A gradient in glucose metabolism regulates hair cell morphology along the tonotopic axis 1 of the developing cochlea 2

61 In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such 62 that complex sounds are broken down into their component frequencies along the basal-to-apical long 63 (tonotopic) axis. Acquisition of frequency-specific morphologies at the appropriate positions along the 64 chick cochlea, the basilar papilla (BP), requires that nascent HCs determine their tonotopic positions 65 during development. The complex signalling within the auditory organ between the developing HC 66 and its local niche along the axis is currently poorly understood. Here we apply NAD(P)H fluorescence 67 lifetime imaging (FLIM) to reveal metabolic gradients along the tonotopic axis of the developing BP. 68 Re-shaping these gradients during development, by inhibiting different branch points of glucose 69 catabolism, alters normal Bmp7 and Chordin like-1 signalling leading to flattening of tonotopic 70 patterning and hair cell morphology along the axis. These findings indicate a causal link between 71 morphogen signalling and metabolic reprogramming in specifying tonotopic morphologies in 72 developing auditory HCs. 73 cell subtypes are determined as a function of morphogen concentration at different 897 positions along them 42,66,67 . We showed previously that reciprocal gradients in Bmp7 and Chd-l1 set 898 up positional identity in HCs along the developing BP 17 . The gradient of Bmp7 is regulated by Shh 899 emanating from the distal BP region 68 . We here identify a proximal-to-distal gradient in glucose 900 metabolism that regulates the morphological properties associated with HCs at different positions 901 along the tonotopic axis. Disrupting this gradient using 2-DOG, SAM or 6-AN mimics the effects of 902 altered Shh, Bmp, or Chd-l1 signalling 17,68 , inducing distal-like HC phenotypes in the proximal BP region. Our findings therefore indicate a causal link between developmental and metabolic pathways along the tonotopic axis during HC formation.

shunting carbons back to glycolysis at glyceraldehyde 3-phosphate and fructose 1,6-bisphosphate. The PPP is a 117 major regulator for cellular redox homeostasis.

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We have previously demonstrated that fluorescence lifetime imaging microscopy (FLIM) can be 123 utilised as a label-free means to identify metabolic alterations in the cell types of the inner ear by 124 spatially resolving differences in the fluorescence decay of the reduced redox cofactors nicotinamide 125 adenine dinucleotide (NADH) and its phosphorylated analogue NADPH (Figure 1

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TMRM is a cell permeable dye that reports mitochondrial membrane potential (mt), giving a direct

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. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2022. ; https://doi.org/10.1101/2022.04.11.487851 doi: bioRxiv preprint

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. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in        for this gradient, and investigate metabolic differences along the tonotopic axis, we exploited existing 423 transcriptional data sets for proximal and distal regions of the developing BP 17 . Data were analysed 424 for differential expression of metabolic mRNAs involved in NADPH regulation at E6.5 and E14 ( Figure   425 5 A). Prior to mRNA isolation for bulk RNA-seq and Affymetrix microarray analysis, BPs were separated 426 into proximal, middle, and distal thirds 17 . Microarray data were analysed to identify transcripts with 427 expression levels at least two-fold higher in the proximal compared to distal half of the BP. From the 428 combined data sets, we identified multiple genes with differential expression between proximal and 429 distal regions involved in regulating cellular NADPH and cellular redox ( Figure 5A).

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Overall, metabolic genes showing the highest differential expression between proximal and distal 432 regions were those involved in cytosolic glycolysis and redox homeostasis ( Figure 4A) and would be 433 consistent with the higher bound reported for the proximal BP region ( Figure 2I). However, as these 434 expression data were obtained from bulk RNA-seq analysis, they do not provide the necessary spatial

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Cytosolic glucose metabolism is necessary for tonotopic patterning in the chick cochlea.

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Since we could not detect an obvious transcriptional basis for the proximal-distal gradient in NADPH,

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we sought to further characterise metabolism in the developing BP by investigating a functional role 452 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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in the BP between E6.5 and E8 17 . To determine whether cytosolic glucose metabolism acts during this 561 same developmental window, we blocked HK activity for defined periods during BP development.

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Explants were established at E6.5 and treated with 2-DOG + sodium pyruvate for either 24 or 48 hours 563 followed by wash out with control medium. These treatments correspond to the developmental 564 window (E6.5-E8) described previously for refinement of tonotopic morphologies along the proximal-  to-distal gradient in NADPH/NADH (bound) observed at E6 and E9 ( Figure 2I).

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To further confirm a role for glucose catabolism in establishing HC positional identity, we employed a 572 second method of modulating its activity that is independent of HK 39 . Glycolysis can also be blocked   CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2022. ; https://doi.org/10.1101/2022.04.11.487851 doi: bioRxiv preprint  . We therefore sought to further dissect the metabolic signalling network during specification of

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However, contrary to all other inhibitor treatments, YZ9 was unique because it did not produce a 662 significant interaction term in our 2-way ANOVA (Figure 6 Supplement 1C, supplementary table 1).

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Because the interaction term indicates the detection of differences in the proximal-distal gradient that 664 are induced by YZ9 treatment relative to the control group, the result demonstrates that YZ9 is 665 significantly less disruptive to the proximal-distal gradient than 2-DOG, SAM and 6-AN treatments. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted September 23, 2022. ; https://doi.org/10.1101/2022.04.11.487851 doi: bioRxiv preprint    . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in