New Results

The Warburg Effect and lactate signaling augment Fgf signaling to promote sensory-neural development in the otic vesicle

Husniye Kantarci, Yunzi Gou, Bruce B. Riley
doi: https://doi.org/10.1101/392548

ABSTRACT

Recent studies indicate that many developing tissues modify glycolysis to favor lactate synthesis, but how this promotes development is unclear. Using forward and reverse genetics in zebrafish, we show that disrupting the glycolytic gene phosphoglycerate kinase-1 (pgk1) impairs Fgf-dependent development of hair cells and neurons in the otic vesicle and other neurons in the CNS/PNS. Focusing on the otic vesicle, we found that Fgf signaling underperforms in pgk1-/- mutants even when Fgf is transiently overexpressed. Wild-type embryos treated with drugs that block synthesis or secretion of lactate mimic the pgk1-/- phenotype, whereas pgk1-/- mutants are rescued by treatment with exogenous lactate. Lactate treatment of wild-type embryos elevates expression of Etv5b/Erm even when Fgf signaling is blocked. Thus, by raising steady-state levels of Etv5b (a critical effector of the Fgf pathway), lactate renders cells more responsive to dynamic changes in Fgf signaling required by many developing tissues.

INTRODUCTION

Development of the paired sensory organs of the head relies on critical contributions from cranial placodes. Of the cranial placodes, the developmental complexity of the otic placode is especially remarkable for producing the entire inner ear, with its convoluted epithelial labyrinth and rich cell type diversity. The otic placode initially forms a fluid filled cyst, the otic vesicle, which subsequently undergoes extensive proliferation and morphogenesis to produce a series of interconnected chambers containing sensory epithelia (Whitfield, 2015). Sensory epithelia comprise a salt-and-pepper pattern of sensory hair cells and support cells. Hair cell specification is initiated by expression of the bHLH factor Atonal Homolog 1 (Atoh1) (Bermingham et al., 1999; Chen et al., 2002; Millimaki et al., 2007; Raft et al., 2007; Woods et al., 2004). Atoh1 subsequently activates expression of Notch ligands that mediate specification of support cells via lateral inhibition. Hair cells are innervated by neurons of the statoacoustic ganglion (SAG), progenitors of which also originate from the otic vesicle. Specification of SAG neuroblasts is initiated by localized expression of the bHLH factor Neurogenin1 (Ngn1) (Andermann et al., 2002; Korzh et al., 1998; Ma et al., 1998; Raft et al., 2007). A subset of SAG neuroblasts delaminate from the otic vesicle to form “transit-amplifying” progenitors that slowly cycle as they migrate to a position between the otic vesicle and hindbrain before completing differentiation and extending processes to hair cells and central targets in the brain (Alsina et al., 2004; Kantarci et al., 2016; Vemaraju et al., 2012).

Development of both neurogenic and sensory domains of the inner ear requires dynamic regulation of Fgf signaling. Fgf-dependent induction of Ngn1 establishes the neurogenic domain during placodal stages, marking one of the earliest molecular asymmetries in the otic placode in chick and mouse embryos (Abello et al., 2010; Alsina et al., 2004; Magarinos et al., 2010; Mansour et al., 1993; Pirvola et al., 2000). Subsequently, as neurogenesis subsides, Fgf also specifies sensory domains of Atoh1 expression (Hayashi et al., 2008; Ono et al., 2014; Pirvola et al., 2002). Moreover, in the mammalian cochlea different Fgf ligand-receptor combinations fine-tune the balance of hair cells and support cells (Hayashi et al., 2007; Jacques et al., 2007; Mansour et al., 2009; Pirvola et al., 2000; Puligilla et al., 2007; Shim et al., 2005). In zebrafish, sensory domains form precociously during the earliest stages of otic induction in response to Fgf from the hindbrain and subjacent mesoderm (Gou et al., 2018; Millimaki et al., 2007). Ongoing Fgf later specifies the neurogenic domain in an abutting domain of the otic vesicle (Kantarci et al., 2015; Vemaraju et al., 2012). As otic development proceeds, the overall level of Fgf signaling increases, reflected by increasing expression of numerous genes in the Fgf synexpression group, including transcriptional effectors Etv4 (Pea3) and Etv5b (Erm), and the feedback inhibitor Spry (Sprouty1, 2 and 4). Sensory epithelia gradually expand during development and express Fgf, contributing to the general rise in Fgf signaling. Similarly, mature SAG neurons express Fgf5. As more neurons accumulate, rising levels of Fgf eventually exceed an upper threshold to terminate further specification of neuroblasts and delay differentiation of transit-amplifying SAG progenitors (Vemaraju et al., 2012). Additionally, elevated Fgf inhibits further hair cell specification while maintaining support cells in a quiescent state (Bermingham-McDonogh et al., 2001; Jiang et al., 2014; Ku et al., 2014; Maier and Whitfield, 2014). Thus, dynamic changes in Fgf signaling regulate the onset, amount, and pace of neural and sensory development in the inner ear.

There is increasing evidence that dynamic regulation of glycolytic metabolism is critical for proper development of various tissues. For example, populations of proliferating stem cells or progenitors, including human embryonic stem cells, neural stem cells, hematopoietic stem cells, and posterior presomitic mesoderm, modify glycolysis by shunting pyruvate away from mitochondrial respiration in favor of lactate synthesis, despite an abundance of free oxygen (Agathocleous et al., 2012; Bulusu et al., 2017; Gu et al., 2016; Oginuma et al., 2017; Sá et al., 2017; Wang et al., 2014; Zheng et al., 2016). This is similar to “aerobic glycolysis” (also known as the “Warburg Effect”) exhibited by metastatic tumors, thought to be an adaptation for accelerating ATP synthesis while simultaneously producing carbon chains needed for rapid biosynthesis (Liberti and Locasale, 2016). In addition, aerobic glycolysis and lactate synthesis appears to facilitate cell signaling required for normal development. For example, aerobic glycolysis promotes relevant cell signaling to stimulate differentiation of osteoblasts, myocardial cells, and fast twitch muscle (Esen et al., 2013; Menendez-Montes et al., 2016; Özbudak et al., 2010; Tixier et al., 2013), as well as maintenance of cone cells in the retina (Aït-Ali et al., 2015).

In a screen to identify novel regulators of SAG development in zebrafish, we recovered two independent mutations that disrupt the glycolytic enzyme Phosphoglycerate Kinase 1 (Pgk1). Loss of Pgk1 causes a delay in upregulation of Fgf signaling in the otic vesicle, causing a deficiency in early neural and sensory development. We show that Pgk1 is co-expressed with Fgf ligands in the otic vesicle, as well as in various clusters of neurons in cranial ganglia and the neural tube. Moreover, Pgk1 acts non-autonomously to promote Fgf signaling at a distance by promoting synthesis and secretion of lactate. Lactate independently activates the MAPK pathway, leading to elevated expression of the effector Etv5b, priming the pathway to render cells more responsive to dynamic changes in Fgf levels.

RESULTS

Initial characterization of sagd1

To identify novel genes required for SAG development, we conducted an ENU mutagenesis screen for mutations that specifically alter the number or distribution of post-mitotic isl2b:Gfp+ SAG neurons. A recessive lethal mutation termed sagd1 (SAG deficient1) was recovered based on a deficiency in the anterior/vestibular portion of ganglion, which are the first SAG neurons to form. Quantification of isl2b:Gfp+ cells in serial sections of sagd1-/- mutants revealed a ~60% deficiency in anterior/vestibular SAG neurons at 24 and 30 hpf (Fig. 1A-C). The number of vestibular neurons remained lower than normal through at least 48 hpf whereas accumulation of posterior/auditory SAG neurons appeared normal (Fig. 1B). Staining with anti-Isl1/2 antibody, which labels a more mature subset of SAG neurons, showed a similar trend: There was a 60% deficiency in anterior/vestibular neurons from the earliest stages of SAG development whereas subsequent formation of posterior/auditory neurons was normal (Fig. S1A-I). Of note, the early deficit of SAG neurons did not result from increased apoptosis (Fig. S1J), and in fact sagd1-/- mutants produced fewer apoptotic cells than normal. sagd1-/- mutants show no overt defects in gross morphology, although sagd1-/- mutants do show deficits in balance and motor coordination indicative of vestibular dysfunction. Mutants typically die by 10-12 dpf.

Figure 1.
Figure 1. Initial screen to identify sagd1.

(A) Lateral views of isl2b:Gfp+ SAG neurons in live wild-type (wt) embryos and sagd1 mutants at the indicated times. The vestibular portion of the SAG (outlined in white) is deficient in sagd1 mutants, as detected in the screen. (B) Number (mean and standard deviation) of isl2b:Gfp+ anterior/vestibular neurons and posterior/auditory neurons in wild-type and sagd1 embryos at 30 hpf. Asterisks, here and in subsequent figures, indicate significant differences (p < .05) from wild-type controls. (C) Cross sections through anterior and posterior portions of the otic vesicle (outlined in white) showing anti-Gfp stained SAG neurons at the indicated times.

Figure S1.
Figure S1. Quantitation of mature Isl1/2+ SAG neurons and TUNEL in sagd1 mutants.

(A-H) Cross sections (dorsal up, medial to the right) through the anterior/vestibular portion of the otic vesicle in control embryos (A-D) and sagd1 mutants (E-H) showing anti-Isl1/2 stained SAG neurons (A,C, E, G) and co-staining for TUNEL (B, D, F, H). (I) Number of Isl1/2+ SAG neurons at 30 hpf counted from whole mount preparations. Anterior/vestibular SAG neurons are under-produced in sagd1 mutants, whereas posterior/auditory neurons develop normally. (J) Number of TUNEL+ apoptotic cells at the indicated times. sagd1 mutants show fewer apoptotic cells than normal at 24 and 30 hpf, indicating that the deficiency in vestibular SAG neurons is not due to cell death.

To further characterize the sagd1-/- phenotype, we examined earlier stages of SAG development. Specification of SAG neuroblasts is marked by expression of ngn1 in the floor of the otic vesicle, a process that normally begins at 16 hpf, peaks at 24 hpf, and then gradually declines and ceases by 42 hpf (Vemaraju et al., 2012). We observed that early stages of neuroblast specification were significantly impaired in sagd1-/- mutants, with only 30% of the normal number of ngn1+ neuroblasts at 24 hpf (Fig. 2A, B, E). However, subsequent stages of specification gradually improved in sagd1-/- mutants, such that the number of ngn1+ neuroblasts in the otic vesicle was normal by 30 hpf (Fig. 2C, D, E). In the next stage of SAG development, a subset of SAG neuroblasts delaminates from the otic vesicle and enters a transit-amplifying phase. Recently delaminated neuroblasts continue to express ngn1 for a short time before shifting to expression of neurod, which is then maintained until SAG progenitors mature into post-mitotic neurons. In sagd1-/- mutants, the number of ngn1+ neuroblasts outside the otic vesicle was reduced by 50-60% at every stage examined (Fig. 2F). Similarly, the number of neurod+ transit-amplifying cells was strongly reduced in sagd1-/- mutants (Fig. 2G-O). This deficit was more pronounced at 30 hpf (~60% decrease) than at 48 hpf (~20% decrease). Moreover, deficit was restricted to anterior transit-amplifying cells that give rise to vestibular neurons, whereas accumulation of posterior/auditory progenitors was normal (Fig. 2O). In summary, the neural deficit observed in sagd1-/- mutants reflects a deficiency in early specification of vestibular neuroblasts. In addition, the continuing deficit in anterior transit-amplifying cells at later stages likely contributes to a long-term deficit in vestibular neurons.

Figure 2.
Figure 2. Early development of SAG neurons is impaired in sagd1 mutants.

(A-D) Cross-sections through the anterior/vestibular portion of the otic vesicle (outlined) showing expression of ngn1 in wild-type embryos and sagd1 mutants at 24 and 30 hpf. (E, F) Mean and standard deviation of ngn1+ cells in the floor of the otic vesicle (E) and in recently delaminated SAG neuroblasts outside the otic vesicle (F), as counted from serial sections. (G-N) Cross-sections through the anterior/vestibular and posterior/auditory regions of the otic vesicle showing expression of neurod in transit-amplifying SAG neuroblasts at 36 and 48 hpf. (O) Mean and standard deviation of neurod+ SAG neuroblasts at 30 and 48 hpf counted from serial sections.

We next examined other aspects of otic development in sagd1-/- mutants. Expression of prosensory marker atoh1a was reduced in both anterior (utricular) and posterior (saccular) maculae at 24 and 30 hpf (Fig. 3A-B’). In agreement, sagd1-/- mutants produced roughly half the normal number of hair cells in the utricle and saccule at 36 and 48 hpf (Fig. 3C-E). Because development of SAG progenitors and sensory epithelia both rely on Fgf signaling, we also examined expression of various Fgf ligands and downstream Fgf-target genes. Expression of fgf3 and fgf8 appeared relatively normal in sagd1-/- mutants from 18-30 hpf (Fig. 3S-T’, and data not shown). However, expression of downstream Fgf-targets etv5b, pax5 and sprouty4 were severely deficient in the otic vesicle of sagd1-/- mutants at 18 hpf (Fig. 3F-H’). Expression of these genes partially recovered by 21 hpf and was only mildly reduced by 24 hpf (Fig. 3I-N’). Despite these changes, markers of axial identity were largely unaffected at 30 hpf, including the dorsal marker dlx3b, the ventrolateral marker otx1b, the anteromedial marker pax5 and posteromedial marker pou3f3b (Fig. 3O-R’). These data suggest that Fgf signaling is impaired during early stages of otic vesicle development but slowly improves during later stages, which could explain the early deficits in sensory and neural specification.

Figure 3.
Figure 3. Sensory development and early Fgf signaling are impaired in sagd1 mutants.

(A-D”, F-T’) Dorsolateral views of whole mount specimens (anterior to the left) showing expression of the indicated genes in the otic vesicle (outlined) at the indicated times in wild-type embryos and sagd1 mutants. (E) Mean and standard deviation of hair cells in the anterior/utricular and posterior/auditory maculae at 36 and 48 hpf in wild-type embryos (black) and sagd1 mutants (red).

Identification of the sagd1 locus: A novel role for Pgk1

To identify the affected locus in sagd1-/- mutants, we used whole genome sequencing and homozygosity mapping approaches (Obholzer et al., 2012). We identified as our top candidate a novel transcript associated with the gene encoding the glycolytic enzyme Phosphoglycerate Kinase-1 (Pgk1). The zebrafish genome harbors only one pgk1 gene, but the locus produces 2 distinct transcripts (Fig. 4A). The primary transcript (pgk1-FL) encodes full-length Pgk1, which is highly conserved amongst vertebrates (nearly 90% identical between zebrafish and human). However, the second pgk1 transcript, termed pgk1-alt, is unique to zebrafish and arises from an independent transcription start site in the first intron of pgk1-FL. There are two short exons (termed exons 1a and 1b) at the start of pgk1-alt that encode a novel peptide with 30 amino acids. pgk1-alt then splices in-frame with exons 2-6, which are identical to pgk1-FL, followed by a splice into a novel exon (termed exon 6a) containing a stop codon (Fig. 4A). Thus pgk1-alt encodes a truncated protein that includes much of the N-terminal half of Pgk1 but presumably lacks glycolytic enzyme activity. In sagd1-/- mutants, pgk1-alt contains two nucleotide substitutions leading to loss of a splice acceptor site as well as the translation start codon in exon 1b (Fig. 4B). Either of these SNPs is predicted to disrupt expression of pgk1-alt protein.

Figure 4.
Figure 4. Two independent transcripts associated with the pgk1 locus.

(A) Exon-intron structure of the primary full-length pgk1 transcript (pgk1-FL); and alternate transcript (pgk1-alt) arising from an independent transcription start site containing two novel exons (1a and 1b) that splice in-frame to exon 2, and a third novel exon (6a) containing to a stop codon. The nucleotide and peptide sequences of exons 1b and 6a are shown. Relative positions of the lesions in sagd1 are indicated (red asterisks). (B) Nucleotide sequence near the 5’ end of exon 1b showing the SNPs detected in sagd1 (red font). (C) Nucleotide sequence of pgk1-FL showing the sgRNA target site (blue font) and the altered sequence of the x55 mutant allele (red font), which introduces a premature stop codon. (D-F’) Cross sections through the otic vesicle (outlined) showing staining with ribo-probe for the entire pgk1 locus (covering both pgk1-FL and pgk1-alt) in wild-type embryos. Note elevated expression in the vestibular ganglion (vg), auditory ganglion (ag), utricular macula (um), and saccular macula (sm). (G, H, M, N) Cross sections through the otic vesicle (outlined) in wild-type embryos and sagd1 mutants stained with ribo-probe for exon 1 (pgk1-FL alone) (G, H) or exon 1b (pgk1-alt alone) (M, N). Accumulation of both transcripts is dramatically reduced in sagd1 mutants. (I-L, O-R) Cross-sections through the hindbrain (dorsal up) showing expression of pgk1-FL (black) plus neurod (red) (I-L) or pgk1-alt alone (O-R). Arrows indicate positions of the trigeminal and facial ganglia. (S-Z) Whole mount staining of the hindbrain (dorsal view, anterior up) showing expression of the pgk1 locus (S-V), pgk1-alt alone (W, X), or staining with anti-acetylated tubulin (Y-Z) in embryos with the indicated genotypes. Positions of the midbrain-hindbrain border (mhb), reticulospinal neurons (rsn), trigeminal ganglion (tg), SAG, and rhombomere centers (white asterisks) are indicated. Embryos in (U-X) were heat shocked at 20 hpf and fixed at 24 hpf. Scale bar, 100 μm.

Although sagd1 does not alter the sequence of pgk1-FL, we tested whether the sagd1 mutation alters accumulation of pgk1-FL transcript. In wild-type embryos pgk1-FL is widely expressed at a low level, but beginning around 18 hpf transcript abundance becomes dramatically elevated in clusters of cells scattered throughout the central and peripheral nervous systems. Examples of cells with elevated pgk1 expression include utricular and saccular hair cells, mature SAG neurons (Fig. 4D-G), trigeminal and facial ganglia, the midbrain-hindbrain border, and reticulospinal neurons in the hindbrain (Fig. 4I, K, S). A similar pattern is observed for accumulation of pgk1-alt transcript (Fig. 4M-R). Local upregulation of pgk1-FL transcript is severely attenuated in sagd1-/- mutants (Fig. 4H, J, L). To test whether Pgk1-alt function is sufficient to upregulate pgk1-FL, we examined the effects of transient misexpression using a heat shock-inducible transgene, hs:pgk1-alt. When hs:pgk1-alt was activated at 20 hpf, accumulation of pgk1-FL was strongly elevated at 24 hpf (Fig. 4U, V). This was in contrast to accumulation of transgenic pgk1-alt transcript, which peaked near the end of the heat shock period at 21 hpf (not shown) but then decayed to background levels by 24 hpf (Fig. 4W, X). We infer that perdurance of Pgk1-alt protein is sufficient to upregulate pgk1-FL. Based on previous studies of yeast and mammalian Pgk1 (Beckmann et al., 2015; Ho et al., 2010; Liao et al., 2016; Ruiz-Echevarria et al., 2001; Shetty et al., 2010; Shetty et al., 2004), we speculate that Pgk1-alt acts to stabilize pgk1-FL transcript (See Discussion).

To directly test the requirement for full length Pgk1, we targeted the first exon of pgk1-FL using CRISPR-Cas9 and recovered an indel mutation leading to a frameshift followed by a premature stop (Fig. 4C). This presumptive null mutation is predicted to eliminate all but the first 19 amino acids of Pgk1. Expression of pgk1 is severely reduced in pgk1-/- mutants, presumably reflecting nonsense-mediated decay (Fig. 4T). The phenotype of pgk1-/- mutants is identical to sagd1-/-, including reduced otic expression of etv5b at 18 hpf, reduced ngn1 at 24 hpf, and deficiencies in neurod+ transit-amplifying cells and mature isl2b-gfp+ SAG neurons (Fig. 5A-H). Additionally, development of the trigeminal ganglion and reticulospinal neurons in the hindbrain is also impaired (Fig. 4Y, Z). When pgk1+/- heterozygotes were crossed with sagd1+/- heterozygotes, all intercross embryos developed normally (Fig. 5Q), confirming that pgk1-alt and pgk1-FL encode distinct complementary functions. We also generated a heat-shock inducible transgene to overexpress pgk1-FL (hs:pgk1). Activation of hs:pgk1 at 18 hpf rescued the SAG deficiency in sagd1-/- mutants (Fig. 5Q). Together, these data show that pgk1-alt is required to locally upregulate pgk1-FL, which in turn is required for proper Fgf signaling and sensory and neural development.

Figure 5.
Figure 5. Initial characterization of pgk1-/- mutants and genetic mosaics.

(A-F) Cross sections through the anterior/vestibular region of the otic vesicle (outlined) showing expression of the indicated genes in wild-type embryos and pgk1-/- mutants at the indicated times. (G-J) Lateral views showing isl2b-Gfp expression in live embryos at 30 hpf (G, H) and fgf3 at 24 hpf (I, J). The otic vesicle is outlined. (K-P) Cross sections through the otic vesicle (outlined) showing positions of lineage labeled wild-type cells (red dye) transplanted into a wild-type host (K, M, O) or a pgk1-/- mutant host (L, N, P). Sections are co-stained with DAPI (M, N) and etv5b probe (O, P). Note the absence of etv5b expression in wild-type cells transplanted into the mutant host (P). (Q) Number of mature Isl1/2+ SAG neurons at 30 hpf in embryos with the indicated genotypes, except for sagd1 x pgk1 intercross expected to contain roughly 25% each of +/+, sagd1/+, pgk1/+ and sagd1/pgk1 embryos. (R) Percent of wild-type donor cells located in the ventral half of the otic vesicle expressing etv5b in wild-type or pgk1-/- hosts. A total of 151 wild-type donor cells were counted in 8 otic vesicles of wild-type host embryos, and 247 wild-type donor cells were counted in 8 otic vesicles of pgk1-/- host embryos.

Pgk1 acts non-autonomously to promote Fgf signaling

To better understand how Pgk1 promotes Fgf signaling during otic development, we generated genetic mosaics by transplanting wild-type donor cells into pgk1-/- mutant host embryos. We reasoned that if Pgk1 acts cell-autonomously, as expected for a glycolytic enzyme, then isolated wild-type cells should be able to respond to local Fgf sources within a pgk1-/- mutant host. Surprisingly, the majority of the wild-type cells located near the utricular Fgf source in the floor of the otic vesicle in pkg1-/- hosts did not express the Fgf-target etv5b (Fig. 5J, L, N, P, R). In control experiments, wild-type cells transplanted into wild-type hosts showed normal expression of etv5b (Fig. 5I, K, M, O, R). Thus, Pgk1 is required non-autonomously to promote Fgf signaling at a distance.

Pgk1 does not act extracellularly through Plasmin turnover

We considered two distinct mechanisms for non-autonomous functions of Pgk1 that have been documented in metastatic tumors. First, tumor cells secrete Pgk1 to promote early stages of metastasis through modulation of extracellular matrix (ECM) and cell signaling (Chirico, 2011; Jung et al., 2009; Wang et al., 2007; Wang et al., 2010). The only specific molecular function identified for secreted Pgk1 is to serve as a disulfide reductase leading to proteolytic cleavage of Plasmin (Lay et al., 2000), a serine protease that cleaves Fgf as well as ECM proteins required for Fgf signaling (Botta et al., 2012; George et al., 2001; Meddahi et al., 1995; Schmidt et al., 2005). This raised the possibility that loss of Pgk1 could elevate Plasmin activity and thereby impair Fgf signaling. To test this, we injected morpholino to block synthesis of Plasminogen (Plg), the zymogen precursor of Plasmin. However, injection of the plg morpholino did not rescue or ameliorate the neural deficiency in sagd1-/- or pgk1-/- mutants (Fig. S2).

Figure S2.
Figure S2. Knockdown of plasminogen does not alter pgk1-/- or sagd1-/- phenotypes.

Mean (green) and standard deviation (red) of the number of Isl1/2+ SAG neurons at 30 hpf in embryos with the indicated genotypes and/or injected with plg-MO. NS, no significant difference between groups indicated in brackets.

Aerobic glycolysis is required for otic development

The second Pgk1-dependent mechanism employed by metastatic tumors is to upregulate and redirect glycolysis to promote lactate synthesis despite abundant oxygen. This process is often called “aerobic glycolysis”, or the “Warburg effect”, and is thought to accelerate synthesis of ATP and provide 3-carbon polymers used for rapid biosynthesis (Liberti and Locasale, 2016). In addition, lactate secretion facilitates cell signaling in cancer cells (San-Millán and Brooks, 2017) as well as during certain normal physiological contexts (Lee et al., 2015; Peng et al., 2016; Yang et al., 2014; Zuo et al., 2015; Reviewed by Philp et al., 2005). We therefore tested whether blocking glycolysis and/or lactate synthesis in wild-type embryos could mimic the phenotype of pgk1-/- and sagd1-/- mutants. Treating wild-type embryos with 2-deoxy-glucose (2DG) (Nirenberg and Hogg, 1958) or 3PO (Clem et al., 2008) to block early steps in glycolysis (Fig. 6A) reduced the number of mature SAG neurons to a level similar to that of pgk1-/- or sagd1-/- mutants (Fig. 6B). Similar results were obtained when these inhibitors were added at 0 hpf or 14 hpf.

Figure 6.
Figure 6. Drugs blocking aerobic glycolysis mimic pgk1-/- mutants.

(A) Diagram of the glycolytic pathway and changes associated with the Warburg Effect in which pyruvate is shunted from mitochondria in favor of synthesis and secretion of lactate. The indicated inhibitors were used to block discrete steps in the pathway. In the Warburg Shunt, mitochondrial Pyruvate Dehydrogenase Complex (PDC) is inhibited by elevated Pyruvate Dehydrogenase Kinase, the activity of which is blocked by DCA. (B, C) Scatter plots showing the mean (green) and standard deviation (red) of the number of Isl1/2+ SAG neurons at 30 hpf in embryos with the indicated genotypes and/or drug treatments. Lactate-treatments and some controls were buffered with 10 mM MES at pH 6.2. (D) Scatter plats showing the mean and standard deviation of the total number of hair cells at 36 hpf in embryos treated with the indicated drugs. Asterisks show significant differences (p ≤ .05) from wild-type control embryos.

We next focused on later steps in the pathway dealing with handling of pyruvate vs. lactate. In cells exhibiting the Warburg Effect, transport of pyruvate into mitochondria is typically blocked by upregulation of Pyruvate Dehydrogenase Kinase (PDK) (Cairns et al., 2011), thereby favoring reduction of pyruvate to lactate (see Warburg Shunt, Fig. 6A). Dichloroacetate (DCA) blocks PDK activity (Kato et al., 2007), allowing more pyruvate to be transported into mitochondria and thereby forestalling lactate synthesis. Treating wild-type embryos with DCA at 14 hpf mimicked the deficiency of SAG neurons seen in pgk1-/- and sagd1-/- mutants (Fig. 6B). To directly block synthesis of lactate from pyruvate, wild-type embryos were treated with Galloflavin, an inhibitor of Lactate Dehydrogenase (Farabegoli et al., 2012; Manerba et al., 2012) (Fig. 6A). Wild-type embryos treated with Galloflavin also displayed a deficiency of SAG neurons similar to pgk1-/- and sagd1-/- mutants (Fig. 6C). Finally, to block transport of lactate across the cell membrane, we treated wild-type embryos with either CHC or UK5099, which block activity of monocarboxylate transporters MTC1, 2 and 4 (Halestrap, 1975; Halestrap and Denton, 1974) (Fig. 6A). Application of either CHC or UK5099 also mimicked the SAG deficiency in pgk1-/- and sagd1-/- mutants (Fig. 6C, and data not shown). Treatment of wild-type embryos with 2DG, Galloflavin or CHC also reduced hair cell production to a level comparable to sagd1-/- or pgk1-/- mutants (Fig. 6D). To further explore the role of lactate, we added exogenous lactate to embryos beginning at 14 hpf and examined accumulation of SAG neurons at 30 hpf. As shown in Fig. 6C, lactate treatment elevated SAG accumulation above normal in wild-type embryos and fully rescued pgk1-/- mutants. Lactate treatment also rescued hair cell production in wild-type embryos treated with 2DG, Galloflavin, or CHC (Fig. 6D). Thus, pharmacological agents that block glycolysis, lactate synthesis or lactate secretion are sufficient to mimic the pgk1-/- and sagd1-/- phenotype, and exogenous lactate is sufficient to reverse these effects.

We next examined whether the above inhibitors and/or exogenous lactate affect Fgf signaling and SAG specification in the nascent otic vesicle. Treatment of wild-type embryos with Galloflavin or CHC reduced the number of cells expressing etv5b and ngn1 at 18 hpf and 24 hpf, respectively (Fig. 7Ag, Ah, Bg, Bh, E), mimicking the effects of pgk1-/- (Fig. 7Ab, Bb, C, D). Treatment with exogenous lactate reversed the effects of Galloflavin and CHC (Fig. 7Ai, Aj, Bj) and rescued pgk1-/- mutants (Fig. Ac, Bc, C, D). Moreover, treating wild-type embryos with lactate increased the number of cells expressing etv5b and ngn1 (Fig. 7Ae, Be, C, D).

Figure 7.
Figure 7. Exogenous lactate reverses the effects of disrupting aerobic glycolysis.

(A, B) Cross sections through the anterior/vestibular portion of the otic vesicle (outlined) showing expression of etv5b at 18 hpf (A) and ngn1 at 24 hpf (B) in embryos with the indicated genotype or drug treatment. Arrows in (A) highlight expression in the ventromedial otic epithelium. (C) Mean and standard deviation of the number of etv5b+ cells in the ventral half of the otic vesicle counted from serial sections of embryos with the indicated genotype or drug treatment. (D, E) Mean and standard deviation of the number of ngn1+ cells in the floor of the otic vesicle counted from serial sections of embryos with the indicated genotype or drug treatment. Asterisks indicate significant differences (p < .05) from wild-type controls, or between groups indicated by brackets.

We also tested whether a deficiency of ATP contributes to the pgk1-/- phenotype. Adding exogenous ATP to pgk1-/- mutants at 14 hpf did not ameliorate the deficiency in expression of etv5b, ngn1, or pax5 (Fig. 7Ad, Bd, C, D; and Fig. S3). Likewise, adding ATP to wild-type embryos at 14 hpf did not significantly affect expression of these genes (Fig. 7Af, Bf, C, D; and Fig. S3). Finally, adding 25 uM sodium azide to wild-type embryos at 14 hpf to block mitochondrial respiration did not significantly impair expression of ngn1 at 24 hpf (Fig. 7Bk, E). Thus, lactate is critical for early Fgf signaling and SAG specification, but exogenous ATP does not affect these functions. Moreover, the deficiencies in otic development observed in pgk1-/- mutants can be attributed to disruption of lactate synthesis and secretion.

Figure S3.
Figure S3. Exogenous ATP does not rescue pgk1-/- mutants.

Dorsolateral view (anterior to the left) of the otic vesicle (outlined) showing expression of etv5b (A-D) or pax5 (E-H) at 18 hpf in wild-type embryos or pgk1-/- mutants with or without exposure to exogenous 1 mM ATP as indicated.

Lactate and Fgf act in parallel to regulate early otic development

Fgf regulates gene expression primarily through the PI3K and MAPK/Erk signal transduction pathways, but it is unknown which is most critical for otic development. Treatment of wild-type embryos with U0126, an inhibitor of the MAPK activator Mek (Hawkins et al., 2008), reduced the number of ngn1+ cells at 24 hpf (Fig. 7Bl, E) and mature Isl1+ SAG neurons at 30 hpf (Fig. S4) to a degree similar to or greater than pgk1-/- mutants. In contrast, treatment of wild-type embryos with the PI3K inhibitor LY294002 (Montero et al., 2003) had negligible effects on these genes (Fig. S4, and data not shown), showing that most of the effects of Fgf are mediated by MAPK/Erk.

Figure S4.
Figure S4. development of SAG neurons requires MAPK/Erk but not PI3K.

Mean (green) and standard deviation (red) of the number of Isl1/2+ SAG neurons at 30 hpf in embryos treated with MEK inhibitor U0126 or PI3K inhibitor LY294002 (LY) at the indicated concentrations. NS, no significant difference from wild-type control.

Recent findings show that lactate can activate the MAPK/Erk pathway through an independent mechanism (Lee et al., 2015, see Discussion), prompting us to investigate whether the ability of lactate to activate Fgf-target genes requires Fgf signaling. Misexpression of dominant negative Fgf receptor from a heat shock-inducible transgene, hs:dnfgfr1, fully suppressed etv5b expression within two hours of heat shock (Fig. 7Ak; Fig. 8C). Importantly, treatment with exogenous lactate partially restored etv5b expression when Fgf signaling was blocked (Fig. 7Al; Fig. 8D). Because developmental defects in pgk1-/- and sagd1-/- mutants appear to stem from reduced Fgf signaling, we tested whether elevating Fgf can rescue pgk1-/- mutants. Activation of hs:fgf8 at a moderate level (37°C for 30 minutes) beginning at 16 hpf increased the level of pax5 expression in the otic vesicle at 18 hpf and expanded the spatial domain (Fig. 8E, G). Activation of hs:fgf8 in pgk1-/- mutants increased expression of pax5 relative to pgk1-/- alone (Fig. 8F, H), but not to the same degree seen in non-mutant embryos. Thus, elevating either Fgf or lactate alone can partially compensate for loss of the other, but full activation of early otic genes requires both Fgf and lactate.

Figure 8.
Figure 8. Fgf and lactate are required together for full activation of early otic genes.

(A-D) Dorsolateral view of the otic vesicle (outlined) showing expression of etv5b at 18 hpf in wild-type embryos or hs:dnfgfr1/+ transgenic embryos with or without exogenous 6.7 mM lactate added at 14 hpf, as indicated. Embryos were heat shocked at 39°C for 30 minutes beginning at 16 hpf. (E-H) Dorsal view (anterior to the top) of the hindbrain and otic region showing expression of the otic domain of pax5 (black arrows) in embryos with the indicated genotypes. Embryos were heat shocked at 37°C for 30 minutes beginning at 16 hpf. (I) A model for how lactate and Fgf signaling converge on MAPK/Erk to increase the pool of phosphorylated activated Etv4/5, enabling cells to respond more quickly and robustly to dynamic changes in Fgf.

DISCUSSION

Enhanced glycolysis and lactate secretion promote Fgf signaling

Metabolic pathways such as glycolysis have traditionally been viewed as “house keeping” functions but are increasingly recognized for their specific roles in development. We discovered that mutations in pgk1 cause specific defects in formation of hair cells and SAG neurons of the inner ear, as well as various cranial ganglia and reticulospinal neurons of the hindbrain, all of which normally show elevated expression of pgk1 (Fig. 4) and require Fgf signaling (Lassiter et al., 2009; Maulding et al., 2014; Millimaki et al., 2007; Nechiporuk et al., 2005; Terriente et al., 2012; Vemaraju et al., 2012). Treatment of wild-type embryos with specific inhibitors of glycolysis or lactate synthesis or secretion mimicked the effects of pgk1-/-, indicting that the affected cell types require “aerobic glycolysis”, similar to the Warburg Effect exhibited by metastatic tumors. Developmental deficiencies seen in pgk1-/- mutants reflect weakened response to Fgf but are fully rescued by treatment with exogenous lactate. Lactate has been shown to bind and stabilize the cytosolic protein Ndrg3, which in turn activates Raf (Lee et al., 2015) thereby converging with Fgf signaling to activate MAPK/Erk. The MAPK/Erk pathway activates transcription of etv4 and etv5a/b genes (Raible and Brand, 2001; Roehl and Nüsslein-Volhard, 2001). Etv4/5 proteins are also phosphorylated and activated by MAPK/Erk and mediate many of the downstream effects of Fgf (Brown et al., 1998; O’Hagan et al., 1996; Znosko et al., 2010). This dual regulation of Etv4/5 implies a feedback amplification step and offers an explanation for why lactate is required to increase the efficiency of early stages of Fgf signaling (Fig. 8I). Specifically, initial activation of MAPK/Erk by lactate would expand the pool of Etv4/5, priming cells to respond more quickly to dynamic changes in Fgf signaling. Without pgk1, the initial response to Fgf is sluggish and causes lasting deficits in vestibular neurons. Later, as Fgf-mediated feedback amplification continues in pgk1-/-, Fgf signaling gradually improves on its own and is sufficient to support normal development of auditory neurons.

It is interesting that sites of pgk1 upregulation correlate with sites of Fgf ligand expression. Fgf expression does not require pgk1 (Fig. 3S, T), and pgk1 upregulation does not require Fgf (not shown), suggesting co-regulation by shared upstream factor(s). Analysis of genetic mosaics showed that isolated wild-type cells transplanted into pgk1-/- host embryos failed to activate etv5b despite close proximity to sensory epithelia, a known Fgf-source. Clearly wild type cells retain the ability to perform glycolysis, but since upregulation of pgk1 in the otic vesicle is limited to sensory epithelia and mature SAG neurons (Fig. 4A-G), other otic cells may be unable to generate sufficient lactate to augment Fgf signaling. This raises the interesting possibility that Fgf and lactate must be co-secreted from signaling sources to support full signaling potential.

Regulation of Pgk1 by Pgk1-alt

The sagd1 mutation does not directly affect full length Pgk1, but instead disrupts a novel transcript arising from an independent downstream transcription start site that appears to reflect an ancient transposon insertion. The first 30 amino acids of Pgk1-alt are novel but downstream the sequence is identical to exons 2-6 of Pgk1-FL. Production of Pgk1-alt is necessary and sufficient for upregulation of pgk1-FL, possibly involving transcript stabilization. Exon 6 of zebrafish Pgk1-FL and Pgk1-alt corresponds closely to the STE (stabilizing element) of yeast Pgk1 (Ruiz-Echevarria et al., 2001). Premature stops within the first half of yeast Pgk1 lead to nonsense-mediated decay (NMD) of the transcript, but later stops (occurring downstream of the STE) do not trigger NMD (Hagan et al., 1995). The STE can also stabilize recombinant transcripts containing foreign sequences that including a 3’UTR that normally destabilizes the transcript independent of NMD. In each case the STE must be translated in order to function, raising the possibility that the corresponding peptide is required for stability (Ruiz-Echevarria et al., 2001). Several recent studies have identified full length Pgk1 in yeast and human as an unconventional mRNA-binding protein (Beckmann et al., 2015; Liao et al., 2016), and human Pgk1 can bind specific mRNAs to affect stability, though often by destabilization (Ho et al., 2010; Shetty et al., 2004). Whether Pgk1 increases or decreases mRNA stability could reflect interactions with specific sequences or secondary structures in the target. In any case, it seems reasonable that the STE of Pgk1-alt has been coopted from an established “moonlighting” function of Pgk1 to locally upregulate pgk1-FL by increasing transcript by stability, thereby facilitating the switch to Warburg metabolism.

Despite the requirement for pgk1-alt in the developing nervous system, it is not required in mesodermal tissues. For example, upregulation of pgk1 in developing somites is not affected in sagd1-/- mutants. Curiously, pgk1 is not appreciably upregulated in presomitic mesoderm, despite the fact that somitogenesis involves a gradient aerobic glycolysis and lactate synthesis that is highest in the tail and declines anteriorly (Bulusu et al., 2017; Oginuma et al., 2017; Özbudak et al., 2010). However, establishment of Warburg-like metabolism does not necessarily require elevated pgk1. Conversely, elevated pgk1 does not necessarily indicate a Warburg-like state. Indeed, upregulation of pgk1 in somites likely provides high levels of pyruvate to favor robust mitochondrial ATP synthesis over lactate synthesis (Özbudak et al., 2010).

Although the sagd1 mutation was recovered in a forward ENU mutagenesis screen, we note that the SNPs detected in the pgk1-alt sequence were previously reported in the genome sequence database. It is therefore likely that familial breeding during our screen allowed generation and detection of homozygous carriers of this preexisting allele. Indeed, we have subsequently detected these SNPs in our wild-type stock, likely accounting for variation in expression levels in early otic genes sometimes detected in specific families. As a cautionary note, the sequence for pgk1-alt was initially represented in older versions of the annotated zebrafish genome but has since been removed, presumably deemed a technical artifact. Had we used more recent versions of the genome as our reference dataset, the SNPs in sagd1 would have been overlooked. Regardless of its origins, identification of the sagd1 mutant allele underscores the continuing utility of forward screens: Had we not recovered sagd1 from our screen, it is highly unlikely that we would have identified pgk1 as a developmental regulatory gene.

MATERIALS AND METHODS

Fish strains and developmental conditions

Wild-type embryos were derived from the AB line (Eugene,OR). For most experiments embryos were maintained at 28.5°C, except where noted. Embryos were staged according to standard protocols (Kimmel et al., 1995). PTU (1-phenyl 2-thiourea), 0.3 mg/ml (Sigma P7629) was included in the fish water to inhibit pigment formation. The following transgenic lines were used in this study: Tg(hsp70:fgf8a)x17 (Millimaki et al., 2010), Tg(Brn3c:GAP43-GFP)s356t (Xiao et al., 2005), Tg(−17.6isl2b:GFP)zc7 (Pittman et al., 2008), Tg(hsp70I:dnfgfr1-EGFP)pdI (Lee et al., 2005), and new lines generated for this study Tg(hsp70I:pgk1-FL)x65 and Tg(hsp70I:pgk1-alt)x66. These transgenic lines are herein referred to as hs:fgf8, brn3c:GFP, Isl2b:GFP, hs:pgk1-FL, hs:pgk1-alt, and hs:dnfgfr1 respectively. Mutant lines sagd1x4ν and pgk1x55 were used for loss of function analysis. Homozygous sagd1-/- and pgk1-/- mutants were identified by a characteristic decrease in the expression of Isl2b-Gfp or brnc3:Gfp, or by PCR amplification of pgk1 DNA using the following primers: Forward primer 1, 5’-AGCAAGTACATCCAATTGCCG-3’; Forward primer 2, 5’ -AAGGAAAGCGGTGATCATGT-3’; Reverse primer, 5’-GGAAGTGTATCTGTCACGCGT-3’. Forward primer 1 produces an amplicon of ~451 bp from both wild-type and mutant DNA, whereas forward primer 2 produces an amplicon of 236 bp from wild-type DNA only.

Gene misexpression and morpholino injections

Heat-shock regimens were carried in a water bath at indicated temperatures and durations. Embryos were kept in a 33°C incubator after the heat-shock. Transgenic carriers were identified by morphological characteristics. Knockdown of Plasminogen was performed by injecting one-cell stage embryos with ~5 ng of morpholino oligomer with the following sequence: 5’-AACTGCTTTGTGTACCTCCATGTCG-3’.

In situ hybridization and immunohistochemistry

Whole mount in situ hybridization and immunohistochemistry protocols used in this study were previously described (Phillips et al., 2001). Whole mount stained embryos were cut into 10 μm thick sections using a cryostat as previously described (Vemaraju et al., 2012). The following antibodies were used in this study: Anti-Islet1/2 (Developmental Studies Hybridoma Bank 39.4D5, 1:100), Anti-GFP (Invitrogen A11122, 1:250), Alexa 546 goat anti-mouse or anti-rabbit IgG (ThermoFisher Scientific A-11003/A-11010, 1:50). Promega terminal deoxynucleotidyl transferase (M1871) was used according to manufacturer’s protocol to perform the TUNEL assay.

Cell Transplantation

Wild-type donor embryos were injected with a lineage tracer (tetramethylrhodamine labeled, 10,000 MW, lysine-fixable dextran in 0.2 M KCl) at one-cell stage and transplanted into non-labeled wild-type or pgk1 mutant embryos at the blastula stage. A total of 151 and 247 transplanted wild type cells were visualized in the ears of wild-type embryos and pgk1 mutants, respectively, (n= 8 otic vesicles each) and analyzed for the expression of etv5b.

Pharmacological treatments

The pharmacological inhibitors used in this study include Hexokinase inhibitor 2DG (2-Deoxy-Glucose, Sigma D8375), PFKFB3 inhibitor 3PO (Sigma SML1343), Pyruvate Dehydrogenase Kinase inhibitor DCA (dichloroacetate, Sigma 347795), Lactate Dehydrogenase inhibitor Galloflavin (Sigma SML0776), MCT1/2/4 inhibitors CHC (á-cyano-4-hydroxycinnamic acid, Sigma C2020) and UK-5099 (Sigma PZ0160), MEK inhibitor U1026 (Sigma U120) and PI3K inhibitor LY294002 (Sigma L9908). All inhibitors were dissolved in a 10 mm DMSO stock solution and diluted into the final indicated concentrations in fish water. Lactate treatments were performed with a 60% stock sodium DL-lactate solution (Sigma L1375) diluted 1 to 1000 in fish water to a final concentration of 6.7 mM, and buffered with 10 mm MES at pH 6.2. Note, lactate treatment has little effect on embryonic development at higher pH (not shown). Treatments were carried in a 24-well plate with a maximum of 15 embryos per well in a volume of 500 μl each.

Statistics

Student’s t-test was used for pairwise comparisons. Analysis of 3 or more samples was performed by one-way ANOVA and Tukey post-hoc HSD test. Sufficiency of sample sizes was based on estimates of confidence limits. Depending on genotype and probe, 2-8 embryos were utilized for the analysis of gene expression in serial sections; and 7-40 embryos were used for counts of Isl1/2+ SAG neurons or brn3c: GFP+ hair cells in whole mount specimens.

Whole Genome Sequencing and Mapping Analysis

Adult zebrafish males derived from the AB line were mutagenized using the alkylating agent N-ethyl-N-nitrosourea (ENU) and outcrossed to Isl2b: GFP + females as previously described (Riley and Grunwald, 1995). sagd1 mutants were identified in the screening process due to the decreased expression of Isl2b:Gfp in vestibular SAG neurons. For the mapping analysis, sagd1 mutants were outcrossed to the highly polymorphic zebrafish WIK line. A genomic pool of 50 homozygous mutants were collected from the intercrosses between 3 pairs of heterozygous sagd1 carriers of the AB/WIK hybrid line and sent for whole genome sequencing at Genomic Sequencing and Analysis Facility (GSAF) at the University of Texas-Austin. Sequence reads were aligned using the BWA alignment software. Bulk segregant linkage and homozygosity mapping of the aligned sequences were performed using Megamapper (Obholzer et al., 2012). Both approaches identified the pgk1 locus as the top candidate lesion in sagd1 mutants.

ACKNOWLEDGEMENTS

We thank Sarah Ferguson for her help in processing whole genome sequence data. This work was funded by NIH-NIDCD grant R01-DC03806.

REFERNCES

  1. ↵
    Aït-Ali, N., Fridlich, R., Millet-Puel, G., Clérin, E., Delalande, F., Jaillard, C., Blond, F., Perrocheau, L., Reichman, S., Byrne, Leah C., Olivier-Bandini, A., Bellalou, J., Moyse, E., Bouillaud, F., Nicol, X., Dalkara, D., van Dorsselaer, A., Sahel, J.-A., Léveillard, T., 2015. Rod-Derived Cone Viability Factor Promotes Cone Survival by Stimulating Aerobic Glycolysis. Cell. 161, 817–832.
    OpenUrlCrossRefPubMed
  2. ↵
    Abello, G., Khatri, S., Radosevic, M., Scotting, P. J., Giraldez, F., Alsina, B., 2010. Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode. Dev Biol. 339, 166–78.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    Agathocleous, M., Love, N. K., Randlett, O., Harris, J. J., Liu, J., Murray, A. J., Harris, W. A., 2012. Metabolic differentiation in the embryonic retina. Nature Cell Biology. 14, 859.
    OpenUrlCrossRefPubMedWeb of Science
  4. ↵
    Alsina, B., Abelló, G., Ulloa, E., Henrique, D., Pujades, C., Giraldez, F., 2004. FGF signaling is required for determination of otic neuroblasts in the chick embryo. Developmental Biology. 267, 119–134.
    OpenUrlCrossRefPubMedWeb of Science
  5. ↵
    Andermann, P., Ungos, J., Raible, D. W., 2002. Neurogenin1 defines zebrafish cranial sensory ganglia precursors. Developmental Biology. 251, 45–58.
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    Beckmann, B. M., Horos, R., Fischer, B., Castello, A., Eichelbaum, K., Alleaume, A.-M., Schwarzl, T., Curk, T., Foehr, S., Huber, W., Krijgsveld, J., Hentze, M. W., 2015. The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs. Nature Communications. 6, 10127.
    OpenUrl
  7. ↵
    Bermingham-McDonogh, O., Stone, J. S., Reh, T. A., Rubel, E. W., 2001. FGFR3 Expression during Development and Regeneration of the Chick Inner Ear Sensory Epithelia. Developmental Biology. 238, 247–259.
    OpenUrlCrossRefPubMedWeb of Science
  8. ↵
    Bermingham, N. A., Hassan, B. A., Price, S. D., Vollrath, M. A., Ben-Arie, N., Eatock, R. A., Bellen, H. J., Lysakowski, A., Zoghbi, H. Y., 1999. Math1: An Essential Gene for the Generation of Inner Ear Hair Cells. Science. 284, 1837–1841.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Botta, A., Delteil, F., Mettouchi, A., Vieira, A., Estrach, S., Négroni, L., Stefani, C., Lemichez, E., Meneguzzi, G., Gagnoux-Palacios, L., 2012. Confluence switch signaling regulates ECM composition and the plasmin proteolytic cascade in keratinocytes. Journal of Cell Science. 125, 4241–4252.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Brown, L. A., Amores, A., Schilling, T. F., Jowett, T., Baert, J. L., de Launoit, Y., Sharrocks, A. D., 1998. Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor. Oncogene. 17, 93–104.
    OpenUrlCrossRefPubMedWeb of Science
  11. ↵
    Bulusu, V., Prior, N., Snaebjornsson, M. T., Kuehne, A., Sonnen, K. F., Kress, J., Stein, F., Schultz, C., Sauer, U., Aulehla, A., 2017. Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Dev Cell. 40, 331–341.e4.
    OpenUrlCrossRef
  12. ↵
    Cairns, R. A., Harris, I. S., Mak, T. W., 2011. Regulation of cancer cell metabolism. Nature Reviews Cancer. 11, 85.
    OpenUrlCrossRefPubMedWeb of Science
  13. ↵
    Chen, P., Johnson, J. E., Zoghbi, H. Y., Segil, N., 2002. The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination. Development. 129, 2495–2505.
    OpenUrlCrossRefPubMedWeb of Science
  14. ↵
    Chirico, W. J., 2011. Protein release through nonlethal oncotic pores as an alternative nonclassical secretory pathway. BMC Cell Biology. 12, 46.
    OpenUrlCrossRefPubMed
  15. ↵
    Clem, B., Telang, S., Clem, A., Yalcin, A., Meier, J., Simmons, A., Rasku, M. A., Arumugam, S., Dean, W. L., Eaton, J., Lane, A., Trent, J. O., Chesney, J., 2008. Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth. Molecular Cancer Therapeutics. 7, 110–120.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Esen, E., Chen, J., Karner, Courtney M., Okunade, Adewole L., Patterson, Bruce W., Long, F., 2013. WNT-LRP5 Signaling Induces Warburg Effect through mTORC2 Activation during Osteoblast Differentiation. Cell Metabolism. 17, 745–755.
    OpenUrl
  17. ↵
    Farabegoli, F., Vettraino, M., Manerba, M., Fiume, L., Roberti, M., Di Stefano, G., 2012. Galloflavin, a new lactate dehydrogenase inhibitor, induces the death of human breast cancer cells with different glycolytic attitude by affecting distinct signaling pathways. European Journal of Pharmaceutical Sciences. 47, 729–738.
    OpenUrlPubMed
  18. ↵
    George, S. J., Johnson, J. L., Smith, M. A., Jackson, C. L., 2001. Plasmin-Mediated Fibroblast Growth Factor-2 Mobilisation Supports Smooth Muscle Cell Proliferation in Human Saphenous Vein. Journal of Vascular Research. 38, 492–501.
    OpenUrlCrossRefPubMedWeb of Science
  19. ↵
    Gou, Y., Vemaraju, S., Sweet, E. M., Kwon, H.-J., Riley, B. B., 2018. sox2 and sox3 Play unique roles in development of hair cells and neurons in the zebrafish inner ear. Developmental Biology. 435, 73–83.
    OpenUrl
  20. ↵
    Gu, W., Gaeta, X., Sahakyan, A., Chan, Alanna B., Hong, Candice S., Kim, R., Braas, D., Plath, K., Lowry, William E., Christofk, Heather R., 2016. Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State. Cell Stem Cell. 19, 476–490.
    OpenUrlCrossRefPubMed
  21. ↵
    Hagan, K. W., Ruiz-Echevarria, M. J., Quan, Y., Peltz, S. W., 1995. Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover. Mol Cell Biol. 15, 809–23.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Halestrap, A. P., 1975. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J. 148, 85–96.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Halestrap, A. P., Denton, R. M., 1974. Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by alpha-cyano-4-hydroxycinnamate. Biochem J. 138, 313–6.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Hawkins, T. A., Cavodeassi, F., Erdélyi, F., Szabó, G., Lele, Z., 2008. The small molecule Mek1/2 inhibitor U0126 disrupts the chordamesoderm to notochord transition in zebrafish. BMC Dev Biol. 8, 42.
    OpenUrlCrossRefPubMed
  25. ↵
    Hayashi, T., Cunningham, D., Bermingham-McDonogh, O., 2007. Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Developmental Dynamics. 236, 525–533.
    OpenUrlCrossRefPubMedWeb of Science
  26. ↵
    Hayashi, T., Ray, C. A., Bermingham-McDonogh, O., 2008. Fgf20 Is Required for Sensory Epithelial Specification in the Developing Cochlea. The Journal of Neuroscience. 28, 5991–5999.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Ho, M.-Y., Tang, S.-J., Ng, W. V., Yang, W., Leu, S.-J. J., Lin, Y.-C., Feng, C.-K., Sung, J.-S., Sun, K.-H., 2010. Nucleotide-binding domain of phosphoglycerate kinase 1 reduces tumor growth by suppressing COX-2 expression. Cancer Science. 101, 2411–2416.
    OpenUrlCrossRefPubMed
  28. ↵
    Jacques, B. E., Montcouquiol, M. E., Layman, E. M., Lewandoski, M., Kelley, M. W., 2007. Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea. Development. 134, 3021–3029.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Jiang, L., Romero-Carvajal, A., Haug, J. S., Seidel, C. W., Piotrowski, T., 2014. Gene-expression analysis of hair cell regeneration in the zebrafish lateral line. Proceedings of the National Academy of Sciences. 111, E1383–E1392.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Jung, Y., Shiozawa, Y., Wang, J., Wang, J., Wang, Z., Pedersen, E. A., Lee, C. H., Hall, C. L., Hogg, P. J., Krebsbach, P. H., Keller, E. T., Taichman, R. S., 2009. Expression of PGK1 by Prostate Cancer Cells Induces Bone Formation. Molecular Cancer Research. 7, 1595–1604.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Kantarci, H., Edlund, R. K., Groves, A. K., Riley, B. B., 2015. Tfap2a Promotes Specification and Maturation of Neurons in the Inner Ear through Modulation of Bmp, Fgf and Notch Signaling. PLoS Genetics. 11, e1005037.
    OpenUrl
  32. ↵
    Kantarci, H., Gerberding, A., Riley, B. B., 2016. Spemann organizer gene Goosecoid promotes delamination of neuroblasts from the otic vesicle. Proceedings of the National Academy of Sciences. 113, E6840–E6848.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Kato, M., Li, J., Chuang, J. L., Chuang, D. T., 2007. Distinct Structural Mechanisms for Inhibition of Pyruvate Dehydrogenase Kinase Isoforms by AZD7545, Dichloroacetate, and Radicicol. Structure. 15, 992–1004.
    OpenUrlCrossRefPubMed
  34. ↵
    Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F., 1995. Stages of embryonic development of the zebrafish. Dev Dyn. 203, 253–310.
    OpenUrlCrossRefPubMedWeb of Science
  35. ↵
    Korzh, V., Sleptsova, I., Liao, J., He, J., Gong, Z., 1998. Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev Dyn. 213, 92–104.
    OpenUrlCrossRefPubMedWeb of Science
  36. ↵
    Ku, Y.-C., Renaud, N. A., Veile, R. A., Helms, C., Voelker, C. C. J., Warchol, M. E., Lovett, M., 2014. The Transcriptome of Utricle Hair Cell Regeneration in the Avian Inner Ear. The Journal of Neuroscience. 34, 3523–3535.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Lassiter, R. N., Reynolds, S. B., Marin, K. D., Mayo, T. F., Stark, M. R., 2009. FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation. Dev Dyn. 238, 1073–82.
    OpenUrlCrossRefPubMed
  38. ↵
    Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., Hogg, P. J., 2000. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase. Nature. 408, 869.
    OpenUrlCrossRefPubMedWeb of Science
  39. ↵
    Lee, Dong C., Sohn, Hyun A., Park, Z.-Y., Oh, S., Kang, Yun K., Lee, K.-m., Kang, M., Jang, Ye J., Yang, S.-J., Hong, Young K., Noh, H., Kim, J.-A., Kim, Dong J., Bae, K.-H., Kim, Dong M., Chung, Sang J., Yoo, Hyang S., Yu, D.-Y., Park, Kyung C., Yeom, Young I., 2015. A Lactate-Induced Response to Hypoxia. Cell. 161, 595–609.
    OpenUrlCrossRefPubMed
  40. ↵
    Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. D., 2005. Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration. Development. 132, 5173–5183.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Liao, Y., Castello, A., Fischer, B., Leicht, S., Föehr, S., Frese, Christian K., Ragan, C., Kurscheid, S., Pagler, E., Yang, H., Krijgsveld, J., Hentze, Matthias W., Preiss, T., 2016. The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease. Cell Reports. 16, 1456–1469.
    OpenUrl
  42. ↵
    Liberti, M. V., Locasale, J. W., 2016. The Warburg Effect: How Does it Benefit Cancer Cells? Trends in Biochemical Sciences. 41, 211–218.
    OpenUrlCrossRefPubMed
  43. ↵
    Ma, Q., Chen, Z., Barrantes, I. d. B., Luis de la Pompa, J., Anderson, D. J., 1998. neurogenin1 Is Essential for the Determination of Neuronal Precursors for Proximal Cranial Sensory Ganglia. Neuron. 20, 469–482.
    OpenUrlCrossRefPubMedWeb of Science
  44. ↵
    Magarinos, M., Aburto, M. R., Sanchez-Calderon, H., Munoz-Agudo, C., Rapp, U. R., Varela-Nieto, I., 2010. RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development. PLoS One. 5, e14435.
    OpenUrlCrossRefPubMed
  45. ↵
    Maier, E. C., Whitfield, T. T., 2014. RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities. PLoS Genetics. 10, e1004858.
    OpenUrl
  46. ↵
    Manerba, M., Vettraino, M., Fiume, L., Di Stefano, G., Sartini, A., Giacomini, E., Buonfiglio, R., Roberti, M., Recanatini, M., 2012. Galloflavin (CAS 568-80-9): a novel inhibitor of lactate dehydrogenase. ChemMedChem. 7, 311–7.
    OpenUrlPubMed
  47. ↵
    Mansour, S. L., Goddard, J. M., Capecchi, M. R., 1993. Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. Development. 117, 13–28.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Mansour, S. L., Twigg, S. R. F., Freeland, R. M., Wall, S. A., Li, C., Wilkie, A. O. M., 2009. Hearing loss in a mouse model of Muenke syndrome. Hum Mol Genet. 18, 43–50.
    OpenUrlCrossRefPubMedWeb of Science
  49. ↵
    Maulding, K., Padanad, M. S., Dong, J., Riley, B. B., 2014. Mesodermal Fgf10b cooperates with other fibroblast growth factors during induction of otic and epibranchial placodes in zebrafish. Dev Dyn. 243, 1275–85.
    OpenUrlCrossRef
  50. ↵
    Meddahi, A., Lemdjabar, H., Caruelle, J. P., Barritault, D., Hornebeck, W., 1995. Inhibition by dextran derivatives of FGF-2 plasmin-mediated degradation. Biochimie. 77, 703–706.
    OpenUrl
  51. ↵
    Menendez-Montes, I., Escobar, B., Palacios, B., Gómez, M. J., Izquierdo-Garcia, J. L., Flores, L., Jiménez-Borreguero, L. J., Aragones, J., Ruiz-Cabello, J., Torres, M., Martin-Puig, S., 2016. Myocardial VHL-HIF Signaling Controls an Embryonic Metabolic Switch Essential for Cardiac Maturation. Dev Cell. 39, 724–739.
    OpenUrlCrossRef
  52. ↵
    Millimaki, B. B., Sweet, E. M., Dhason, M. S., Riley, B. B., 2007. Zebrafish atoh1 genes: classic proneural activity in the inner ear and regulation by Fgf and Notch. Development. 134, 295–305.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Millimaki, B. B., Sweet, E. M., Riley, B. B., 2010. Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear. Dev Biol. 338, 262–9.
    OpenUrlCrossRefPubMed
  54. ↵
    Montero, J.-A., Kilian, B., Chan, J., Bayliss, P. E., Heisenberg, C.-P., 2003. Phosphoinositide 3-Kinase Is Required for Process Outgrowth and Cell Polarization of Gastrulating Mesendodermal Cells. Current Biology. 13, 1279–1289.
    OpenUrlCrossRefPubMed
  55. ↵
    Nechiporuk, A., Linbo, T., Raible, D. W., 2005. Endoderm-derived Fgf3 is necessary and sufficient for inducing neurogenesis in the epibranchial placodes in zebrafish. Development. 132, 3717–3730.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Nirenberg, M. W., Hogg, J. F., 1958. Inhibition of Anaerobic Glycolysis in Ehrlich Ascites Tumor Cells by 2-Deoxy-D-Glucose. Cancer Research. 18, 518–521.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    O’Hagan, R. C., Tozer, R. G., Symons, M., McCormick, F., Hassell, J. A., 1996. The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades. Oncogene. 13, 1323–1333.
    OpenUrlPubMedWeb of Science
  58. ↵
    Obholzer, N., Swinburne, I. A., Schwab, E., Nechiporuk, A. V., Nicolson, T., Megason, S. G., 2012. Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing. Development. 139, 4280–4290.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Oginuma, M., Moncuquet, P., Xiong, F., Karoly, E., Chal, J., Guevorkian, K., Pourquié, O., 2017. A Gradient of Glycolytic Activity Coordinates FGF and Wnt Signaling during Elongation of the Body Axis in Amniote Embryos. Dev Cell. 40, 342–353.e10.
    OpenUrlCrossRef
  60. ↵
    Ono, K., Kita, T., Sato, S., O’Neill, P., Mak, S.-S., Paschaki, M., Ito, M., Gotoh, N., Kawakami, K., Sasai, Y., Ladher, R. K., 2014. FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation. PLoS Genetics. 10, e1004118.
    OpenUrl
  61. ↵
    Özbudak, E. M., Tassy, O., Pourquié, O., 2010. Spatiotemporal compartmentalization of key physiological processes during muscle precursor differentiation. Proceedings of the National Academy of Sciences. 107, 4224–4229.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    Peng, M., Yin, N., Chhangawala, S., Xu, K., Leslie, C. S., Li, M. O., 2016. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism. Science. 354, 481–484.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Phillips, B. T., Bolding, K., Riley, B. B., 2001. Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev Biol. 235, 351–65.
    OpenUrlCrossRefPubMedWeb of Science
  64. ↵
    Philp, A., Macdonald, A. L., Watt, P. W., 2005. Lactate – a signal coordinating cell and systemic function. Journal of Experimental Biology. 208, 4561–4575.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Pirvola, U., Spencer-Dene, B., Xing-Qun, L., Kettunen, P., Thesleff, I., Fritzsch, B., Dickson, C., Ylikoski, J., 2000. FGF/FGFR-2(IIIb) Signaling Is Essential for Inner Ear Morphogenesis. The Journal of Neuroscience. 20, 6125–6134.
    OpenUrlAbstract/FREE Full Text
  66. ↵
    Pirvola, U., Ylikoski, J., Trokovic, R., Hébert, J. M., McConnell, S. K., Partanen, J., 2002. FGFR1 Is Required for the Development of the Auditory Sensory Epithelium. Neuron. 35, 671–680.
    OpenUrlCrossRefPubMedWeb of Science
  67. ↵
    Pittman, A. J., Law, M. Y., Chien, C. B., 2008. Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points. Development. 135, 2865–71.
    OpenUrlAbstract/FREE Full Text
  68. ↵
    Puligilla, C., Feng, F., Ishikawa, K., Bertuzzi, S., Dabdoub, A., Griffith, A. J., Fritzsch, B., Kelley, M. W., 2007. Disruption of fibroblast growth factor receptor 3 signaling results in defects in cellular differentiation, neuronal patterning, and hearing impairment. Developmental Dynamics. 236, 1905–1917.
    OpenUrlCrossRefPubMedWeb of Science
  69. ↵
    Raft, S., Koundakjian, E. J., Quinones, H., Jayasena, C. S., Goodrich, L. V., Johnson, J. E., Segil, N., Groves, A. K., 2007. Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development. Development. 134, 4405–15.
    OpenUrlAbstract/FREE Full Text
  70. ↵
    Raible, F., Brand, M., 2001. Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development. Mech Dev. 107, 105–117.
    OpenUrlCrossRefPubMedWeb of Science
  71. ↵
    Riley, B. B., Grunwald, D. J., 1995. Efficient induction of point mutations allowing recovery of specific locus mutations in zebrafish. Proc Natl Acad Sci U S A. 92, 5997–6001.
    OpenUrlAbstract/FREE Full Text
  72. ↵
    Roehl, H., Nüsslein-Volhard, C., 2001. Zebrafish pea3 and erm are general targets of FGF8 signaling. Current Biology. 11, 503–507.
    OpenUrlCrossRefPubMedWeb of Science
  73. ↵
    Ruiz-Echevarria, M. J., Munshi, R., Tomback, J., Kinzy, T. G., Peltz, S. W., 2001. Characterization of a General Stabilizer Element That Blocks Deadenylation-dependent mRNA Decay. Journal of Biological Chemistry. 276, 30995–31003.
    OpenUrlAbstract/FREE Full Text
  74. ↵
    Sá, J. V., Kleiderman, S., Brito, C., Sonnewald, U., Leist, M., Teixeira, A. P., Alves, P. M., 2017. Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis. Neurochem Res. 42, 244–253.
    OpenUrl
  75. ↵
    San-Millán, I., Brooks, G. A., 2017. Reexamining cancer metabolism: lactate production for carcinogenesis could be the purpose and explanation of the Warburg Effect. Carcinogenesis. 38, 119–133.
    OpenUrlPubMed
  76. ↵
    Schmidt, A., Echtermeyer, F., Alozie, A., Brands, K., Buddecke, E., 2005. Plasmin-and Thrombin-accelerated Shedding of Syndecan-4 Ectodomain Generates Cleavage Sites at Lys114–Arg115 and Lys129–Val130 Bonds. Journal of Biological Chemistry. 280, 34441–34446.
    OpenUrlAbstract/FREE Full Text
  77. ↵
    Shetty, P., Velusamy, T., Bhandary, Y. P., Liu, M. C., Shetty, S., 2010. Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase. Mol Cell Biochem. 335, 235–47.
    OpenUrlPubMed
  78. ↵
    Shetty, S., Muniyappa, H., Halady, P. K. S., Idell, S., 2004. Regulation of Urokinase Receptor Expression by Phosphoglycerate Kinase. Am J Respir Cell Mol Biol. 31, 100–106.
    OpenUrlCrossRefPubMedWeb of Science
  79. ↵
    Shim, K., Minowada, G., Coling, D. E., Martin, G. R., 2005. Sprouty2, a Mouse Deafness Gene, Regulates Cell Fate Decisions in the Auditory Sensory Epithelium by Antagonizing FGF Signaling. Dev Cell. 8, 553–564.
    OpenUrlCrossRefPubMedWeb of Science
  80. ↵
    Terriente, J., Gerety, S. S., Watanabe-Asaka, T., Gonzalez-Quevedo, R., Wilkinson, D. G., 2012. Signalling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis. Development. 139, 2978–87.
    OpenUrlAbstract/FREE Full Text
  81. ↵
    Tixier, V., Bataillé, L., Etard, C., Jagla, T., Weger, M., DaPonte, J. P., Strähle, U., Dickmeis, T., Jagla, K., 2013. Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proceedings of the National Academy of Sciences. 110, 18982–18987.
    OpenUrlAbstract/FREE Full Text
  82. ↵
    Vemaraju, S., Kantarci, H., Padanad, M. S., Riley, B. B., 2012. A spatial and temporal gradient of Fgf differentially regulates distinct stages of neural development in the zebrafish inner ear. PLoS Genet. 8, e1003068.
    OpenUrlCrossRefPubMed
  83. ↵
    Wang, J., Wang, J., Dai, J., Jung, Y., Wei, C.-L., Wang, Y., Havens, A. M., Hogg, P. J., Keller, E. T., Pienta, K. J., Nor, J. E., Wang, C.-Y., Taichman, R. S., 2007. A Glycolytic Mechanism Regulating an Angiogenic Switch in Prostate Cancer. Cancer Research. 67, 149–159.
    OpenUrlAbstract/FREE Full Text
  84. ↵
    Wang, J., Ying, G., Wang, J., Jung, Y., Lu, J., Zhu, J., Pienta, K. J., Taichman, R. S., 2010. Characterization of Phosphoglycerate Kinase-1 Expression of Stromal Cells Derived from Tumor Microenvironment in Prostate Cancer Progression. Cancer Research. 70, 471–480.
    OpenUrlAbstract/FREE Full Text
  85. ↵
    Wang, Y.-H., Israelsen, William J., Lee, D., Yu, Vionnie W. C., Jeanson, Nathaniel T., Clish, Clary B., Cantley, Lewis C., Vander Heiden, Matthew G., Scadden, David T., 2014. Cell-State-Specific Metabolic Dependency in Hematopoiesis and Leukemogenesis. Cell. 158, 1309–1323.
    OpenUrlCrossRefPubMed
  86. ↵
    Whitfield, T. T., 2015. Development of the inner ear. Current Opinion in Genetics & Development. 32, 112–118.
    OpenUrl
  87. ↵
    Woods, C., Montcouquiol, M., Kelley, M. W., 2004. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nature Neuroscience. 7, 1310.
    OpenUrlCrossRefPubMedWeb of Science
  88. ↵
    Xiao, T., Roeser, T., Staub, W., Baier, H., 2005. A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection. Development. 132, 2955–67.
    OpenUrlAbstract/FREE Full Text
  89. ↵
    Yang, J., Ruchti, E., Petit, J.-M., Jourdain, P., Grenningloh, G., Allaman, I., Magistretti, P. J., 2014. Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons. Proceedings of the National Academy of Sciences. 111, 12228–12233.
    OpenUrlAbstract/FREE Full Text
  90. ↵
    Zheng, X., Boyer, L., Jin, M., Mertens, J., Kim, Y., Ma, L., Hamm, M., Gage, F. H., Hunter, T., 2016. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. eLife. 5, e13374.
    OpenUrlCrossRef
  91. ↵
    Znosko, W. A., Yu, S., Thomas, K., Molina, G. A., Li, C., Tsang, W., Dawid, I. B., Moon, A. M., Tsang, M., 2010. Overlapping functions of Pea3 ETS transcription factors in FGF signaling during zebrafish development. Dev Biol. 342, 11–25.
    OpenUrlCrossRefPubMed
  92. ↵
    Zuo, R.-J., Gu, X.-W., Qi, Q.-R., Wang, T.-S., Zhao, X.-Y., Liu, J.-L., Yang, Z.-M., 2015. Warburg-like Glycolysis and Lactate Shuttle in Mouse Decidua during Early Pregnancy. Journal of Biological Chemistry. 290, 21280–21291.
    OpenUrlAbstract/FREE Full Text
Back to top
Posted August 15, 2018.
Download PDF
Email
The Warburg Effect and lactate signaling augment Fgf signaling to promote sensory-neural development in the otic vesicle
Husniye Kantarci, Yunzi Gou, Bruce B. Riley
bioRxiv 392548; doi: https://doi.org/10.1101/392548
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools

Subject Area