Figueiredo AL et al. (2017), PFKFB4 control of AKT signaling is essential fo...

XB-ART-54151

Development 2017 Nov 15;14422:4183-4194. doi: 10.1242/dev.157644.

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PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

Figueiredo AL, Maczkowiak F, Borday C, Pla P, Sittewelle M, Pegoraro C, Monsoro-Burq AH.

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Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate n-cadherin and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

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Species referenced: Xenopus laevis
Genes referenced: acta1 akt1 bmpr1a casp3.2 cdh2 ctnnb1 eef1a1 fgf8 fn1 foxd3 grn h2bc21 hes4 hk1 hoxb9 itk krt12.4 mapk1 msx1 myc myod1 odc1 otx2 pax3 pfkfb1 pfkfb2 pfkfb3 pfkfb4 pfkp pik3ca psmd6 slc12a3 snai2 sox10 sox2 sox9 twist1 wnt7b zic1
GO keywords: neural crest cell migration [+]
???displayArticle.antibodies??? Acta1 Ab6 Akt1 Ab1 Akt Ab10 Casp3 Ab1
???displayArticle.morpholinos??? ctnnb1 MO1 fgf8 MO1 pax3 MO1 pfkfb4 MO1 pfkfb4 MO2 sox9 MO1 tfap2a MO1

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	Fig. 1. PFKFB4 promotes NB and NC formation. (A) NC is induced during neural plate stage (st.12.5-14). Expression of NB specifiers (red) is robustly established lateral to the neural plate, expression of early NC specifiers (snail2, foxd3) is faintly initiated (light pink). (B,C) During neural fold elevation (st.14-17, B), future NC cells progressively acquire their definitive specification and specific cellular properties (e.g. survival, expression of late NC specifiers, cadherin switch) enabling them to undergo EMT and migration (st.18-19, C). (D-F,H,I) pfkfb4 is enriched in the NB/NC in neurulas and tadpoles. (J) pfkfb3 is expressed in the neural tube. Sense probes are shown inG( pfkfb4) and K ( pfkfb3). Neurula stages: late gastrula/early neural plate stage (st.12, D); end of neural plate stage (st.14, E); neural fold stage (st.16, F); neural tube closure/end of neurulation (st.19, H); tailbud stage (st.22, I). E-H,J,K: dorsal views, anterior to the right. D,I: side views. Scale bar: 1 mm. (L-N) Cross-sections through the mid-neurula anterior neural plate (st.14) show snail2 and pfkfb4 expression in NB/NC. (L) The notochord, neural plate and paraxial mesoderm are outlined on Hematoxylin and Eosin-stained sections. (M,N) Vibratome sections. Arrow indicates the midline. Arrowheads indicate the st.14 NB (red bar). Scale bars: 200 Î¼m. (O-S) pfkfb4 gain-of-function expanded the st.14 NB (O,P) and premigratory NC (st.14, Q; st.18, R, S). Migrating NC streams were expanded (st. 22-24, T, Z). In contrast, pan- or regional neural plate (sox2, otx2, hoxb9), ectoderm (ep. ker.) or paraxial mesoderm (myod) markers were unaffected (U-Y). O-S,U-X: dorsal views; T,Z: side views; Y: dorsal-posterior view. Scale bar: 500 Î¼m. co, control side; inj, injected side. Phenotype scores are shown in Table S6.
	Fig. 2. Moderate PFKFB4 depletion results in severe craniofacial defects in tadpoles. (A) pfkfb4 mRNA levels were quantified on neurula dorsal tissues by RT-qPCR. Injections at â€˜high concentrationâ€™ (10 ng/cell in 4-cell-stage embryos) reduced pfkfb4 levels by 52% and caused developmental arrest. A â€˜milderâ€™ concentration (5 ng/cell in 4-cell-stage embryos) decreased pfkfb4 mRNA by 32% (low-level depletion), and allowed development to swimming tadpoles. (B-E) Craniofacial morphology in tadpoles st.45-46. Low-level PFKFB4MO depletion reduced the size of craniofacial structures on the injected side. The branchial arch cartilages area was severely diminished on the injected side. Lower doses did not significantly reduce cartilage area, or affect head morphology. n.s., not significant. (C) Head morphology, dorsal view; arrowhead indicates the injected side. (D) Dissected jaw and branchial arches cartilages, ventral view. inj, injected side. (E) Schematic of the image shown in D. Dashed red line indicates midline; light blue, Meckelâ€™s cartilage; mid blue, ceratohyal cartilage; dark blue, branchial cartilage. Scale bar: 500 Î¼m. Phenotype scores are shown in Table S7.
	Fig. 3. PFKFB4 low-level depletion delays NC early specification, causes retention of NB character, and impairs NC late specification and migration. (A) At st.14, snail2 expression was severely reduced, or abolished, on the injected side. (B) At st.18, sibling embryos had recovered snail2 expression. (C,D) Whereas sox10 expression was mainly unaffected, twist1 was severely impaired. (E-H) In contrast, expression of the immature NC marker hes4 was expanded, as were some NB markers, either strongly ( pax3) or moderately (zic1). Other NB markers were unperturbed (msx1). (I-K) Neural plate (sox2), non-neural ectoderm (ep. ker.) and paraxial mesoderm (myod) seemed to be unaffected. A-K: dorsal views. (L) Percentage of embryos with each phenotype, i.e. diminished, increased or normal expression. Snail2 score at st.14 is indicated as the first bar, then several gene scores at st.18 are indicated in the following ten bars. (M-R) St.24 tailbud embryos exhibited a severe NC migration defect (M-Q). Sox2 expression appeared grossly unaffected, despite marginal reduction of optic vesicle size (R). The injected side (inj) is compared with the control side (co) in side views (M,N,P,Q, anterior to the right) or frontal views (O,R; red arrow on injected side). (S) Co-injection of pfkfb4 mRNA with PFKFBMO rescued both sox10/twist1 alterations of expression and NC migration defects in a significant proportion of the embryos, compared with PFKFB4MO injections alone. sox10 and twist1 expression were restored or increased a majority of the embryos. Scale bar: 500 Î¼m. Phenotype scores are shown in Table S8.
	Fig. 4. Inducible depletion demonstrates that PFKFB4 has independent roles in NC specification and migration.We co-injected a UV-cleavable senseMO (PhMO) to block PFKFB4MO until the desired developmental stage and monitored twist1 expression at st.18. (A-H) Strategy of phMO validation: lacZ control injections (A), unmasked PFKFB4MO, â€˜highâ€™ dose (B), PhMO/MO without UV illumination (C; uninduced, u.i.); UV illumination at early NC specification st.12.5 (D-G). D-F show two examples of diminished phenotype and one example of abolished phenotype at st.14;Gshows an example of diminished phenotype at st.19. (H) Percentage of embryos with each phenotype, i.e. abolished, diminished, increased or normal twist1 expression. lacZ injections did not alter twist1 pattern. PFKFB4MO severely blocked twist1 expression. twist1 expression was decreased in PhMO/MO-injected embryos UV illuminated immediately after injection (a.i.) compared with uninduced PhMO/MO-injected embryos. Pfkfb4 depletion, activated at st.12.5, decreased twist1 expression strongly or moderately. This effect persisted until EMT and early NC emigration (G; st.20). (A-G): dorsal views. (I-W) To deplete pfkfb4 during EMT/early migration, st.18 neurulas were UV illuminated and analyzed at tadpole st.24. twist1 expression was normal in embryos injected with lacZ (I,J), or with PhMO/MO but not illuminated (M,N). Cells injected with PFKFB4MO alone died as expected (K,L). PhMO/MO-injected embryos UV illuminated at st.18 exhibited three phenotypes classes: â€˜migrationâ€™ (O, P), â€˜staining and migrationâ€™ (Q,R) and â€˜severe stainingâ€™ defects (S,T). We compared the percentage of each phenotype (U), of migration distance (V) and of NC stream area (W; twist1-expressing area) with the contralateral side. Scale bars: 500 Î¼m. All phenotype differences were statistically significant between illuminated PhMO/MO-injected embryos, and either lacZ-injected, or uninduced PhMO/MO-injected embryos (P<0.05; P<0.01; **P<0.001; ns, not significant). Error bars represent s.e.m. Phenotype scores are shown in Table S9.
	Fig. 5. AKT signaling mediates the effect of PFKFB4 on premigratory NC maturation. (A) In embryos, PFKFB4 regulates AKT signaling in addition to glycolysis. Dissected morphant st.14 NB or st.17 NC displayed decreased AKT signaling. (B) AKT regulates many aspects of cell homeostasis including cell proliferation, cell survival, and cell metabolism. However, EdU incorporation showed that cell proliferation rate was normal after PFKFB4MO injections. Error bars represent s.e.m. n.s., not significant. (C,D) PFKFB4MO affected late NC specifier twist1 expression, whereas the NC stem cell marker cmyc was normally activated. (E-H) Pharmacological treatment during neural fold stage (st.14-18) showed that blocking MAPK signaling (E,G) did not affect NC development, but blocking PI3K-AKT signaling (F,H) affected twist1 but not cmyc, thus phenocopying the PFKFB4MO effect. (I,J) Co-injection of PFKFB4MO with a constitutively AKT (caAkt; blue arrows) rescued the morphant twist1 phenotype (red arrowheads): two sibling embryos for each injection are shown. St.18 pfkfb4 morphants presented diminished twist1. In contrast, siblings co-injected with PFKFB4MO and caAkt mRNA had normal twist1 expression in the majority of cases. Scale bars: 500 Î¼m. Phenotype scores are shown in Tables S8, S13.
	Fig. 6. PFKFB4 morphant NC progenitors fail to undergo EMT and migration. (A,B) When transplanted into a wild-type host embryo, GFPlabeled wild-type NC efficiently migrates and populates the host branchial arches, regardless of the size of the grafted tissue, i.e. large graft (A) or small graft (B). Scale bar: 500 Î¼m. (C,D) In contrast, PFKFB4 morphant NCexhibited defective healing, resulting in small-size grafts, which yielded few, if any, migratory NCC into the host craniofacial area. (C) Small graft without migratory cells; (D) small graft with few cells reaching the branchial arches. In D, gfp was detected by WISH to enhance individual cell visualization. (E) The effect of PFKFB4MO on N-cadherin expression (RT-qPCR on dissected explants), either prior to EMT (NB, st.14), or in premigratory NC (st.17). Error bars represent s.e.m. *Pâ‰¤0.05. (F-J) Wild-type (F,G; gfp only, no MO) or morphant (G,I; MO 10 ng) cells were lineage traced in vivo, on embryos injected in the prospective neural fold unilaterally. At tailbud stage, control cells (G) efficiently populated branchial arches and were also found in brain and eye (J). In contrast, morphant cells failed to populate branchial arches (I) in a dosedependent manner (J), but were normally distributed in the brain and eye (J). (K-R) NB tissue was dissected prior to EMT (st.14) and plated onto fibronectin. Cell behavior was followed by time-lapse videomicroscopy.Wild-type NC (K-N) adheres efficiently, and undergoes EMT (L), cell scattering and migration (M, N). Morphant NC (O-R) presented poor adherence, delayed (P) and inefficient EMT (Q), and few emigrating cells (R). Time after plating is indicated (h:min). Scale bar: 160 Î¼m. Phenotype scores are indicated in Table S10.
	Fig. 7. Glycolysis and PI3K-AKT signaling impact NC migration similarly to PFKFB4 low-level depletion, and activating AKT signaling rescues PFKFB4 downregulation. (A-H) When glycolysis (2DG) or PI3K-AKT pathway (LY294002) were blocked during EMT and migration (st.18-24) both treatments severely affected NC migration at st.24 (as revealed by twist1 and sox10 expression). (I-L) At st.45, tadpoles treated during NC migration then grown in control medium, exhibited general head morphology defects, including eye defects and smaller branchial cartilages. (I,K) Sibling controls; (J) 2DG; (L) LY294002. A-H show side views; I-L dorsal views. (M-P) At tadpole st.45, morphant sides were severely affected (M,P), whereas activation of Akt signaling (N,P) did not affect overall craniofacial morphogenesis. Tadpoles coinjected with PFKFB4MO and caAkt (O,P) were largely rescued, with 66% of embryos with injected side symmetrical to contralateral side. (M) Red bar indicates eye distance from the midline on the morphant side; blue bar indicates control distance. Both bars are aligned below for comparison. (N,O) On both sides, the same blue bar measures eye distance from the midline. Arrowheads indicate the injected side. Scale bar: 500 Î¼m. Phenotype scores are shown in Tables S11, S12.
	Fig. 8. Pfkfb4 activation during neural fold stage is regulated by the NCGRN. (A-X) Unilateral depletion of members of the NC-GRN modulated pfkfb4 activation at the NB (st.14) and NC (st.18). (A-H) Blockade of WNT and FGF signaling strongly decreased snail2 and pfkfb4 expression. (I-L). Conversely, BMP signaling downregulation upregulated snail2 and pfkfb4 expression, laterally to the NB. (M-T) Knockdown of the NC specifiers PAX3 and TFAP2a resulted in loss of snail2 and pfkfb4. (U-X) Knockdown of the NC specifier SOX9 depleted snail2 and pfkfb4. Dorsal views, injected side on the right. Scale bar: 500 Î¼m. Phenotype scores are shown in Table S13.
	Fig. 9. Model of PFKFB4-controlled AKT signaling in NC maturation, EMT and migration. During neurulation, in controls (left side), NC is induced at the neural plate stage with weak snail2 expression. This induction is then strengthened during neural fold stage, under the action of the NC-GRN, which notably activates pfkfb4 expression in NC progenitors. This second phase allows acquisition of cellular ability to undergo EMT and migration upon neural tube closure. When AKT signaling is defective (right side), either after PFKFB4 low-level depletion, or using pharmacological inhibitors, delays in NC specification cascade occur, incomplete maturation is observed, and NC fails to undergo EMT. When PFKFB4 and AKT functions are prevented after EMT, fewer NCCs migrate resulting into reduced craniofacial skeletal elements. Hence, each step of NCearly development relies upon continuous and elevated AKT signaling, sustained by PFKFB4, itself triggered by the NC-GRN.
	Fig. S1. Schematics of glycolysis. Glucose phosphorylation by hexokinase is the first irreversible step of glycolysis. The second and rate-limiting step of glycolysis is catalyzed by PFK1. This step is tightly controlled in cells, by the levels of Fructose-2.6-bisphosphate, potent allosteric regulator of PFK1. Fructose-2.6-bisphosphate is synthesized by PFKFB1-4 enzymes, which thus control the rate of glycolysis in the cells. PFKFB3 and PFKFB4, with a strong kinase activity over their phosphatase activity, promote Fructose-2.6-bisphosphate synthesis and stimulate glycolysis. In addition to their classic role in glycolysis regulation, recent unconventional roles of the PFKFB enzymes have been described in cancer or development (see text). Development 144: doi:10.1242/dev.157644: Supplementary information Development • Supplementary information
	Fig. S2. Pfkfb4 is specifically upregulated in the neural border and neural crest during neurulation. (A-J). Transverse sections through the anterior neural plate at mid and late neurula stages (stage 14 and 18, respectively) allow comparing Snail2 and Pfkfb4 expression in the neural crest (Snail2 and Pfkfb4), neural plate (Pfkfb3) and somites (Pfkfb1). (A,F): The notochord, neural plate and paraxial mesoderm are outlined on hematoxylin-eosin stained paraffin sections. (B-E,G-J) Vibratome sections of WISH-stained embryos. The arrow indicates the midline and the notochord. The blue arrowheads indicate in situ hybridization staining position. Red bar positions the neural border at stage 14. Scale bar = 200Î¼m. (K) A quantitative analysis of Xenopus laevis Pfkfb1-4 expression levels shows that Pfkfb4 is specifically enriched at the neural border of frog neurulas (red), compared to the anterior neural fold (yellow). All values are normalized to Odc expression and to the average expression in a sibling whole embryo lysed at late neurula stage 18 (blue, value = 1). Snail2 expression is used to monitor the quality of dissected tissues and shows high and specific expression at the neural border.
	Fig. S3. Patterning defects in NC are not due to increased cell death, after low-level PFKFB4 depletion in vivo. In this study, we use a low-level depletion of PFKFB4, with reduced MO concentrations, in order to bypass the earlier developmental arrest observed in previous study, when PFKFB4 severely depleted (above 60% mRNA depletion) and prevents gastrulating ectoderm specification (Pegoraro et al., 2015). Importantly, this severe depletion was rescued by adding back a MO-resistant pfkfb4 mRNA, assessing both MO specificity and lack of non-specific toxicity (Pegoraro et al., 2015). (A) Here, we check that the morphant cells do not undergo elevated cell death at later stages of development by analyzing mid-neurulas at stage 14 and the end of neurulation stage 18. (BC) We find less than 30% of embryos with caspase-positive cells in the lacZ-injected area (B), in conditions when over 70% of sibling embryos show deficient twist expression (C, either "strong", "moderate" or "mild" decrease). The number of caspase-positive cells is slightly more elevated than in uninjected or lacZ-only-injected embryos, or in ventrally-injected morphants, but it remains limited to few cells and cannot account for the incidence of the patterning defects. Representative exemples of the majoritary phenotypes are shown for each condition, i.e. "rare" or "+/-" phenotypes. Embryos analyzed in this experiments: uninjected, n=49; lacZ-injected, n=46; PFFB4misMO-injected, n=16; PFKFB4MO-injected, n=43.
	Fig. S4. PFKFB4 is required for neural crest migration. (A,D) WISH analysis of neural crest migration at tadpole stages. When Pfkfb4 MO is injected, embryos display decreased Twist expression and neural crest migration defects (B), when compared to control embryos (A). (C) In contrast, the coinjection of Pfkfb4 mRNA with Pfkfb4 MO restores Twist expression and NC migration, showing that the neural crest migration defects are due to PFKFB4 knowckdown. A-C are side views, anterior is to the right.
	Fig. S5.PFKFB4 depletion and 2DG treatment block glycolysis, LY294002 blocks AKT phosphorylation. (A) PFKFB4 low-level depletion affects lactate levels in embryos, indicating efficient glycolysis blockade. (B, C) Lactate concentration and Akt phosphorylation were decreased following 2- DG or LY294002 treatment (LY, two doses 60-80 Î¼M), respectively. Error bars represent s.e.m (t-test p<0.05). (C,D) Blocking glycolysis during neurulation, between st. 14 and st. 18 does not affect twist expression in vivo. (D) control and (E) 2DG-treated embryos displayed normal twist expression (n=15 each).
	Fig. S6. Glycolysis and PI3K-Akt signaling control neural crest migration. Glycolysis blockade (AJ) and PI3K-Akt blockade (K-T) during EMT and neural crest migration (stage 18 to stage 24) affects neural crest migration as shown by the expression of Sox9, Sox10 and Twist. Embryos rinsed at stage 24 and grown until stage 45 display craniofacial and eye development defects, albeit the presence of all individual cartilage elements. (A-C,F-H,K-M,P-R, U-W) Side views, anterior is to the left. (D,I,N,S,X) dorsal views st 45. (E,J,O,T,Y) Ventral views of alcian blue stained visceral cartilages, st 45. Scale bar= 500Î¼m.
	Fig. S7. Pfkfb4 is regulated by the neural crest gene regulatory network. Unilateral overexpression of members of the neural crest gene regulatory network modulates Pfkfb4 expression at mid and late neurula stages (stage 14 and 18, respectively). (A-D) Overexpression of WNT signals increases both Snail2 and Pfkfb4 expression. (E-H) FGF8 overexpression results in complete loss of pfkfb4, while snai2 is expanded. (I-L) BMP overexpression with a constitutively active form of BMPR1 results in loss of Snail2 and Pfkfb4 expression in the NC and ectopic expression in the neural plate. (M-T) PAX3 and TFAP2a positively regulate Pfkfb4 expression, similarly to Snail2. Dorsal views, anterior is to the top. Scale bar = 500Î¼m.