Katada T and Sakurai H (2019), Xenopus slc7a5 is essential for notochord funct...

XB-ART-55603

Mech Dev 2019 Feb 01;155:48-59. doi: 10.1016/j.mod.2019.01.001.

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Xenopus slc7a5 is essential for notochord function and eye development.

Katada T , Sakurai H .

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slc7a5 (also known as LAT1), largely accepted as an amino acid transporter, has been shown to play important roles in cancer and developmental processes. Because knockout mice of Slc7a5 are embryonically lethal due to placental defects, it is difficult to evaluate its role in early development. In this study, expression and function of slc7a5 were evaluated in Xenopus laevis embryos that develop without a placenta. Expression of slc7a5 was detected in the notochord and in the eye and it was not co-localized with slc3a2, which helps slc7a5 to localize at the plasma membrane, before the late neurula stage. Loss-of-function experiment with a morpholino antisense oligonucleotide led to defect of neural and non-neural patterning, inhibition of primary neurogenesis, and disruption of eye development. Disruption of neural development and primary neurogenesis was likely due to impaired notochord development as sonic hedgehog (shh) signaling pathway was compromised in slc7a5-inhibited embryos. These results suggest that slc7a5 is required for notochord development and subsequent primary neurogenesis via shh/gli signaling and for eye development. These novel developmental roles of slc7a5 appeared to be independent of transport function at least before the late neurula stage.

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Species referenced: Xenopus laevis
Genes referenced: aga agt chrd chrd.2 foxa2 fubp1 gaa gca gcg ggt1 gli1 gli2 krt12.4 mag nrl pax6 pcyt1a ptch2 rpe shh slc25a20 slc3a2 slc7a5 sox2 tgm2 tnf tubb2b ugt8
GO keywords: eye development [+]
???displayArticle.morpholinos??? hbg1 cMO1 slc7a5 MO1

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	Fig. 1.Comparison of amino acid sequences between slc7a5.S and slc7a5.L inXenopus laevis.Two amino acid sequences, slc7a5.S (NM_001096373) and slc7a5.L (NM_001090065), were aligned by clustalW. Asterisks represent identical amino acid residues.Dots show similar residues. Shaded cysteine is predicted to be participated in a disulfide bond with slc3a2 (4F2hc or CD98) protein.
	Fig. 2.Temporal expression ofslc7a5.Sandslc7a5.Linthe developmental stages.Semi-quantitative RT-PCR was performed to examine the expression ofslc7a5 mRNA in the developmental stages.slc7a5.Sstarted to express from the gastrula stage(st.10.5) and increased thereafter.slc7a5.Lexpressionwas found maternally and also detected in the zygoticstage.slc3a2(4F2hcorCD98) expression was detected from st.20.Histone H4was used as a loading control.−RT represents a negative control without reverse transcriptase. Numbers show developmental stages de-fined by Nieuwkoop and Faber (1994). U: unfertilized egg.
	Fig. 3.Spatial expression of slc7a5.S and slc3a2 in the developmental stages. Whole-mount in situ hybridization was performed to analyze spatial expression ofslc7a5.S(shown in A–F, I, J, M–Q) and slc3a2(shown inG, H, K and L) in the developmental stages. (A)st.10.5. Lateral view. slc7a5.S expressed in theanimal hemisphere. Black arrowheads indicatethe blastopore lip (bl). (B) st.12. Lateral view.slc7a5.S expression was observed throughoutthe animal hemisphere at st.10.5. (C, D) st.15.(C) Anterior view.slc7a5.Slocalized at the eyeanlagen (black arrows). (D) Posterior view.slc7a5.Swas detected in the notochord from theblastopore to anterior along midline as indicatedby white arrowheads. (E–H) st.20. (E) Anteriorview. More definite expression ofslc7a5.Swasdetected at the eyefield in comparison to st.15embryo in (C) (black arrows). A yellow brokenline indicates the plane of section in panel (F).(F) Transverse section of st.20 embryo shown inpanel (E). Strong expression ofslc7a5.Swasobserved in the notochord (nc). (G) Anteriorview. Weak expression ofslc3a2was detected inthe cement grand (white arrow). A yellowbroken line indicates the plane of section inpanel (H). (H) Transverse section of st.20 em-bryo shown in panel (G). Spotted expression ofslc3a2was observed in the neural tube (yellowarrowheads), but no expression was detected inthe notochord unlikeslc7a5.Sexpression in st.20embryo. (I–L) st.25. (I) Lateral view.slc7a5.Sexpression was intensely detected in the eye(yellow arrow) and also observed in the neuraltube (yellow arrowheads) and the notochord(white arrowheads). Two yellow broken linesrepresent the plane of section in panel (J) and(M). (J) Transverse section of st.25 embryo attrunk region shown in panel (I).slc7a5.Swasobserved in the dorsal part of the neural tube(nt, yellow arrowheads) and the notochord (nc).(K) Lateral view.slc3a2expression was intenselydetected in the eye (yellow arrow) and also ob-served in the neural tube (yellow arrowheads)and the notochord (white arrowheads) asslc7a5.Sexpression shown in panel (I). (L)Transverse section of st.25 embryo shown inpanel (K).slc3a2was observed in the ventralpart of the neural tube (nt, yellow arrowheads)and the notochord (nc). (M) Transverse sectionof st.25 embryo at trunk region shown in panel(I).slc7a5.Sexpressed in the eye vesicle (ev) andthe neural tube (nt). (N–Q) st.35. (N) Lateralview.slc7a5.Sexpression was maintained in theeye (yellow arrow), the neural tube (yellow ar-rowhead) and the notochord (white arrowhead).A yellow broken line indicates the plane of sec-tion in panel (P). (O) Negative control with a sense probe ofslc7a5.S. (P) Transverse section of st.35 embryo. A rectangle with red broken line represents a regionmagnified in panel (Q). (Q) Higher magnification of photomicrograph of rectangle (Q) in panel (P).slc7a5.Sexpressed in the lens (le), CMZ of the neural retina (redarrow),the border of INL and ONL (red asterisks) and RPE (yellow asterisks). Scale bars: 1 mm in A–E and G; 0.1 mm in F, H, J and L; 1 mm in I, K, N and O; 0.1 mm inM; 0.1 mm in P; 0.1 mm in Q. bl: blastopore lip, CMZ: ciliary marginal zone of the neural retina, ev: eye vesicle, nc: notochord, nt: neural tube, so: somite, GCL:ganglion cell layer, INL: inner nuclear layer, le: lens, ONL: outer nuclear layer, RPE: retinal pigment epithelium. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)
	2.3. RT-qPCRTotal RNA extraction from slc7a5 MO- or control MO-injected em-bryos (both sides) at st.15 was performed with ISOGEN (NIPPON GENE,Tokyo, Japan) and reverse transcription was carried out withSuperScript III (Thermo Fisher Scientific, Yokohama, Japan) using0.1μg of total RNA and oligo dT primer. Sequences of primer sets wereas follows:chrd.1forward, 5′-TGG ATG GTC TGC ACA ATG TT-3′;chrd.1reverse, 5′-GAA TCC AAG GTG GCA AGG TA-3′;shhforward, 5′-GCA CCA GGT CGT TCA AGT CT-3′;shhreverse, 5′-GGC AGT TAG AGGCGC ATA AG-3′;ptch2forward, 5′-GCC AGC AAG GAT CCA AAT TA-3′;ptch2reverse, 5′-CTT CTG CTC TTG GCA GGT TC-3′;foxa2forward, 5′-TCA TGG ACC TGT TCC CTT TC-3′;foxa2reverse, 5′-GAA TCA GGGTGT AGG GTC CA-3′;gli1forward, 5′-ACC AAC AGT GGG GAT GAT GT-3′;gli1reverse, 5′-TCT TGA GAG CTT GGG CTC AT-3′;gli2forward, 5′-CTC CAT GCT CAC AAC ATT GG-3′;gli2reverse, 5′-CCC AGG ACC CTAACG TCT TT-3′;Histone H4forward, 5′-TAT CAC TAA ACC CGC CATCC-3′;Histone H4reverse, 5′-TCT TCC TCT TGG CGT GTT CT-3′.Reactionwas performed with SYBR Select Master Mix and 7300 RealTime PCR System (Applied Biosystems) according to the manufacturer'sinstruction. The data was analyzed using 7300 System SequenceDetection Software Version 1.4.2.4. Whole-mount in situ hybridizationWhole-mountin situhybridization was performed as describedpreviously (Katada and Sakurai, 2016). Briefly, the method of Shainand Zuber was employed (Shain and Zuber, 1996) and embryos aftersignal detection were bleached with a solution containing hydrogenperoxide (Koga et al., 2007; Mayor et al., 1995).slc7a5.S,slc3a2.L,shh,ptch2,foxa2(also known asHNF-3β),gli1andgli2were obtained byPCR cloning using following primer sets after reverse transcription oftotal RNAs fromXenopusembryos and these amplified PCR fragmentswere subcloned intopBIISK+vector.slc7a5.Sforward, 5′-GGG GGA TCC ATG GCA GCG GGC AGC GTGAA-3′;slc7a5.Sreverse, 5′-GGG CTC GAG TTA AGA CTC CTG AGG GACAG-3′;slc3a2.Lforward, 5′-GGG GAA TTC CAA GAC ACC AGG ACTCCG AC-3′;slc3a2.Lreverse, 5′-GGG CTC GAG TTA TCC ACT ATA AGGGTA TT-3′;shhforward, 5′-GGG GGA TCC ATG CTG GTT GCG AAC TCGAA-3′;shhreverse, 5′-GGG GAA TTC TCA ACT GGA TTT CGT TGC CA-3′;ptch2forward, 5′-GGG CTC GAG CCG AAA CCA ACG TTC AGT AC-3′;ptch2reverse 5′-GGGTCT AGA CTA GTG TTG AAC AGA CAA GT-3′;foxa2forward, 5′-GGG GAA TTC ATG CTT GGG GCT GTG AAA AT-3′;foxa2reverse, 5′-GGG CTC GAG CAT GGG ACA GGA CAG AAT GG-3′;gli1forward, 5′-GGG GAA TTC TGT GAT TAC CAA GGG CAA CA-3′;gli1reverse, 5′-GGG CTC GAG ACA TTG TGT TTA GGT ACT TA-3′;gli2forward, 5′-GGG GGA TCC GTC TGT ACA GAG GAA TAT CA-3′;gli2reverse, 5′-GGG GAA TTC TTA GGA CAT CAG ATT GAG AA-3′.sox2,xk81a1,tubb2b,chrd.1andpax6are kind gifts from Dr. TsutomuKinoshita (Rikkyo University, Tokyo). The antisense probes forslc7a5.S(BamHI/T7),slc3a2.L(EcoRI/T7)sox2(EcoRI/T7),xk81a1(EcoRI/SP6),tubb2b(BamHI/T3),chrd.1(EcoRI/T7),shh(BamHI/T7),ptch2(XhoI/T3),foxa2(EcoRI/T7),gli1(EcoRI/T7),gli2(BamHI/T7) andpax6(EcoRI/T7) were prepared with restriction enzyme and RNA poly-merase shown in the parenthesis in presence of digoxygenin-UTP(RöcheDiagnostics).2.5. HistologyEmbryos after whole-mountin situhybridization were dehydratedand embedded in paraffin, Parabett (Muto Pure Chemicals, Tokyo,Japan). Sections were cut at 10μm. Slides were deparaffinized andmounted with 50% glycerol in PBS. For hematoxylin and eosin staining,sections were cut at 10μm and slides were processed according to themanufacturer's instruction (Wako Pure Chemical Industries, Osaka,Japan).Fig. 4.slc7a5 MO specifically inhibits translation ofslc7a5.Sandslc7a5.LmRNA.(A) The sequence of slc7a5 MO used in this study. slc7a5MO completely matches toslc7a5.S, but has three mis-matches toslc7a5.L. Two rescue forms,slc7a5.S rescueandslc7a5.L rescue, have 10 or 11 mismatches to slc7a5MO with silent mutations. ATG in red represents a startcodon and double dots represent the mismatch of thebase. (B–J) Validation assay of slc7a5 MO with GFP fu-sion constructs,slc7a5.S-GFPandslc7a5.L-GFP.GFPconstruct (0.5 ng) with or without MO (20 ng) was in-jected into dorsoanimal region of 4-cell stage embryos.GFPfluorescence was observed in the embryos injectedwithGFPonly orGFPand control MO, but slc7a5 MOabolished thefluorescence in bothslc7a5.S-GFPandslc7a5.L-GFPco-injection. (For interpretation of the re-ferences to color in thisfigure legend, the reader is re-ferred to the web version of this article.)
	Fig. 5.slc7a5 depletion impaired neural tube closure and eye development.Loss-of-function experiment was performed with slc7a5 MO. (A, B) st.15 embryo. Anterior view. White arrowheads represent the neural fold. (A) Control MO-injectedembryo. (B) slc7a5 MO-injected embryo. The neural fold in the injected side was not distinct compared with that in the uninjected side. (C, D) st.20 embryo. Anteriorview. White arrowheads show the neural fold. (C) Control MO-injected embryo. (D) slc7a5 MO-injected embryo. The neural fold in the injected side did not fuse atthe midline and neural tube closure was not seen in the injected side. (E–X) st.35 embryo. (E–I) Control MO-injected embryo. (E) Uninjected side of control MO. (F)Injected side of control MO. Yellow broken line shows the plane of section in panel (G). (G) Transverse section of control MO-injected embryo. Two rectangles withred broken line represent regions magnified in panel (H) and (I). (H, I) Higher magnification of photomicrograph of rectangle (H) and (I) in panel (G). (J–S) Embryosinjected with slc7a5 MO. (J–N) Mild phenotype. (J) Uninjected side of slc7a5 MO-injected embryo. (K) Injected side of slc7a5 MO-injected embryo. Yellow brokenline shows the plane of section in panel (L). (L) Transverse section of slc7a5 MO-injected embryo with small eye. Two rectangles with red broken line representregions magnified in panel (M) and (N). (M, N) Higher magnification of photomicrograph of rectangle (M) and (N) in panel (L). (O–S) Severe phenotype. (O)Uninjected side of slc7a5 MO-injected embryo. (P) Injected side of slc7a5 MO-injected embryo. Yellow broken line shows the plane of section in panel (Q). (Q)Transverse section of slc7a5 MO-injected embryo with no apparent eye. Two rectangles with red broken line represent regions magnified in panel (R) and (S). (R, S)Higher magnification of photomicrograph of rectangle (R) and (S) in panel (Q). (T–X) Rescue by coinjection ofslc7a5.SmRNA andslc7a5.LmRNA with slc7a5 MO.(T) Uninjected side (i.e.no rescue). (U) Injected side of rescue constructs. Eye structure was rescued. Yellow broken line shows the plane of section in panel (V). (V)Transverse section of rescue constructs-injected embryo. Two rectangles with broken line represent regions magnified in panel (W) and (X). (W, X) Higher mag-nification of photomicrograph of rectangle (W) and (X) in panel (V). Arrow indicates the injected side. White brackets indicate the size of the eye.β-GalactosidasemRNA (1 ng) was used as a tracer (blue). Scale bars: 1 mm in A–D; 1 mm in E, F, J, K, O, P, T, U; 0.1 mm in G, L, Q, V; 0.1 mm in H, I, M, N, R, S, W, X. le: lens. (Forinterpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)
	Fig. 6.Depletion of slc7a5 showed decrease ofpax6andnrl(L-maf) in eye development.Eye structure was investigated in slc7a5-depleted embryoby whole-mountin situhybridization withpax6andnrl(L-maf) probes. (A–D) Control MO-injected embryo. (A,B)pax6expression in control MO-injected embryo. (A)Uninjected side of control MO-injected embryo. (B)Injected side of control MO-injected embryo. (C, D)nrl(L-maf) expression in control MO-injected embryo. (C)Uninjected side of control MO-injected embryo. (D)Injected side of control MO-injected embryo. (E–H)slc7a5 MO-injected embryo. (E, F)pax6expression inslc7a5 MO-injected embryo. (E) Uninjected side of slc7a5MO-injected embryo. (F) Injected side of slc7a5 MO-in-jected embryo.pax6expression was repressed. (G, H)nrl(L-maf) expression in slc7a5 MO-injected embryo. (G)Uninjected side of slc7a5 MO-injected embryo. (H)Injected side of slc7a5 MO-injected embryo.nrl(L-maf)expression was decreased. (I–L) Rescue effect in coinjec-tion ofslc7a5.SmRNA andslc7a5.LmRNA with slc7a5MO. (I, J)pax6expression in rescue constructs-injectedembryo. (I) Uninjected side of rescue constructs-injectedembryo. (J) Injected side of rescue constructs-injectedembryo.pax6expression was rescued. (K, L)nrl(L-maf)expression in rescue contents-injected embryo. (K)Uninjected side of rescue constructs-injected embryo. (L)Injected side of rescue constructs-injected embryo.nrl(L-maf) expression was rescued. Yellow brackets indicatethe size of the eye.β-GalactosidasemRNA (1 ng) was usedas a tracer (blue). Scale bars: 1 mm. (For interpretation ofthe references to color in thisfigure legend, the reader isreferred to the web version of this article.)
	Fig. 7.Inhibition of slc7a5 led to the disorganization of neural- and non-neural patterning and primary neurogenesis.Neurula stage embryos (st.15 and st.20) were surveyed to analyze neural- and non-neural patterning in slc7a5-depleted embryos. Whole-mountin situhybridizationwas performed withsox2,xk81a1,tubb2b(N-tubulin) probes in embryos injected with control MO or slc7a5 MO. Black and white arrow represents the injected side.Yellow brackets indicate the width of the neural region in the embryo. (A–E)sox2expression. Anterior view. Broader expression domain was observed in slc7a5 MO-injected side. (F–J)xk81a1expression. Anterior view.xk81a1-negative domain was observed in the anterior part of slc7a5 MO-injected side even after st.20. (K–O)tubb2bexpression. Dorsal view. Motoneuron in medial region and interneuron in more lateral region were eliminated in slc7a5 MO-injected embryo. (E, J, O)Coinjection ofslc7a5.Sandslc7a5.LmRNA with slc7a5 MO rescued these phenotypes.β-GalactosidaseRNA (1 ng) was used as a tracer. Scale bars: 1 mm. l: lateralneurons (sensory neurons or Rohon-Beard neurons), i: intermediate neurons (interneurons), m: medial neurons (motoneurons). (For interpretation of the referencesto color in thisfigure legend, the reader is referred to the web version of this article.)
	Fig. 8.Inhibition of slc7a5 disturbed notochord forma-tion and diminished target genes in sonic hedgehog sig-naling.Notochordal markers and downstream molecules in sonichedgehog signaling were analyzed in slc7a5-depletedembryos at st.15. Arrow indicates the injected side. (A, B,E, F)chrd.1expression. (A, B) Dorsal view. (E, F)Transverse section. (A) Control MO-injected embryo. (B)slc7a5 MO-injected embryo. Expression ofchrd.1was notextended enough along the anteroposterior axis and dif-fused laterally in slc7a5 MO-injected side (white arrow-heads). (E) Transverse section of control MO-injectedembryo. (F) Transverse section of slc7a5 MO-injectedembryo. Expression ofchrd.1diffused laterally (whitearrowheads). (C, D, G, H)shhexpression. (C, D) Dorsalview. (G, H) Transverse section. (C) Control MO-injectedembryo. (D) slc7a5 MO-injected embryo. Faint expressionofshhwas observed similar tochrd.1expression (whitearrowheads). (G) Transverse section of control MO-in-jected embryo. (H) Transverse section of slc7a5 MO-in-jected embryo. Expression ofshhdiffused laterally similartochrd.1expression (white arrowheads). (I, J, M, N)ptch2expression. (I, J) Dorsal view. (M, N) Transversesection. (I) Control MO-injected embryo. (J) slc7a5 MO-injected embryo.ptch2was vanished in the injected side(yellow arrowheads). (M) Transverse section of controlMO-injected embryo. (N) Transverse section of slc7a5MO-injected embryo. Expression ofptch2was diminishedin the injected side (yellow arrowhead). (K, L, O, P)foxa2(HNF-3β) expression. (K, L) Dorsal view. (O, P)Transverse section. (K) Control MO-injected embryo. (L)slc7a5 MO-injected embryo. Expression offoxa2wasweakened in the injected side (white arrowheads). (O)Transverse section of control MO-injected embryo. (P)Transverse section of slc7a5 MO-injected embryo.Expression offoxa2was diffused in the injected side(white arrowhead). (Q, R)gli1expression. Anterior view.slc7a5 depletion impairedgli1expression (yellow ar-rowheads). (S, T)gli2expression. Anterior view.Expression ofgli2was abolished in the slc7a5 MO-in-jected side similar to that ofgli1(yellow arrowheads).β-GalactosidaseRNA (1 ng) was used as a tracer. (U) RT-qPCR was performed with slc7a5-depleted embryos.Expression level of these genes was down-regulatedespecially in shh signaling family genes. Scale bars: 1 mmin A–D, I–L, Q–T; 0.1 mm in E–H, M–P. (For interpreta-tion of the references to color in thisfigure legend, thereader is referred to the web version of this article.)