Crosstalk between glucose metabolism and morphogen signalling specifies tonotopic identity in developing hair cells

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along the proximal-to-distal long (tonotopic) axis. Acquisition of frequency-specific morphologies at the appropriate positions along the chick cochlea, the basilar papilla (BP), requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between the developing HC and its local niche along the axis is currently poorly understood. Here we apply NAD(P)H fluorescence lifetime imaging (FLIM) to reveal metabolic gradients along the tonotopic axis of the developing BP. Re-shaping these gradients during development, by inhibiting different branch points of cytosolic glucose catabolism, alters normal morphogen signalling and abolishes tonotopic patterning, normalising the graded differences in hair cell morphology along the BP. These findings highlight a causal link between morphogen signalling and metabolic reprogramming in specifying tonotopic identity in developing auditory HCs.


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Hearing relies upon the life-long function of mechanosensory hair cells (HCs) and their associated glial-32 like supporting cells (SCs) within the cochlea. In both mammals and birds, different frequencies 33 stimulate HCs located at different positions along the basal-to-apical long axis of the auditory 34 epithelium to separate complex sounds into their spectral components. This phenomenon, known as 35 tonotopy, underlies our ability to differentiate between the high pitch of a mosquito and the low 36 rumbling of thunder. The specific factors regulating the development of tonotopy remain, largely, 37 unclear. As high frequency HCs show increased vulnerability to insults, including aging 1 , noise damage 38 2,3 and ototoxicity 4 , awareness of the mechanisms underlying the formation of frequency-specific HC 39 properties is crucial to understanding both acquired auditory defects, HC repair and regeneration.

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Enhanced knowledge of the pathways that drive specification of HC phenotypes at different frequency 41 positions could identify novel strategies to preserve and restore high frequency hearing loss.

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Reprogramming between glycolytic and oxidative pathways has also been reported in developing 50 tissues , including migratory neural crest cells 8 , the zebrafish otic vesicle 9 , trophectoderm in the 51 mouse embryo 10 and the presomitic mesoderm [11][12][13][14] . Nevertheless, a regulatory role for metabolism 52 has not been explored in the context of cell fate and patterning in developing inner ear epithelia. This 53 is, in part, because the classic biochemical approaches from which our knowledge of metabolism has 54 formed involve the destructive extraction of metabolites from a sample. Probing metabolism in this 55 manner, although valuable, means that any spatial organisation of metabolic pathways in complex 56 tissues is lost. As the cochlea contains multiple cell types, investigating the regulation of their 57 development by metabolism requires experimental approaches capable of interrogating metabolic 58 pathways in live preparations with single cell resolution.

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We have previously demonstrated that fluorescence lifetime imaging microscopy (FLIM) provides a 61 label-free method to identify metabolic differences between inner ear cell types 15

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To further probe the biochemical basis for the gradient in bound we exploited existing transcriptional 149 data sets generated from proximal and distal regions of the developing BP 18 . Prior to mRNA isolation 150 for bulk RNA-seq and Affymetrix microarray analysis, BPs were separated into proximal, middle, and 151 distal thirds. Data were then analysed for differential expression of metabolic mRNAs involved in

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The metabolic function of PKM2 is determined by whether the enzyme exists as a tetramer or a dimer.

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In its dimeric form, PKM2 functions as a metabolic switch, diverting glucose towards the PPP for 177 biosynthesis or towards pyruvate for energy production. 33 . Allosteric modifications regulating the 178 ratio between the tetrameric and dimeric forms of PKM2 are driven by factors in the surrounding 179 environment including intermediate metabolites and pH 33,34 . Given the pH-dependent nature of 180 PKM2 allostery and that the main rate-limiting enzymes driving PPP-linked glucose metabolism display 181 optimal activity at alkaline cytosolic pH 35 , we next investigated differences in intracellular pH (pHi) 182 along the tonotopic axis using the indicator pHrodo Red. When using this probe, low pHrodo Red 183 fluorescence reflects an alkaline pH and high fluorescence a more acidic pH.

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Explants were dual-loaded with the pHi indicator pHrodo Red ( Figure 5) and the live probe SIR-actin 186 to distinguish HCs from SCs ( Figure 5 Supplement 1, 2). We identified opposing proximal-to-distal 187 gradients in pHi in HCs and SCs along the tonotopic axis, using pHrodo Red, which reported a more 188 alkaline pHi in HCs at the proximal compared to distal end of the organ ( Figure 5). The higher pHi in 189 the proximal region reflects a metabolic phenotype consistent with higher PPP activity and dimeric 190 PKM2. Overall, the higher pH and PKM2 expression levels and the possible dimeric confirmation are 191 consistent metabolically with a longer bound (NAD(P)H lifetime). To investigate whether the proximal-7 to-distal gradient in pH was maintained at later developmental stages, we also quantified the pHrodo 193 Red signal in HCs and SCs at E14. At later developmental stages, we find the pH gradients to be 194 reversed ( Figure 5 Supplement 3). As tonotopic patterning and positional identity are specified 195 between E6-E7.5 18 the gradient at E14 is unlikely to impact the gradient in HC morphology.

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

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Since we could not detect an obvious transcriptional basis for the proximal-distal gradient in NAD(P)H 199 bound, we sought to investigate a functional role for metabolism in tonotopic patterning by

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Increased proliferation is therefore unlikely to account for the higher cell density observed in the 227 proximal region following the inhibition of glycolysis. Further studies are needed to determine the 228 specific mechanisms underlying this frequency-specific increase in HC density.

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As shown in our previous work, reciprocal morphogen gradients of Bmp7 and Chdl1 establish HC 231 positional identity at the morphological level along the developing BP between E6.5 and E8 18 . To 232 determine whether cytosolic glucose metabolism acts during this same developmental window, we 233 blocked hexokinase activity for defined periods during BP development using 2-DOG. Explants were 234 established at E6.5 and treated for either 24 or 48 hours followed by wash out with control medium.

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These treatments correspond to the developmental window (E6.5-E8) described previously for 236 refinement of tonotopic morphologies in developing HCs along the proximal-to-distal axis 18 . The

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To determine whether this effect was specific to glucose flux through the PPP we also blocked 262 phosphofructokinase (PFK), a rate limiting enzyme further down in the glycolytic pathway, using 1 mM 263 YZ9 (Figure 7 Supplement 2). Blocking PFK activity inhibits the glycolytic cascade involved in pyruvate 264 production but does not change the activity of G6PD in the PPP 10 . YZ9 treatment resulted in a 265 reduction in HC size, especially in the proximal region leading to a reduction in the HC gradient when 266 analysed using pairwise comparisons (Sidak's multiple comparisons, p=0.17). However, contrary to all 267 other metabolic inhibitor treatments, YZ9 was unique in that it did not produce a significant

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Having identified graded differences in Pkm2 expression (

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In the developing BP, live imaging of mitochondrial activity using TMRM revealed no apparent 303 difference in OXPHOS along the tonotopic axis. To determine whether mitochondrial metabolism 304 influences tonotopic patterning during development, we blocked uptake of glycolytically-derived

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In many developing systems, gradients of one or more morphogen act to regulate cell fate, growth 320 and patterning along a given axis 41,42 . In the chick cochlea, reciprocal gradients of Bmp7 and its

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Taking both descriptive and experimental approaches, we characterised regional differences in 377 metabolism along the developing chick cochlea and explored a role for coordinated signalling between

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Here we identify a gradient in glucose metabolism that regulates the morphology of developing HCs

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All data were assessed for normality prior to application of statistical tests, with a threshold of p < 0.05 610 used for determining significance. When comparing between proximal and distal regions within the 611 same tissue explant, paired t-tests with unequal variance were used. This statistical approach was 612 chosen given that measurements were made from different regions within the same sample and were 613 therefore not independent from each other. Comparisons made between different developmental 614 stages were assumed independent from one another and thus here, independent t-tests and 2-way 615 ANOVAs were used. For bulk RNA-seq analysis, all genes with a Log2 P-value > 1 were considered significantly expressed in 636 the distal BP region and all genes with a Log2 < 1 significantly expressed in the proximal BP region.

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Statistical significance levels were calculated by one-way ANOVA. For a gene to be considered 638 'differential', at least one region of the BP (proximal, middle or distal) was required to be ≥ 0.5 RPKM.

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A fold change of ≥ 2 was imposed for the comparison between distal and proximal regions.