DIS3L2 is essential for neural crest survival by modulating Akt signaling

DIS3L2 is essential for neural crest survival by modulating Akt signaling | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results DIS3L2 is essential for neural crest survival by modulating Akt signaling Sian D’Silva , Tuhina Prasad , View ORCID Profile Megha Kumar doi: https://doi.org/10.1101/2024.12.26.630456 Sian D’Silva 1 CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB) , Habsiguda, Uppal road, Hyderabad-500007, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tuhina Prasad 1 CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB) , Habsiguda, Uppal road, Hyderabad-500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad-201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Megha Kumar 1 CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB) , Habsiguda, Uppal road, Hyderabad-500007, India 2 Academy of Scientific and Innovative Research (AcSIR) , Ghaziabad-201002, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Megha Kumar For correspondence: meghakumar{at}ccmb.res.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract DIS3 like 3’-5’ exoribonuclease 2 (DIS3L2), an exoribonuclease is known to preferentially degrade uridylated RNA substrates, miRNAs, and ncRNAs. Recent reports show that DIS3L2 also plays a key role in cell proliferation and tumor growth. Mutations in DIS3L2 are associated with congenital disorders such as Perlman syndrome, yet the developmental functions of DIS3L2 remain unknown. We report the developmental role of dis3l2 in neural crest specification, patterning, and survival in the zebrafish embryo. Dis3l2 morphants exhibited reduced expression of neural crest specifier genes coupled with extensive apoptosis in the neural tissue. Our study demonstrates that DIS3L2 regulates neuronal apoptosis and progenitor functions through the Akt –GSK3β signaling pathway. Additionally, we show that DIS3L2 is essential for early mitoses in the zebrafish blastula and plays a key role in maintaining spindle length at metaphase, chromosome congression, spindle pole integrity, and cytokinesis. In summary, we identify new functions of exoribonuclease DIS3L2 in cell fate specification, neural progenitor survival, and mitosis during embryogenesis which form the underlying basis of DIS3L2-associated overgrowth Perlman syndrome. Plain English summary DIS3 like 3’-5’ exoribonuclease 2 (DIS3L2) degrades mRNAs and various RNA substrates in the eukaryotic cells. DIS3L2 mutations are associated with congenital overgrowth syndromes like Perlman syndrome and Wilm’s tumor. The role of DIS3L2 in regulating the embryonic processes remains poorly understood. Our study delineates the developmental functions of DIS3L2 in cell fate determination, survival, and proliferation during vertebrate embryogenesis using zebrafish embryos as a model. We show that DIS3L2 plays a key role in neural crest survival and patterning by modulating Akt-GSKβ signaling. We also report unique molecular functions of DIS3L2 in mitotic fidelity and cytokinesis during embryonic mitoses. Our study provides novel insights into the molecular functions of DIS3L2 in regulating neuronal apoptosis during embryonic brain morphogenesis and associated CNS disorders. Background DIS3 like 3’-5’ exoribonuclease 2 (DIS3L2), is a member of the RNase II family of 3’-5’ exoribonucleases which binds to the core RNA exosome complex. DI3L2 can degrade both single and double-stranded RNA substrates in vitro and is known to preferentially degrade uridylated RNA substrates in addition to degradation of mature miRNAs, aberrant mRNAs, and non-coding RNAs ( 1 ). A recent report shows that DIS3L2 can induce apoptotic mRNA decay and inhibition of translation ( 2 ). It has many biological functions including cell division and differentiation. In vitro studies show that DIS3L2 knockdown promotes cell proliferation and cancer cell growth by regulating cell cycle progression ( 3 ). Particularly, siRNA-mediated knockdown in HeLa cells shows that depletion of DIS3L2 results in mitotic errors, and multiple spindle poles. DIS3L2 regulates size and proliferation in the developing imaginal discs in Drosophila by upregulating Igdf2 signaling ( 4 – 6 ). The DIS3L2 -depleted mice show Perlman syndrome-associated features and the homozygous DIS3L2 null mice failed to survive the first postnatal day due to underinflated lungs, hypotonia, bradykinesia, and abnormal spine curvature ( 1 , 3 ). However, the developmental functions of DIS3L2 remain unknown to date. Our study delineates the functional role of DIS3L2 in cranial neural crest specification, patterning, and survival during early embryogenesis. We demonstrate that DIS3L2 functions via Akt-GSK3β signaling to modulate cell fate specification, apoptosis, and cell survival in the neural crest cells. Further, DIS3L2 is vital for early mitotic events in the zebrafish blastulae, ensuring proper chromosome alignment, spindle pole stability, metaphase spindle length, and cytokinesis. Methods Zebrafish lines, MO injection, and characterization of phenotypes Tubingen strain (TU-AB) zebrafish were raised according to standard protocols described earlier ( 51 ). All experiments were performed according to protocols approved by the Institutional Animal Ethics Committee of the Council of Scientific and Industrial Research Centre for Cellular and Molecular Biology, India. Embryos were obtained from the natural spawning of adult fish, kept at 28.5°C, and staged according to hours and days after fertilization ( 52 ). The endogenous DIS3L2 levels were depleted by microinjecting 0.1mM, 0.5mM or 1mM DIS3L2 translation blocker morpholino (Gene Tools), 5’-ATGCCATAATCTCGTCTACAGTTAC-3′ at the one-cell stage. The embryos were then analyzed for gross morphological defects, and survival at later stages of development (24hpf). RNA isolation, Semi□quantitative RT□PCR, and sequencing Total RNA was isolated from embryos using the RNA isolation kit (MN; 740955.50) as per the manufacturer’s protocol. 1 μg of total RNA was taken to synthesize cDNA by using PrimeScript 1st strand cDNA synthesis kit (Takara, Cat. No. 6110A). RT-PCR was carried out for the multiple developmental stages for amplification of zebrafish DIS3L2 with beta-actin as a control. PCR was performed in BioRad C1000 Touch thermal cycler with, Taq DNA Polymerase (TaKaRa, cat. no. R500A). The following PCR conditions were used for amplification of dis3l2 : initial denaturation was at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 secs, annealing at 60°C for 30 secs, and extension at 72 °C for 30 sec. The final extension was for 10 min at 72 °C. Amplified PCR products were examined and pictures were captured using the Vilber-Lourmat Gel Documentation system (Germany). Real-time PCR For quantitative real-time PCR (qPCR), we performed PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) with ViiA 7 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. 100 ng/μL cDNA of control and injected embryos were used along with PowerSYBR Green PCR master mix (Applied Biosystems, Cat. No. 4367659), and the following program was used: 40 cycles of 95 °C−10 min, 95 °C−15 s, 60 °C−1 min. Quantification data are represented as means ± SEM of three independent experiments. Analyses on normalized data were performed using the 2−δδCT algorithm (the delta−delta-Ct or ddCt algorithm). All genes were normalized against beta-actin unless mentioned otherwise. The GraphPad Prism software was used for the analysis. For the statistical significance of the data, p-values were calculated by performing an unpaired Student t-test. All primers used in this study are listed in Fig.S1 table. Antisense riboprobe preparation The total RNA was used to prepare cDNA using the PrimeScript 1 st strand cDNA synthesis kit (TaKaRa, Cat. # 6110A). Sequence-specific primers were used to amplify the specific gene fragment and cloned into the pGEMT easy vector and the sequence was verified. The sequence-verified plasmid was linearised using Nco1 and antisense digoxigenin (DIG)--labeled riboprobes were synthesized using the DIG RNA labeling kit (Roche #11175025910). ( 51 ). Whole Mount RNA in Situ Hybridization Control and injected embryos at 1dpf were fixed in 4% paraformaldehyde overnight followed by methanol fixation. Methanol-fixed embryos were rehydrated with 50% MeOH/PBT (1× PBS and 0.1% Tween 20). Embryos were rinsed with PBT, a 1:1 PBT/hybridization wash, and then a hybridization wash (50% formamide, 1.3 × SSC, 5 mM EDTA, 0.2%, Tween-20, H2O). Embryos were incubated with a hybridization mix (50% formamide, 1.3 × SSC, 5 mM EDTA, 0.2%, Tween-20, 50 μg/mL yeast t RNA, 100 μg/mL heparin, H2O) for 1 h at 55 °C. The riboprobes prepared in the hybridization mix were added and incubated for 16 h at 55 °C. Then, embryos were rinsed with prewarmed hybridization wash for 30 min (2 times) at 55 °C and followed by 1 × TBST wash (5 M NaCl, 1 M KCl, 1 M Tris, pH 7.5, and 10% Tween 20) at room temperature. Then, blocking was done with 10% FBS heat-inactivated fetal bovine serum (Gibco; 16210-064) for 1 h at room temperature. The embryos were then incubated with an Anti-DIG-AP antibody (Roche 11093274910) with a dilution of 1:5000 overnight at 4°C. The next day, embryos were transferred into a 6-well plate and washed twice with 1X NTMT (0.1M NaCl, 0.1 M Tris-Cl at pH 9.5, 0.05M MgCl2, 1% Tween-20 and H2O) for 10 min; then, incubated with 1:1 NBT-BCIP solution (Cat no. PI34042) NTMT solution for the color development. The embryos were monitored during this color reaction and stopped with 1× PBT as a stop solution. Images of embryos were captured using a Zeiss Stemi 508 stereomicroscope. Western Blot analysis Zebrafish embryos (n=100) were manually dechorinated and deyolked with a deyolking buffer and gentle pipetting. The deyolked embryos were then washed followed by 100ul of Laemmli buffer and homogenized with a 2ml syringe. The lysates were boiled and then subjected to SDS-PAGE. 20 μg of total protein was loaded in each lane on 8% SDS-PAGE gel for detection of the desired proteins. Proteins were transferred to PVDF membranes (Merck Milipore, ISEQ00010) by electro-blotting. Membranes were blocked for 2 h at room temperature in 5% Blotto non-fat dry milk/TBST (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% TWEEN 20) and probed overnight with primary antibodies. After three washes with TBST, the blots were incubated with HRP-conjugated secondary antibody for 1 hr, and then washed in TBST. The blots were developed for chemiluminescence signal using the chemiluminescence (Bio-Rad) and captured in the Vilber-Lourmat Chemiluminescence System (Germany). The indicated antibody dilutions were used for western blot. Rabbit antibodies: anti-DIS3L2 (1:2000) (Thermo, #PA5-31273), anti-CASPASE 3 (1:1000) (CST# 9662), anti-CASPASE 9 (1:000) (Thermo # PA5-22252), anti-GSK3β (1:1000) (CST#1256), anti-pGSK3β ser9 (1:1000) (CST#5558). Mouse antibodies: anti-CDK1 (1:1000), (Invitrogen #33-1800), anti-Mek1 (1:1000) (Invitrogen#13-3500), anti-GAPDH (1:2000) (Invitrogen #MA5-15738), anti-β tubulin (1:2000) (Thermo#MA5-16308), anti-AKT (1:1000) (CST#40D4, 2920), anti-pAKT thr308 (1:1000) (CST#D25E6, 13038). Immunohistochemistry of zebrafish embryos Embryos at 256 cell stage were fixed in 4% paraformaldehyde overnight followed by methanol at −20 °C. Methanol-fixed embryos were rehydrated with 50% MeOH/PBST (1X PBS and 0.1% Triton X-100) followed by incubation in blocking solution 1% BSA/PBST for 2 h at room temperature. Then the embryos were incubated with primary antibody overnight at 4 °C. The following day embryos were washed with 1X PBST and then incubated overnight at 4 °C with the appropriate secondary antibody. The next day embryos are washed with 1 X PBS and incubated in DAPI (1µg/ml) for 10 min at RT and then washed with 1X PBS and stored at 4°C until confocal/fluorescence imaging. ( 53 ) The mitotic phenotypes were quantified using 3D reconstruction of the confocal images using the IMARIS software ( 54 ). Rabbit antibodies: anti-γ tubulin (1:2000) (Thermo#PA5-34815), Mouse antibodies: anti-α tubulin (1:100) (CST#3873). Akt Activator assay and acridine orange staining DIS3L2 morpholino injected embryos at the shield stage (6hpf) were exposed to 1uM of Akt activator SC79 (Tocris, # 4635). Exposure to 0.5% DMSO (vehicle) was used as a control. The control and DIS3L2 injected embryos treated with SC79 were phenotyped at 24hpf and stained with acridine orange to detect the apoptotic cells in the embryos. For acridine orange staining, the embryos were dechorinated, and stained with 5mg/L AO for 1 hour in the dark followed by washing with embryo water. Then the embryos were anesthetized with tricaine and were photographed by a Zeiss AxioZoom.V16 microscope, the bright green dots in the embryos indicate apoptotic cells. The number of apoptotic cells in the head region was counted using Image J software ( 27 , 48 , 51 ). Skeletal preparations by Alcian blue and Alizarin red staining Wild-type control and morphants 5-day-old larvae were fixed in 4% PFA/PBS overnight at 4 0 C. The embryos were washed with 1XPBS and stored overnight in 100% methanol at - 20 0 C. The following staining solutions were prepared - Solution A (0.04% Alcian blue, 125mM MgCl2 in 70% ethanol) and Solution B (1.5% Alizarin Red S). 1ml of solution A and 10ul of solution B were added to the larvae and kept overnight at room temperature with constant rocking. The staining solution was removed and the larvae were washed two times with water, followed by bleach solution for 1 hour at room temperature and a gradation of Glycerol-KOH. The embryos were then stored in 50% glycerol/ 0.25% KOH for 2 hours at room temperature. The stained larvae were then imaged using a Zeiss Stemi508 stereomicroscope ( 51 ). Results Dis3l2 depletion results in neurodevelopmental defects To ascertain the developmental functions of DIS3L2, we first examined the gene expression profile of dis3l2 across developmental stages of zebrafish ( Fig. 1 ). We observed dis3l2 transcript expression in the cleavage stages (2 cell), gastrula stage (50% epiboly), somitogenesis (6-18 somites) and in 24hpf by RT-PCR analysis ( Fig. 1A ). We also examined the spatiotemporal expression pattern of dis3l2 in early development, somite stages and in 24hpf embryos by in situ hybridization ( Fig. 1A ). We observed that dis3l2 is expressed ubiquitously during early embryogenesis and the expression is restricted to the anterior part of the embryo, particularly the brain (red asterisk, Fig. 1A ) and in the eye in 24hpf embryos (red arrowhead, Fig. 1A ). At the cellular level, DIS3L2 localizes to the nucleus (white asterisk, Fig.1B ) and cortex in the blastula stage (256 cell stage) (white arrowhead, Fig. 1B ). To probe the function of DIS3L2 in embryogenesis, we used the antisense morpholino-based gene knockdown approach ( Fig.1C ). The dis3l2 -depleted embryos showed a range of phenotypes. About 18% of morpholino-injected embryos showed normal morphology and were termed as P0 ( Fig. 1C ). About 58% of the dis3l2 -depleted embryos that exhibited neurodevelopmental defects, poorly formed brains, and disrupted brain architecture were classified as P1 (red asterisk, Fig. 1C ). The severe phenotype termed P2 (22%), showed acute neurodevelopmental abnormalities (double red asterisk, Fig.1C ) and a complete lack of midbrain-hindbrain boundary (MHB) (red arrowhead, Fig. 1C ). The severity of the phenotypes P0, P1, and P2 was observed in a morpholino concentration-dependent manner, as the number of P1 and P2 phenotypes increased in the embryos injected with higher morpholino concentration ( Fig. 1D ). Western blot analysis of control and dis3l2 morpholino-injected embryonic lysates showed a significant reduction in DIS3L2 protein levels, thus confirming the DIS3L2 specific depletion ( Fig. 1E ). Further, the dis3l2 -depleted embryos showed an overall increase in mortality by 20% ( Fig. 1F ). The morphants that survived to larval stages, exhibited craniofacial defects, as shown by Alcian blue and Alizarin red S staining of bone and cartilage (red asterisk, Fig. 1G, H ). The morphants larvae also exhibited cardiac edema (red arrowhead, Fig. 1G ). These observations suggest that dis3l2 mRNA is maternally inherited in the embryo and plays a key role in early brain development. Download figure Open in new tab Figure 1: Dis3l2 depletion results in neurodevelopmental defects A: RT-PCR analysis of dis3l2 transcripts in different developmental stages. Zebrafish actin was used as the loading control ( left ). Whole-mount RNA in situ hybridization showing dis3l2 transcripts during early development. Scale bars, 100µm ( right ). B: Sum projection confocal image of 256 cell stage control embryos showing endogenous DIS3L2 localization. Blue/DAPI - DNA, green – DIS3L2, red – γ tubulin. Scale bars, 50µm. C: Gross morphological analysis of dis3l2 depleted embryos by morpholino in comparison to control embryos at 24hpf showing forebrain defects (red asterisks), midbrain-hindbrain boundary defects (red arrowhead) Quantification of percent phenotype of 1mM dis3l2 morphants as compared to stage-matched control. Data are shown as mean+ SEM, *p<0.05, **p<0.01, ***p<0.001. n=3 for each experiment. D: Percent phenotype of dis3l2 morphants in different concentrations of morpholino. E: Western blot analysis showing DIS3L2 levels by western blot in dis3l2 morpholino injected morphants. GAPDH was used as the loading control. F: Percent mortality of dis3l2 morphants with respect to control at 24hpf. G: Bright field image of dis3l2 depleted larvae as compared to control larvae at 5dpf. H: Skeletal preparations of dis3l2 morphants by bone and cartilage staining using Alizarin red S and Alcian blue respectively. Scale bars 50µm. Dis3l2 is crucial for the specification of neural crest cells To investigate the developmental basis of neural crest-derived craniofacial abnormalities in DIS3L2-depleted embryos ( Fig.1G , H), we examined the expression dynamics of neural crest specifier genes foxd3 , crestin , twist1a and sox10 ( Fig.2 ) ( 7 ). Foxd3 is essential for survival of hindbrain neural crest cells and plays a key role in specification of the premigratory neural crest cells into the neuronal, glial or cartilage fate by inducing expression of lineage specific transcription factors ( 8 , 9 ). Foxd3 is downregulated in the cranial neural crest of the morphants (red asterisk, Fig.4A ). Intriguingly, foxd3 expression is retained in the caudal neural crest and somites. On the other hand, crestin expression in the post otic trunk neural crest increased in the DIS3L2 morphants (red arrow, Fig. 4B ). Crestin is a pan neural crest marker which is expressed after the neural crest cells are specified until they undergo overt differentiation ( 10 – 12 ). Twist1a is a neural crest specifier expressed in the premigratory cranial neural crest in the diencephalon and is essential for the migration and differentiation ( 13 ). Sox10 is also expressed in undifferentiated neural crest cells and is required for neural crest specification, migration, and survival ( 12 – 16 ). The DIS3L2 morphants showed downregulation of both twist1a and sox10 (red asterisks, Fig.4C , D). The loss of sox10 expression results in cell death and failure to undergo differentiation to give rise to the neural crest-derived craniofacial skeleton ( 17 , 18 ). The down regulation of these specifiers attribute the craniofacial anomalies observed in the dis3l2 morphants. We also examined the neural tube patterning and differentiation genes like pax2a and fgf8 . Pax2a is expressed in the optic stalk, MHB, eye, otic placode and promotes otic differentiation ( 19 – 22 ). Fgf8 is also expressed in the MHB and eye ( 23 , 24 ). Pax2a (red arrow, Fig.4E ) and fgf8 (red asterisk, Fig.4F ) are downregulated in dis3l2 morphants. The loss of structures in the midbrain and anterior hindbrain in the dis3l2 morphants may be attributed to the reduced expression of fgf8 and loss of inductive signals from the Fgf8 expressing hindbrain tissue. Thus, DIS3L2 is essential to initiate the neural differentiation program, and its precocious depletion results in the loss of differentiation-inducing signals in the embryo. Download figure Open in new tab Figure 2: Dis3l2 is crucial for the specification of neural crest cells A, B: Gene expression analysis of foxd3 (red asterisk) and crestin (red arrow) in dis3l2 morphants with respect to control. C, D: Expression of twist1a (double red asterisks) and sox10 (single red asterisk) in dis3l2 morphants as compared to control. E, F: Gene expression of pax2a (red arrow) and fgf8 in dis3l2 morphants and control. Scale bars, 100µm. DIS3L2 is essential for neural progenitor survival In addition to craniofacial defects, the DIS3L2 morphant P1 larvae also exhibited smaller head size at 5dpf ( Fig.3A ). To determine the cellular basis of the smaller head phenotype, we examined the status of cell death in the DIS3L2 P1 morphants by acridine orange staining ( Fig. 3B ). Intriguingly, we observed a massive surge of apoptosis in the developing brain and neural tube of the dis3l2 morphants, indicating that DIS3L2 associated head size reduction might be due to increased apoptosis (white asterisk, Fig. 3B ) and reduced expression of neural tube markers such as pax2a ( Fig.4E , F). As the intrinsic apoptosis pathway responds to developmental cues, we first examined the pro-apoptotic Bcl2 family, initiator protease CASPASE 9, and associated effector CASPASE 3 ( 25 – 27 ). Remarkably, the expression of pro-apoptotic Bcl2 members BIM , puma, and foxo increased in the DIS3L2 morphants ( Fig. 3C ). However, the pro-survival Bcl2 levels remain unchanged in the DIS3L2 morphants (Fig3C). BIM , puma , and foxo activate CASPASE9 and CASPASE3 upon apoptotic stimulation ( 27 – 30 ). We also observed an increase in CASPASE3 and CASPASE9 levels in the DIS3L2-depleted embryonic lysates ( Fig. 3D ). Elevated PUMA levels are known to promote neuronal cell death by activating both intrinsic and extrinsic apoptotic pathways in the zebrafish embryonic neural tissue ( 27 , 31 , 32 ). Thus, the over-expression of these proapoptotic genes may be the underlying basis of increased cell death in the dis3l2 morphants. And the depletion of DIS3L2, induces pro-death signals, leading to activation of these BH3-only class of Bcl2 proteins and elevated CASPASE levels. Reports show that transcriptional repression of BIM is a key cell survival signal and its activity is regulated through phosphorylation by Akt signaling ( 27 , 29 , 30 ). To further probe into the mechanism of DIS3L2-mediated apoptosis, we therefore examined the Akt signaling pathway. Akt signaling has been shown to regulate brain size in the zebrafish embryo and reduced Akt signaling results in a reduction in brain size. Further, over-expression of the constitutive form of Akt inhibits neuronal apoptosis ( 7 , 19 , 33 – 35 ). We used immunoblotting assay to determine the phosphorylation levels of Akt in control and DIS3L2-depleted embryos. AKT (pAKT-Thr308) phosphorylation levels decreased in DIS3L2 morphants, while the total AKT remained unchanged. ( Fig.3E ). Phosphorylation at Thr308 is critical for the activation loop of the kinase by protein kinase PDK1 and activation of AKT signaling. Akt in turn, phosphorylates GSK3β, which also affects cell survival, learning, and memory in the brain ( 33 , 36 ). As the phosphorylation status of GSK3β at ser9 position dictates its kinase activity and the phosphorylated form is pro-survival, we examined the phosphorylated GSK3β levels in DIS3L2-depleted embryonic lysates. We observed that phosphorylated GSK3β (pGSK3β Ser9) was reduced in DIS3L2 morphants and the total GSK3β levels did not change. ( Fig. 3E ). Thus, decreased GSK3β levels are concomitant with a significant decrease in Akt signaling in the DIS3L2 morphants. To ascertain direct DIS3L2-mediated regulation of apoptosis by Akt signaling, we treated the DIS3L2 morphants to Akt activator SC79. SC79 binds to Akt, enhances its phosphorylation, and activates cytosolic Akt ( 37 , 38 ). The embryos treated with the Akt activator showed a significant reduction in DIS3L2 phenotypes P1 and P2 ( Fig. 3F ) and apoptosis, marked by acridine orange staining (white arrowhead, Fig. 3G ). These data strongly suggest that DIS3L2 is essential for neural progenitor survival and regulating apoptosis during CNS morphogenesis via Akt signaling. Download figure Open in new tab Figure 3: Dis3l2 is essential for neural progenitor survival A: Skeletal preparation and head measurements of dis3l2 depleted larvae showing smaller head area with respect to control. B: Cell death analysis in the dis3l2 morphants by acridine orange staining. Acridine orange positive cells (white asterisk) shown in the head. Scale bars, 200µm. C: Relative expression of Bcl2 family members – bim , puma, foxo, and bcl2 in dis3l2 morphants. Data are shown as mean+ SEM, *p<0.05, **p<0.01, ***p<0.001. n=3 for each experiment. D: Western blot analysis to show CASPASE3 levels in morphants embryos. GAPDH was used as loading control. E: Western blot analysis showing phosphorylated AKT with respect to total AKT levels and phosphorylated GSK3β with respect to total GSK3β levels in dis3l2 depleted embryos. GAPDH was used as loading control. F: Rescue experiments showing phenotype P1 and P2 upon treatment with Akt activator SC79 in dis3l2 morpholino injected embryos. G: Cell death analysis by acridine orange staining in dis3l2 morpholino injected embryos treated with SC79 and WT embryos treated with SC79. DIS3L2 is required for early embryonic mitoses As DIS3L2 plays an important role in regulating apoptosis in the developing CNS, we wanted to investigate its possible functions in regulating mitosis and the cell cycle ( Fig. 4 ). Indeed, dis3l2 morphants showed a myriad of mitotic defects during the early blastula stage ( Fig. 4A ). The morphants exhibited elongated spindles (white dotted line, Fig. 4A ) and chromosome mis-congression at metaphase ( Fig. 3A, B, C ), suggesting that DIS3L2 is essential to maintain spindle length and aids in chromosome congression during metaphase. The morphants also exhibited unfocused spindle poles with multiple γ-tubulin positive foci during interphase and mitosis (white asterisk, Fig. 3A, B, C ). The morphant embryos also showed an increased cytokinetic index, indicating its role in cytokinetic progression (white arrowhead, Fig.3B, C ). Hence, our results show that DIS3L2 is essential for mitotic spindle organization, spindle pole integrity and mitotic conformity during early embryonic divisions. We also examined the effect of DIS3L2 depletion on cell cycle regulators p21 and p27. These are key cell cycle inhibitors that suppress cell cycle progression by inhibiting cyclin-dependent kinases (CDKs) and arresting cells in the G1 phase. p21 is also a cellular marker of senescence, which is characterized by cell cycle arrest and loss of function ( 39 – 42 ). The morphants showed an overall upregulation of p21 , p27a, and cyclinD1 ( Fig. 4D ). The upregulation of p21 and cyclinD transcripts may be the underlying cause of impaired cell cycle progression, increased cell death, and growth retardation in the dis3l2 morphants. Further, cell cycle progression is tightly regulated by cyclins and CDKs complexes ( 41 , 43 , 44 ). The mitotic CDK, CDK1, a downstream target of p21, is essential for cell division and recently reported for cell survival. CDK1 promotes cell death by activating Bcl2 family members like BAD and phosphorylates MCL1, suppressing its anti-apoptotic functions ( 43 – 45 ). Hence, the upregulation of CDK1 and pro-apoptotic BIM and Puma transcripts strongly corroborates with the surge of apoptosis in the dis3l2 morphants ( Fig. 4E ). Also, the Mek/Erk (MAPK) pathway is a key signaling cascade that regulates cell division, differentiation, and apoptosis. MEK signaling is also essential for cranial neural crest specification, formation of Meckel’s cartilage, ceratohyal cartilage, and neural crest-derived craniofacial skeletogenesis ( 46 – 49 ). Interestingly, we observed a downregulation of MEK1 (MAP2K1) in the DIS3L2 morphants ( Fig.4E ), which also contributes to the observed craniofacial defects ( Fig.1G , H). Together, we demonstrate that DIS3L2 plays a key role in cell cycle regulation and mitotic progression during early embryogenesis. Download figure Open in new tab Figure 4: DIS3L2 is required for early embryonic mitoses A: Sum projection confocal image of 256 cell stage control and dis3l2 morphants embryos showing mitotic defects. Blue/DAPI - DNA, red – alpha-tubulin, green – γ tubulin. B: Enlarged sum projection confocal image of dis3l2 morphants showing cytokinetic index (white arrowhead), spindle length (white dotted line), and spindle poles (white asterisks) during early mitoses. Scale bars, 50µm. C: Quantification of spindle length (white dotted line), chromosome congression, spindle pole (white asterisk), and cytokinesis (white arrowhead) in the dis3l2 morphants as compared to control. Scale bars, 50µm. All data are shown as mean + SEM. *p<0.05, **p<0.01, ***p<0.001. n=3 for each experiment. D: Relative expression of cell cycle regulators – p21 , p27, and cyclinD1 in dis3l2 morphants. Data are shown as mean+ SEM, *p<0.05, **p<0.01, ***p<0.001. n=3 for each experiment. E: Western blot analysis showing CDK1 and MEK1 levels in dis3l2 depleted embryos as compared to control. GAPDH was used as the loading control. Discussion DIS3L2 is an exoribonuclease that can degrade single and double-stranded RNA substrates ( 1 , 3 , 50 ). In vitro studies show that DIS3L2 negatively regulates cell cycle progression and its depletion results in mitotic aberrations and tumor growth ( 4 ). A recent report shows that DIS3L2 regulates cell proliferation and tissue growth by Idgf2 signaling pathway coupled with increased Akt phosphorylation levels ( 6 ). Yet, the molecular mechanism of DIS3L2 function in cell proliferation and differentiation during vertebrate embryogenesis remains elusive. This study delineates novel developmental functions of DIS3L2 in early embryonic mitoses and neurodevelopment. Depletion of DIS3L2 results in upregulation of cell cycle regulators p21, p27 and cyclinD1 and mitotic aberrations such as elongated metaphase spindles, diffused spindle poles, chromosome mis-congression, and cytokinetic defects. These results show that DIS3L2 is required for mitotic progression during early embryonic development and is essential for proper chromosome congression, maintenance of spindle pole integrity, and cytokinesis. In the context of embryogenesis, we show that DIS3L2 is maternally expressed during early embryonic stages and plays a key role in brain morphogenesis and architecture. Our study reveals that dis3l2 plays a key role in cranial neural crest specification. The reduced expression of neural crest specifier genes foxd3 , twist1a, and sox10 in morphants indicates that DIS3L2 plays a key role in specifying progenitor cell fate, migration, and differentiation of neural crest cells. The reduced expression of neural crest specifiers like sox10 and decreased MEK signaling contribute to observed craniofacial skeletal abnormalities. Thus, dis3l2 is critical for the fate determination of neural crest and its derivatives. Further, dis3l2 depletion resulted in massive cell death and upregulation of pro-apoptotic genes bim , puma, and foxo3a in the embryonic neural tissue. Previous studies show that neuronal survival and growth in the developing embryo are attributed to the phosphoinositide 3 kinase (PI3K) / Akt kinase cascade. PI3K stimulates the activation of Akt which in turn, regulates cell size, proliferation, and survival. Our results demonstrate that DIS3L2 is essential for neural progenitor survival by regulating the Akt signaling. DIS3L2 depletion showed down-regulation of Akt-GSK3β signaling, and the administration of Akt activator SC79 rescued cell death. Akt signaling also regulates neural crest gene expression in vivo, which further potentiates our DIS3L2-mediated Akt and neural crest specifier genes down-regulation ( 7 ). These findings are summarised in Fig.5 . Download figure Open in new tab Figure 5: Summary of developmental functions of dis3l2 Schematic representation to synopsize role of dis3l2 in early embryogenesis. Dis3l2 depletion results in reduced Akt and GSK3β signaling, resulting in upregulation of apoptosis pathways involving bim , puma , foxo and effector CASPASES. This results in massive surge of apoptosis in the embryonic neural tissue. Concomitantly, dis3l2 depletion also results in upregulation of cell cycle regulators such as CDK1, p21 and p27, which in turn further upregulate the apoptotic mechanisms. Hence, the embryos show myriad of mitotic defects during early embryogenesis. Additionally, the dis3l2 morphants exhibit reduced expression of neural crest specifier genes, resulting in severe craniofacial anomalies at larval stages. Limitations of the study The current study is limited to cell fate specification and survival of neural progenitor cells in the embryonic CNS. The DIS3L2-mediated mechanism of cell survival in non-CNS tissues such as mesodermal tissue has not been addressed. It is imperative to also determine how DIS3L2 recruits its targets to perform its diverse cellular functions. Lastly, the mechanism by which DIS3L2 interacts with the mitotic machinery remains to be elucidated. Conclusions Our study provides fresh perspectives on DIS3L2-mediated neuronal apoptosis and its neurodevelopmental functions, particularly in cranial neural crest specification, patterning, and survival. DIS3L2 mutations are associated with Perlman syndrome and Wilm’s tumor ( 1 , 3 , 50 ). Future studies to establish a DIS3L2-based Perlman syndrome model using zebrafish embryos will provide mechanistic insights into the pathophysiology and cellular basis of the affected neural tissue. Delineating such molecular mechanisms governing neuronal apoptosis and attainment of normal brain size will also provide a better understanding of neurological diseases like Alzheimer’s and Parkinson’s disease. Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors have read the manuscript and given consent for publication. Availability of data and materials Not applicable. Competing interests The authors have no competing financial and non-financial interests. Funding The authors thank the CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Govt. of India, and the Department of Science and Technology, Govt. of India (DST/INSPIRE/04/2016/001436) for supporting and funding this research. Author contributions SD and TP contributed equally to this work. SD performed all the zebrafish experiments, analyzed the data, and assisted in manuscript writing and figure preparation. 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Share DIS3L2 is essential for neural crest survival by modulating Akt signaling Sian D’Silva , Tuhina Prasad , Megha Kumar bioRxiv 2024.12.26.630456; doi: https://doi.org/10.1101/2024.12.26.630456 Share This Article: Copy Citation Tools DIS3L2 is essential for neural crest survival by modulating Akt signaling Sian D’Silva , Tuhina Prasad , Megha Kumar bioRxiv 2024.12.26.630456; doi: https://doi.org/10.1101/2024.12.26.630456 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (8106) Biochemistry (18938) Bioengineering (15088) Bioinformatics (44943) Biophysics (22841) Cancer Biology (19966) Cell Biology (27177) Clinical Trials (138) Developmental Biology (14076) Ecology (21276) Epidemiology (2067) Evolutionary Biology (25670) Genetics (16275) Genomics (23695) Immunology (18890) Microbiology (42946) Molecular Biology (18234) Neuroscience (94416) Paleontology (709) Pathology (3012) Pharmacology and Toxicology (5147) Physiology (8212) Plant Biology (16164) Scientific Communication and Education (2106) Synthetic Biology (4610) Systems Biology (10331) Zoology (2408) window.__CF$cv$params={r:'a48e088e0a5b4ebe',t:'MTc5MTcyMzQ3Ng==',u:'01a12b0a95ca70d9827423982b79185c',ut:'T_bsRLhVDsNqRXwiAW_8OoLA.80JUkzSyRPZ_8gBcGk-1791723476-1.2.1.1-AvLhgDosTJEkYOMhi69wXH5KNU9q7NitknYq71uK.1wchq0tAW2NmiWX6Ll0akV0CgJQynKOH7vNLVF7kYIFIvjP5rMPUQorep60uw6B.t0',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();