes5.3⁺ cells. Likewise, we noted a slight decrease in the distance between mitochondria but no change in the circularity (Fig. S3 F, G).

Overall, mitochondria density increased because of reduced cytoplasmic areas (Fig. S3 D, E). Labeling mitochondria with MitoTracker Green FM revealed no change of mitochondrial mass in Hes5.3⁺ cells from E4 to E5, and a modest decrease from E5 to E6 (Fig. S3 A, B). Finally, we compared the expression of genes involved in mitochondria biogenesis and mitophagy in cells identified with Hes5.3-GFP at E4 and E5 (Fig. 5E). The accumulation of Hes5.3 and Atoh7 transcripts was significantly lower at E4 than at E5 confirming that our assay discriminated between early and late Hes5.3⁺ pre-committed progenitors. The master regulator of mitochondria biogenesis PGC1 was modestly down-regulated at E5, while Nrf and Tfam, and the mitochondrial autophagy receptor Nix remained unchanged. Taken

together our data suggest that the spectacular decrease in the abundance of MitoDsRed2-labelled mitochondria in Hes5.3⁺ cells was not matched by a decrease in the number of mitochondria per cell.

Inhibition of MitoDsRed2 import could result from a loss of membrane potential leading to a decrease in mitochondrial activity (Hood et al., 2003; Rehling et al., 2001). To determine whether HES5.3 plays a role in this process, E5 chick retinas were electroporated with CMV-MitoDsRed2 and a β actin-Hes5.3:GFP expression vector or a CMV-GFP control vector. Forced expression of Hes5.3 led to a significant decrease in the proportion of cells with MitoDsRed2-labelled mitochondria 24 h later (Fig.

5D). Hes5.3 is one of the earliest genes activated by ATOH7, and while transcription of the Atoh7 gene is repressed by the Notch effector HES1 (Hairy1, ENSGALG00000002055) in early retina, HES5.3 has an inhibitory effect on Hes1 (Hernandez et al., 2007; Matter-Sadzinski et al., 2005; Rodrigues et al., in preparation). Consistent with the functional properties of this transcriptional network, the forced expression of HES1 in a subset of cells identified with Hes5.3-GFP at E5 led to an increase in the proportion of MitoDsRed2-positive cells 8 h later (Fig. 5F). We conclude that ATOH7, through the activation of Hes5.3, impedes MitoDsRed2 import by mitochondria in pre-committed progenitors.

Figure 5: MitoDsRed2-labelled mitochondria content vs mitochondria count in Hes5.3⁺ pre-committed progenitors

(A, B) Decrease in the proportion of Hes5.3⁺ progenitors with fluorescent mitochondria. E4 (HH22-23), E5 (HH25-26) and E6 (HH28-29) retinas were co-electroporated with Hes5.3-GFP and CMV-MitoDsRed2. (A) Retinas (4 for each condition, processed as one biological replicate) were disaggregated 8 h later and GFP⁺ cells were selected by FACS for confocal imaging. (B) The proportions

of Hes5.3⁺ cells with MitoDsRed2-labelled mitochondria are shown. (***p<0.001; Chi square test; E4 n=1126 cells, E5 n=3953, E6 n=3965). (B, inset) Accumulation of Hes5.3 transcripts measured by RT-qPCR analysis as mean±SD from biological triplicates (C) Morphometric measurements of mitochondria in individual Hes5.3⁺ progenitors. E4 and E6 retinas (52 at E4; 16 at E6) were electroporated with Hes5.3-GFP. Dissociated GFP⁺ cells were selected by FACS 8 h later and centrifuged. The pellet was processed for TEM. Boxplot show the number of individual mitochondria per cell (left), the total mitochondrial area per cell (center), and the average size of individual mitochondria (right). The box comprises median (central line) surrounded by 25^th and 75^th percentiles, and mean indicated as cross. Whiskers show min and max values, and dots show all values including outliers (left p=0.0158, center p=0.7431, right p=0.0008; unpaired t-test; E4 n=51 cells, E6 n=55). (D) Effect of Hes5.3 overexpression on the accumulation of MitoDsRed2-labelled mitochondria. E5 or E6 retinas (4-8 for each condition, processed as one biological replicate) were electroporated with reporter plasmids or with a β actin-Hes5.3:GFP expression vector. Dissociated GFP⁺ cells were selected by FACS. The proportions of cells with MitoDsRed2-labelled mitochondria are shown as the mean±SD.

All pairwise comparisons with p<0.001 (Chi square test, Chrna7-GFP n=164 cells, Hes5.3-GFP n=3965, CMV-GFP n=8480, β actin-Hes5.3:GFP n=1421). (E) Transcript profiles in Hes5.3⁺ progenitors. E4 and E5 retinas (20 at E4; 8 at E5) were electroporated with Hes5.3-GFP. Retinas were disaggregated 8 h later, GFP⁺ cells were sorted by FACS and RNA was isolated and processed for RT-qPCR. Data are from three biological replicates, and presented as mean±SD, (*p<0.05, **p<0.01; unpaired t-test; n=3). (F) Effect of Atoh7 inhibition on the accumulation of MitoDsRed2-labelled mitochondria in pre-committed progenitors. E5 retinas (10 for each condition) were electroporated with Hes5.3-GFP alone or in combination with a Hes1 expression vector (EMSV-Hes1). Dissociated GFP⁺ cells were selected by FACS.

The proportions of Hes5.3⁺ progenitors with MitoDsRed2-labelled mitochondria are presented as mean±SD, (***p<0.001; Chi square test; ctrl n=4993 cells, EMSV-Hes1 n=5605).

85 Figure S3: mitochondria in Hes5.3⁺ progenitors

(A, B) E4, E5 and E6 retinas (2 for each condition, pooled and processed as one biological replicate) were electroporated with Hes5.3-RFP and stained with MitoTracker Green FM 24 h later. (A) Fluorescence intensity proportional to total mitochondrial mass per cell reported as boxplot with median (central line), mean (cross), 25^th and 75^th percentiles, min and max as whiskers, as well as internal and outliers as dots (E4 vs E5 p=n.s., E5 vs E6 p<0.01; unpaired t-test; E4 n=135 cells, E5 n=631, E6 n=629). (B) An example of confocal image used for the quantifications shown in panel A. (C-G) Hes5.3-GFP⁺ cells were selected by FACS, pelleted and processed for TEM. (C) An example of a TEM image processed for quantifications shown in panels D-G and Fig. 7C. Yellow arrowheads point to regions enriched with mitochondria. (D-G) Morphometric measurements of mitochondria in individual Hes5.3⁺ progenitors (E4 n=51 cells, E6 n=55). (D) The average cytoplasmic area is significantly lower at E6 than at E4 (p<0.001; unpaired t-test; n=51-55 cells). (E) Difference in panel D is consistent with the

increased proportion of cytoplasm filled with mitochondria at E6 (p<0.001; unpaired t-test; n=51-55 cells). (F) Average distance between mitochondria in µm, measured as the distance from centers of fitted ellipses (p<0.05; unpaired t-test; n=51-55). (G) Mitochondria circularity, measured as the ratio of major to minor lengths of fitted ellipse, remains constant (p=n.s.; unpaired t-test; n=51-55).

6. Active mitochondria in cells that misexpress Atoh7

While fluorescent mitochondria decreased in pre-committed cells expressing Hes5.3, 90% of cells identified with the Atoh7-RFP reporter, i.e., cells expressing Atoh7 at high level, contain MitoDsRed2-labelled mitochondria (Figs. 4C; 5D). The up-regulation of Atoh7 marks the transition from Hes5.3⁺ pre-committed progenitors to cells committed to the RGC fate, and Hes5.3 is no longer expressed in these cells (Chiodini et al., 2013). The forced expression of Atoh7 lead to a modest increase in the proportion of RGCs (Liu et al., 2001). The reason might be that only cells that passed through the stage of pre-commitment can be induced by the forced expression of Atoh7 to enter the RGC lineage. This could explain why a late target of Atoh7 like Chrnb3 was downregulated in cells that misexpressed Atoh7 (Fig. 6D). Likewise, we asked whether cells that misexpress Atoh7 have a higher content of active mitochondria. Forced expression of Ngn2 or Atoh7 in E5 retinas induced strong ectopic activity of the Atoh7 promoter region (Fig. 6A; Matter-Sadzinski et al., 2005; Skowronska-Krawczyk et al., 2004) and led to an increase in the proportion of cells that contain MitoDsRed2-labelled mitochondria (Fig. 6B, C). In these cells, like in RGC-committed cells expressing Atoh7 at high level (Fig. 4E), genes involved in mitochondria biogenesis were not activated (Fig. 6D). It appears that a high content of active mitochondria and the expression of Atoh7 at high levels are not sufficient for cells to enter the RGC lineage. It strengthens the idea that Atoh7⁺ cells must pass through the stage of pre-commitment during which they express Hes5.3 and have a low content of active mitochondria, before they commit to the RGC fate.

Figure 6: ATOH7 and NGN2 promote the accumulation of MitoDsRed2-labelled mitochondria (A-C) E5 retinas were electroporated with Atoh7-GFP and CMV-mito-DsRed2 in combination with expression vectors. Each condition represents 10 pooled retinas processed as one biological replicate.

Cells were dissociated 24 h later. (A) Proportion of cells that up-regulate Atoh7 as mean±SD, (***p<0.001; Chi square test; ctrl n=4692 cells, EMSV-Atoh7 n=7241, EMSV-Ngn2 n=13’228). (B) Proportion of Atoh7⁺ cells with MitoDsRed2-labelled mitochondria as mean±SD, (***p<0.001; Chi square test; ctrl n=4652, EMSV-Atoh7 n=7240, EMSV-Ngn2 n=12’839). (C) Confocal images of cells electroporated with Atoh7-GFP and CMV-MitoDsRed2. (D) Effects of Atoh7 misexpression on gene expression. E5 retinas were electroporated with Atoh7-GFP alone or in combination with an Atoh7 expression vector (EMSV-Atoh7). Retinas were disaggregated 24 h later, GFP⁺ cells were selected by FACS and RNA was isolated and processed for RT-qPCR. Data are from three biological replicates, each

including 20-30 retinas, and presented as mean±SD, (*p<0.05, **p<0.01, ***p<0.001; unpaired t-test;

n=3).

7. Mitochondria content decreases as development proceeds

Current knowledge suggests that proliferating embryonic cells rely mainly on glycolysis to produce their energy, while upon differentiation cells switch to oxidative phosphorylation for increasing efficiency in energy production (Folmes et al., 2012a). However, the fact that uncommitted progenitors have high content of active mitochondria (Fig. 5D) suggests that the situation could be different in the avian retina. Only a few quantifications of mitochondria in developing retina have been reported so far (Buono and Sheffield, 1991; Ruggiero and Sheffield, 1998). We measured the mitochondria content in the developing chick and pigeon retinas. In both species, the ratio of mt-DNA to g-DNA steadily decreased between E3 and E15 (Fig. 7A). However, the ~4-fold decrease between E4 and E8 in both species was relatively modest in comparison with the >100-fold increase in the total number of cells in chick and pigeon retinas during the same period (Rodrigues et al., 2016) indicating that mitochondria biogenesis keeps pace with the rapid expansion of the pool of retinal progenitors in both species. The steady decrease of the ratios of mt-DNA to g-DNA between E3 and E7 is consistent with the slight decrease in expression of genes involved in mitochondria biogenesis during this period (Fig. 7E). The modest increase in the level of Nix2 transcript between E3 and E7 confirms that mitophagy does not play a major role during this period. At E15, retinal cells complete their differentiation and the ratios of mt-DNA to g-DNA are ~4-fold (chick) and ~9-fold (pigeon) smaller than in early progenitors (Fig. 7A). The decrease measured between E7 and E15 reflects, at least in part, the translocation of mitochondria in RGC axons forming the optic nerve. In pigeon, the ratio of mt-DNA to g-mt-DNA increased ~5-fold between E15 and one-month old pigeon and remained stable up to 6 years (Fig. 7B). Interestingly, in cells identified with Atoh7-RFP, i.e., cells committed to the RGC fate and newborn RGCs, the ratio of mt-DNA to g-DNA was in the same range than in early progenitors (Fig. 7C). This result indicates that the mitochondria content is higher in RGCs than in other retinal cell types. In pigeon retina, neurogenesis starts 3 days later than in chick (Rodrigues et al., 2016).

Consistent with these different schedules, in chick there was a ~10-fold decrease in the fraction of MitoDsRed2-labelled cells between E3 and E6, while in pigeon, high proportions of cells with fluorescent mitochondria were maintained up to E6 (Fig. 7D). This interspecies comparison demonstrates that the sharp decrease in the proportion of cells with MitoDsRed2-labelled mitochondria is related to neurogenesis rather than to eye growth.

Figure 7: mitochondrial content in the chick and pigeon retinas

(A-C) Ratio±SD of mt-DNA to g-DNA during retina development from biological triplicates with 4 targets (chick) or 2 targets on mt-DNA (pigeon). Mitochondrial DNA and genomic DNA isolated from:

(A) whole embryonic chick and pigeon retinas, (B) whole adult pigeon retinas, 1M = 1 month, 6Y = 6

years, (*p<0.05, ***p<0.001; unpaired t-test; n=3), (C) whole E2.5 (HH18) chick retina and a subset of chick cells identified with Atoh7-RFP and selected by FACS (p=0.134; unpaired t-test; n=3). (D) Proportion±SD of cells that contain MitoDsRed2-labelled mitochondria during development of the chick and pigeon retinas, including 3-10 pooled retinas for each condition and processed as one biological replicate. All pairwise comparisons give p<0.001 (Chi square test; Chick E3 n=370 cells, E4 n=9754, E5 n= 8480, E6 n=4109; Pigeon E4 n=827, E5 n=4861, E6 n=3808, E7 n=2516). (E) Accumulation of transcripts measured by RT-qPCR analysis during development of the chick retina. At each developmental stage, data are from three biological replicates including 3-10 retinas and presented as mean±SD.

8. 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

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