Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum

Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum | 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 Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum Ryota Noji , Mari Kaneko , Takaya Abe , Hiroshi Kiyonari , Yukihiro Nishikawa , Takuma Kumamoto , Hitoshi Gotoh , Chiaki Ohtaka-Maruyama , Katsuhiko Ono , Tatsuya Yoshizawa , Tadashi Nomura doi: https://doi.org/10.1101/2024.12.03.625459 Ryota Noji 1 Cell Biology, Kyoto Prefectural University of Medicine, INAMORI Memorial Building , 1-5 Shimogamo-hangi cho, Sakyoku, Kyoto 606-0823 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: noji0928{at}koto.kpu-m.ac.jp tadnom{at}kit.ac.jp Mari Kaneko 2 Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research , 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takaya Abe 2 Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research , 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hiroshi Kiyonari 2 Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research , 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yukihiro Nishikawa 3 Materials Properties Control, Kyoto Institute of Technology , 1 Hashikamicho, Matsugasaki, Sakyoku, Kyoto 606-8585, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takuma Kumamoto 4 Developmental Neuroscience Project, Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science , 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hitoshi Gotoh 1 Cell Biology, Kyoto Prefectural University of Medicine, INAMORI Memorial Building , 1-5 Shimogamo-hangi cho, Sakyoku, Kyoto 606-0823 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chiaki Ohtaka-Maruyama 4 Developmental Neuroscience Project, Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science , 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katsuhiko Ono 5 Developmental Neurobiology, Kyoto Prefectural University of Medicine, INAMORI Memorial Building , 1-5 Shimogamo-hangi cho, Sakyoku, Kyoto 606-0823 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tatsuya Yoshizawa 1 Cell Biology, Kyoto Prefectural University of Medicine, INAMORI Memorial Building , 1-5 Shimogamo-hangi cho, Sakyoku, Kyoto 606-0823 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tadashi Nomura 6 Biomedical Center, Applied Biology, Kyoto Institute of Technology , 1 Hashikamicho, Matsugasaki, Sakyoku, Kyoto 606-8585, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: noji0928{at}koto.kpu-m.ac.jp tadnom{at}kit.ac.jp Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF SUMMARY The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. In these animal groups, the spatiotemporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure, (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of CRISPR-mediated gene-targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation. INTRODUCTION The corpus callosum (CC) is the prominent axon bundle connecting the two cerebral hemispheres and plays crucial roles in the integration of interhemispheric information processes. The CC is formed exclusively in placental (eutherian) mammals, and comparable anatomical structures do not exist in other extant mammalian taxa (marsupials and monotremes) or in non-mammalian amniotes, such as reptiles and birds 1 , 2 . Notably, in these animal groups the two cerebral hemispheres are interconnected with the anterior commissure and the hippocampal commissure 3 , which are highly conserved commissural pathways in all amniote species 2 , 4 . However, it remains unclear how the new route of interhemispheric connections appeared evolved in eutherian lineages. Several studies have demonstrated multiple developmental steps for mammalian CC formation 2 , 5 – 7 . In early stages of mammalian brain development, the two cerebral hemispheres are separated by interhemispheric fissure (IHF), which is composed of leptomeningeal cells and an extracellular matrix 8 . As development progresses, specific astroglial cells in the telencephalic midline, such as midline zipper glia (MZGs), indusium griseum glia, and glial wedges, play essential roles in the retraction of the IHF 9 , 10 , through the degradation of leptomeningeal cells and the intercalation of midline tissue 11 , 12 . The remodeling process of the IHF is crucial for CC formation because it involves bridging the two hemispheres before pioneer callosal axons reach the midline 12 – 15 . Defects in interhemispheric remodeling are strongly associated with congenital agenesis of the CC in humans 7 , 16 . However, how the dynamic process of midline remodeling prior to CC development evolved in ancestral mammals remains unclear. Consistent with the lack of CC formation, IHF retraction is not detected in marsupials 11 , while the presence of midline zipper glia in these animal groups obscures the evolutionary origin of IHF remodeling and the underlying molecular mechanisms. Special AT-rich sequence-binding protein 2 (Satb2) plays crucial roles in mammalian neocortical development. In the developing murine neocortex, Satb2 regulates the differentiation of upper cortical neurons that contribute to CC bundle formation 17 – 20 . The loss of Satb2 function results in the fate conversion of upper cortical neurons to deep layer neurons, whereby callosal axons misroute to subcortical regions 17 – 19 , 21 . Furthermore, the differential timing of Satb2 expression in the developing neocortex provides a route of species-specific commissural connections, suggesting that heterochronic expression of Satb2 contributes to eutherian and marsupial-specific interhemispheric projections 22 . Notably, several studies implicated that agenesis of CC in Satb2-deficient mice is also associated with defects in midline fusion 23 . However, the functional contribution of Satb2 to the midline glial has not been examined in detail. Here we report a remarkable degree of conservation in the interhemispheric remodeling process in non-mammalian amniotes, geckoes, turtles, and birds, despite the lack of a CC in these animal groups. By performing a detailed histological analysis, we observed that distinct developmental mechanisms confer rostral and caudal IHF remodeling, which is tightly associated with the route of other commissural pathways: the rostral pallial commissure (RPC) and HC. Furthermore, using CRISPR-mediated gene targeting, we observed that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes. Our findings suggest that developmental mechanisms for interhemispheric remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation. RESULTS Interhemispheric remodeling is highly conserved in the developing amniote brain To address the evolutionary conservation of the interhemispheric remodeling process in non-mammalian brains, we first compared midline morphogenesis of the telencephalon in various amniote species, including mice ( Mus musculus ), Chinese softshell turtles ( Pelodiscus sinensis ), domestic chicks ( Gallus gallus ), and Madagascar ground geckoes ( Paroedura picta ). Hematoxylin and Eosin (H&E) staining of serial horizontal sections throughout development confirmed that in the early embryonic stages of all the examined species, telencephalic hemispheres are connected only by thin epithelial tissue (telencephalic hinge), whereas the rest of the midline region is completely separated by the IHF ( Figure 1A-D ). In mice, after E14, the IHF is progressively retracted and both sides of the septal tissues are fused to bridge the telencephalic hemispheres ( Figure 1A ). Notably, these interhemispheric remodeling processes (i.e., IHF retraction and septal fusion) were also detected in turtles, chicks, and geckoes, as telencephalic development progressed ( Figure 1B-D ). Exceptionally, in chicken telencephalon the extent of IHF retraction was limited to the caudal part of the septum, and the rest part of the septal midline remained to be separated ( Figure 1C ). In mice, interhemispheric remodeling occurs from the caudal-ventral to the rostral-dorsal direction during development 12 . The ratio of the IHF length to total midline length revealed that stage-dependent IHF retraction occurs progressively in ventral to dorsal direction in both the mouse and the gecko telencephalon ( Figure 1E-G ), suggesting that the spatial and temporal progression of interhemispheric remodeling is highly conserved in amniotes. Download figure Open in new tab Figure 1. Interhemispheric remodeling is common in the developing amniote brains (A-D) Horizontal sections of developing mouse (A), Chinese softshell turtle (St. 16 and St. 26; B), chick (C), and Madagascar ground gecko (D) forebrains. All sections were stained with hematoxylin and eosin. (E-G) Progressive regression of interhemispheric fissure (IHF) from the dorsal to ventral planes in the mouse (E) and gecko (F) forebrains. (G) Quantitative analysis of the IHF regression in a mouse (upper panel) and gecko (lower panel) at different embryonic stages (mouse: E15, N=3 animals and P0 N=4 animals; gecko: 15 dpo N=3 animals and 25 dpo N=4 animals). The formula for simple linear regression to various groups (mouse: E15, y=0.9359841−0.0002687x: R 2 =0.06353; P0, y=0.6630286−0.0024960x, R 2 =0.2498; Geckos:15dpo, y=0.9430885−0.0010498x, R 2 =0.1184; 25dpo, y=0.8253532−0.0045618x, R 2 =0.1938). Horizontal axis: IHF/total IHF, Vertical axis: anatomical positions. The graph shows the ratio of IHF length to total midline length along the dorsal-ventral axis of the forebrain based on anatomical landmarks. For each individual, a dot represents a section and is color coded; early developmental stages represent warm colors, while late developmental stages represent cold colors. IHF : Interhemispheric fissure. Chp : Choroid plexus. LF: Longitudinal Fissure. MZG-dependent degradation of leptomeningeal components occurs in reptiles To address whether the interhemispheric remodeling in non-mammalian amniotes is dependent on midline glial activities, we next examined the expression of several glial markers in the reptilian and avian midline regions. We detected the accumulation of GFAP-positive cells and phosphorylated vimentin (Ser55) -positive mitotic radial glia 24 in the IHF and telencephalic hinge in developing turtles, geckoes and chicks ( Figure 2A ), suggesting that interhemispheric remodeling in non-mammalian species is mediated by specific glial components, such as the MZG and glial wedge, as in the case of eutherian mammals. It has been shown that MZG-dependent degradation of leptomeninges is crucial for the retraction of the IHF in the developing mouse brain 12 . We detected the degenaration of laminin-positive leptomeninges and its degradation during astroglial interculation(GFAP-positive glial fibers) in the developing gecko ( Figure 2B ). In addition, it was suggested that astroglial interculation occurs in conjunction with the aforementioned regression of the IHF from st.21 to st.26 in the developing turtle( Figure 2C ). Furthermore, we corroborated the activity of pan-matrix metalloproteinase (MMP) in the laminin-positive leptomeninges adjacent to GFAP-positive glial fibers in the developing turtle midline region (Figure2D, Supplementary Movie1), suggesting that MMP-mediated degradation of leptomeningeal components occurs during interhemispheric remodeling in reptiles, as reported in mice 12 . Download figure Open in new tab Figure 2. MZG-dependent degradation of leptomeningeal cells in non-mammalian amniotes (A) GFAP-positive or phosphorylated vimentin-positive midline glial cell components in the developing mouse, turtle, chick, and gecko forebrains. (B) Distribution of laminin-positive leptomeninges and GFAP-positive midline zipper glia (MZG)-like fibers in the gecko forebrain. (C) GFAP-positive fibers before IHF regression (left) and during IHF regression (right) in the turtle forebrain by immunohistochemistry. The lower panels show high magnification of each image. (D) Detection of pan-matrix metalloproteinase (MMP) activity during IHF remodeling of the turtle forebrain in slice culture(DIV: Day In Vitro) and schematic drawings. IHF : Interhemispheric fissure. Epithelial remodeling occurs during caudal-ventral interhemispheric remodeling As observed histologically, telencephalic hemispheres are joined with the lamina terminalis, which gives rise to the choroid plexus during embryogenesis. We confirmed that at early embryonic stages the two hemispheres are connected with the primordial choroid plexus in all examined amniote species ( Figure 1A-D ). However, at later stages the choroid plexus is disconnected from the telencephalic hinge that bridges the caudal-ventral part of the septum. We examined serial oblique sections of the septum from the dorsal to ventral planes, which represent the temporal process of interhemispheric remodeling, and corroborated the gradual disconnection of the choroid plexus from the hinge tissue in the mouse, turtle, chick, and gecko midlines ( Figure 3A-E ). We also confirmed the accumulation and invasion of phosphorylated vimentin-positive glial fibers into the lamina terminalis, which coincided with the disruption of the laminin-positive leptomeninges, during the remodeling process in chick embryos ( Figure 3F , G, Supplementary Movie2). These lines of evidence suggest that dynamic rearrangement of epithelial tissues occurs at the caudal-ventral part of the septum. In the developing chicken brain, the distributions of vimentin-positive fibers and GFAP-positive MZG-like cells were clearly separated in the interhemispheric midline, which were coincided with remodeling (caudal) and non-remodeling (rostral) regions of the interhemispheric fissure ( Figure 2A ). This finding suggests that caudal and rostral interhemispheric remodeling are mediated by distinct glial components, and despite the presence of MZG-like cells, IHF retraction does not occur in the rostral part of the septum in chicken brains. Download figure Open in new tab Figure 3. Epithelial remodeling during the caudal-ventral interhemispheric remodeling (A) Schematic drawings of caudal remodeling procedures. (B) Serial sections of the dorsal to ventral plane demonstrating epithelial remodeling of hinge regions in the developing mouse (B), turtle (C), gecko (D), and chick (E) forebrains. (F) Distribution of laminin and phosphorylated vimentin at the lamina terminalis of chick forebrain. (G) A confocal image showing invasion of phosphorylated vimentin-positive glia into the leptomeninges in the chick forebrain. Histological images of turtles (C), geckos (D), and chicks (E) were obtained from the same individuals as in Figure 1 . IHF : Interhemispheric fissure. Chp : Choroid plexus. LF: Longitudinal Fissure. Vas : Vasculature. Rostral and caudal remodeling bridge the pathway of distinct pallial commissures To determine whether interhemispheric remodeling in non-mammalian amniotes provides substrates for distinct commissural pathways, we traced commissural axons in gecko hemispheres and compared them with those in murine brains. Unilateral deposition of a lipophilic tracer on the gecko dorsal cortex resulted in retrograde labeling of cells in the contralateral cortex ( Figure 4B ), as in the case of the labeling of callosal projections in the mammalian neocortex ( Figure 4A ). Thus, the reptilian telencephalic hemispheres are connected with commissural axons at positions similar to those of the mammalian CC. To compare topographic patterns of commissural pathways in mice and geckoes, we combined immunostaining with an anti-GAP43 antibody, a conserved marker for commissural axons, and anterograde axon tracing. We confirmed that distinct routes of commissural axons that connect the rostral and caudal parts of gecko hemispheres, which correspond to the rostral and caudal pallial commissure (RPC and CPC), respectively ( Figure 4C ). Notably, the positions of RPC and CPC in geckoes is coincided well with the anatomical positions of the CC and HC in mice ( Figure 4C ). Furthermore, we found that GAP43-positive axons do not cross the midline before IHF remodeling in the developing chicken and turtle brains (Figure S1). These data suggest that rostral and caudal interhemispheric remodeling play crucial roles in the routing of distinct commissural pathways; in particular, rostral midline remodeling provides substrates for both the CC and RPC, whereas the caudal remodeling bridges the route of the HC in both the mammalian and reptilian brain. Download figure Open in new tab Figure 4. Rostral and caudal remodeling bridge the pathways of distinct pallial commissures (A) A schematic drawing showing the position of plane I and plane II in the developing forebrain. (B) Retrograde tracing of callosal projection neurons in the mouse and pallial commissural neurons in the gecko (arrowheads in the left and right panels, respectively). The images correspond to plane I. (C) Immunohistochemistry and anterograde axon tracing showing the position of the corpus callosum (CC) and the hippocampal commissure (HC) in the mouse forebrain (left panels) and rostral and caudal pallial commissures (RPC and CPC, respectively) in the gecko forebrain (right panels). The absence of rostral IHF remodeling coincides with the lack of the RPC in chicks Previously we reported that the minimal retraction of the IHF results in the absence of rostral interhemispheric remodeling in the developing chicken brain. To examine the route of pallial commissures that connect chicken telencephalic hemispheres, we performed anterograde axon tracing of distinct regions of the chicken pallium. The deposition of a lipophilic tracer on the hyperpallium apicale (HA), a homologous region to the mammalian neocortex, labeled axons that projected medially toward the rostral septum, whereas the tracer deposition on the hippocampus labeled discrete axonal projections direct to the caudal midline of the septum ( Figure 5A-C ). To further examine the projection patterns of HA-derived axons, we performed unilateral electroporation of GFP expression vector into the chicken brain. At E10, GFP-labeled axons derived from the HA extended ventrally toward the septum, routing to the ipsilateral ventral telencephalon via the tractus septomesencephalicus (TSM, Figure 5D ). Importantly, these axons never crossed the midline of the septum in the developing chicken forebrain. Immunostaining with the GAP43 antibody also revealed ipsilateral axonal projections in the rostral septum ( Figure 5D ). To address whether the TSM is derived from specific neuronal subtypes, we performed retrograde axon tracing followed by immunostaining for Satb2, a transcription factor expressed in excitatory projection neurons. Injection of CTB488 into the rostral septum retrogradely labeled SATB2-positive neurons in the HA ( Figure 5E ). In developing chicken brains, caudal interhemispheric remodeling occurred at approximately E10 ( Figure 1C ). In contrast, the number and density of retrogradely labeled cells were not significantly different before (E9) or after (E13) remodeling. ( Figure 5F ), suggesting that caudal remodeling does not provide substrates for midline crossing of HA-derived axons. Using anterograde tracing and immunostaining with anti-GAP43 antibody, we confirmed that the CPC instead crossed the midline after caudal interhemispheric remodeling ( Figure 5G, H ). These data indicated unique trajectories of pallial axons in chicken hemispheres, and in line with the absence of rostral remodeling, axons derived from the HA do not cross the midline, unlike to mammalian CC and reptilian RPC. Download figure Open in new tab Figure 5. The absence of rostral IHF remodeling coincides with the lack of the RPC in the chick forebrain (A) Schematic drawings showing injection sites of the lipophilic tracer in chicken brain sections. (B, C) Anterograde tracing of Hyperpallium apicale (HA)-derived axons in the rostral septum (B) and HC-derived axons crossing the caudal septum. (D) Anterograde tracing of HA-derived axons by electroporation of a GFP-expression vector Images were coronal sections. GFP-labeled axons projected toward the ipsilateral septum and contributed to the TSM. Image was coronal sections. Immunostaining with an anti-GAP43 antibody also revealed axons running through the septum Image was horizontal section. (E) Retrograde tracing of Satb2-positive neurons by injecting CTB488 into the septum. (F) Proportions of Satb2-positive soma in CTB-labeled soma (left panel) and CTB-labeled soma density (right panel) in chicken brain sections at different developmental stages. Soma located in HA or Mesopallium 54 were labeled in green or red, respectively. E9+1DIV:N=7; E13+1DIV:N=4 (G) Anterograde tracing of the CPC at the caudal septal midline in the E14 chicken brain. GAP43-positive CPC was detected only the ventral septum where the IHF remodeling occurs. Genes for interhemispheric remodeling are expressed in the developing chicken brain To further investigate the evolutionary conservation of gene expression associated with interhemispheric remodeling, we performed spatial gene expression profiling on the Visium platform (10x Genomics). We collected data from coronal sections of the forebrain hemisphere of a E14 chick. The gene expression of individual spots was automatically classified into 8 clusters and visualized by dimension reduction with t -distributed stochastic neighbor embedding ( t -SNE, Figures S2A and S2B). Each cluster was mapped to a specific anatomical region in the chicken hemisphere (Figure S2A). We focused on cluster 8, which corresponded to the midline region as well as the margin of the hemisphere, presumably containing leptomeninges (Figure S2C). We found that several genes associated with midline glial components or interhemispheric remodeling, such as Vimentin (Figure S2D), MMP2 (Figure S2E), and Draxin (Figure S2F), were also expressed in cluster 8, suggesting that molecular basis for caudal interhemispheric remodeling is highly conserved between mice and chicks, despite the lack of rostral remodeling and the RPC in chick brains. Satb2-dependent rostral remodeling is associated with CC and RPC formation in both mice and geckoes Our comparative analyses suggest that discrete developmental mechanisms confer the rostral and caudal interhemispheric remodeling in amniotes. It has been shown that the rostral part of CC formation was severely compromised in cortex-specific Satb2 mutant mice, in accordance with aberrant projection of callosal axons toward the ipsilateral septum 20 . Furthermore, several histological data from Satb2 knockout mice implied incomplete IHF retraction in the rostral part of the septum 18 , 23 . These lines of evidence prompted us to re-examine the interhemispheric remodeling phenotype in Satb2 mutants. To test this hypothesis, we generated Satb2 -targeting mice using CRISPR-mediated gene editing (Figure S3A-S3C). Detailed histological examination revealed that callosal agenesis was closely associated with the disconnection of the rostral septal midline in the mice with only Satb2 deletion alleles ( Satb2 del/del ), whereas callosal formation and midline morphology were not affected in the mice with the wild-type allele ( Satb2 WT/del , Figure 6A ). We detected altered accumulation of the GFAP-positive glial population including the MZG, indusium griseum glia, and glial wedge, on both sides of the midline in Satb2 del/del mice ( Figure 6B ). Concomitantly, an altered direction of GFAP-positive glial fibers was evident in Satb2 del/del mutants, suggesting that disorganized glial components contribute to the failure of IHF remodeling ( Figure 6B, C ). Download figure Open in new tab Figure 6. Satb2-dependent rostral remodeling is associated with CC and RPC formation in both mice and geckoes (A) Horizontal sections of Satb2 WT/del and Satb2 del mouse forebrains. Arrowheads indicate the cellular accumulations corresponding to MZGs. (B) Distributions of GFAP-positive glial fibers in the Satb2 WT/del (upper panels) and Satb2 del (lower panels) IHF regions. Threshold processed images in higher magnification are indicated in black and white. White boxes indicate ROI (Region of Interest) for quantification. Schematic drawing showing how the direction of GFAP+ fiber is quantified. (C) Quantification of GFAP-positive fiber directions in the Satb2 WT/del (upper panels) and Satb2 del (lower panels) IHF regions. The different colors of each dot indicate different sections from the same individual. (D) Horizontal sections of wild-type (WT) and Satb2 del gecko forebrains. An asterisk indicates the remanent of IHF. (E) Distributions of GFAP-positive glial fibers in the dorsal (left) and ventral (right) septum of WT and Satb2 del geckos. Threshold processed images are indicated in black and white. Threshold processed images in higher magnification are indicated in black and white. Blue or red boxes indicate ROI (Region of Interest) for corresponding quantification. (F) Quantification of GFAP-positive fiber directions in the dorsal and ventral septum of WT and Satb2 del geckoes. CC: Corpus Callosum. HC: Hippocampal Commissure. LVChP: Lateral ventricle Choroid Plexus. Next, we investigated whether Satb2 contributes to rostral interhemispheric remodeling and RPC formation in non-mammalian amniotes. We have previously shown that Satb2 is expressed in the developing gecko pallium 25 , 26 . Thus, we performed CRISPR-mediated gene targeting to disrupt Satb2 in geckoes (Figure S4A, S4B). Compared with wild-type embryos, mutant gecko embryos with only Satb2 deletion alleles ( Satb2 del/del ) exhibited a shortened mandible (Figure S4C), which resembled Satb2-deficient mice 27 . Microcomputed tomography (micro-CT) analysis indicated abnormal skeletal formation and altered distribution of pan-collagen in the midline of Satb2 del/del embryos (Figure S4C). Histological analysis revealed that Satb2 del/del mutant embryos exhibited the disconnection of hemispheres and RPC agenesis ( Figure 6D ). The failure of IHF retraction was specific to the rostral septum, whereas the midline closure and CPC projection were not compromised at the caudal septum ( Figure 6D ). We examined whether the distribution of midline glial components was affected in the Satb2 del/del mutant. In contrast to those in mice, GFAP-positive fibers extended in the rostral-caudal direction in wild-type gecko ( Figure 6E, F ). However, the vertical projection of glial fibers was less prominent in Satb2 del/del geckoes ( Figure 6E, F ). These data suggest that the role of Satb2 in the rostral interhemispheric remodeling is highly conserved in eutherian mammals and reptiles, depending on species-specific distribution of midline glial cells in the dorso-ventral axis. Satb2 affects astrocyte characteristics in the developing mouse pallium SATB2 is expressed in postmitotic neurons and plays an essential role in the specification of upper-layer neurons in the developing mouse neocortex 17 , 18 , 20 . Intriguingly, the loss of callosal projection identity also affects laminar-specific astrocyte identity in Satb2 knockout mice 3 . To address the functional link between Satb2 and midline glial development, we performed gene expression profiling in E17.5 Satb2 knockout mice based on published datasets 28 . The expression of callosal development-associated genes, such as Epha4 29 , Ephb1 29 , Zeb2 30 , 31 , Robo2 32 , and Slit3 15 , Unc5C 19 were significantly altered in the cerebral cortex of Satb2 mutant mice compared with than in wild-type mice (Figure S5A-C). To further investigate the contribution of Satb2 functions in glial cell characteristics, we secondary analyzed a previously published single-cell RNA-seq datasets of E15.5 mouse cortex 33 . We classified total 7 clusters (#0-6) with distinct cell states and identified lineage trajectories from cluster 4 toward cluster 1 and 3 (Figure S5D), which contained cells expressing genes associated with midline glial cells or interhemispheric fusions (Figure S5E, F). Intriguingly, Satb2 was highly expressed in Zeb2 -enriched cluster 4, and gradually decreased along with pseudotime (Figure S5D-F). By immunohistochemistry, we confirmed that SATB2 is expressed in the midline subventricular zone, adjacent to the glial wedge that produces GFAP-positive midline glial sling in the developing mouse brain (Figure S5G). Taken together, these results suggest that Satb2 contributes to the establishment of midline glial cell characteristics. DISCUSSION Here we demonstrate that interhemispheric remodeling, an initial step for CC formation in eutherian mammals, is also detected in non-mammalian amniotes. Spatio-temporal remodeling procedures coincide well with the routes of distinct pallial commissures, such as the RPC and CPC, in the reptilian forebrains. The reptilian and avian pallial commissures are considered to connect the hippocampus, therefore these axonal bundles are homologous to the HC 2 , 34 , 35 . However, several histological reports have suggested that the reptilian pallial commissure consists of the rostral and caudal pathways, and the former carries axons derived from the dorsal cortex, a homolog of the mammalian neocortex, whereas the latter connects the medial pallium, a homologue of the mammalian hippocampus 36 . In the present study we identified that the RPC and the CPC exhibit a high degree of similarity with the mammalian CC and HC, in terms of anatomical and developmental aspects. Thus, we propose that developmental mechanisms for interhemispheric connections are highly conserved in amniotes, and that the regulatory components for the RPC, probably derived from the ancestral amniotes, may correspond to evolutionary precursors for the development of the CC in eutherian mammals. Our comparative analyses suggest that the rostral and caudal interhemispheric remodeling is conferred by distinct developmental mechanisms. In particular, caudal remodeling is mediated by the rearrangement of the lamina terminalis, leading to separation from the telencephalic hinge and a part of choroid plexus. The caudal remodeling occurs prior to midline crossing of the CPC or HC, suggesting that bridging mechanisms for the caudal septum midline provide substrates for the evolutionarily conserved commissural pathways. Notably, the rostral interhemispheric remodeling and subsequent formation of the RPC were not detected in chicken forebrain. Instead, the axons derived from the HA, a homologous region of the mammalian neocortex, extend to the ipsilateral septum and contribute to the TSM. We propose that avian TSM corresponds to the reptilian RPC, and that the rostral degeneration of IHF and midline crossing of RPC may have been secondarily lost during the evolution of avian lineages. Intriguingly, IHF degeneration is not detected in marsupials or monotremes that lack CC formation 11 . Whether these animal characteristics are the result of secondary modifications or are derived from ancestral traits awaits further analysis. Disruption of Satb2 function results in impaired rostral interhemispheric remodeling in both mice and geckoes, suggesting evolutionary conservation of Satb2-dependent pallial rostral commissure development in amniotes. It has been considered that CC agenesis in Satb2 mutant mice is due to misrouting of callosal axons to subcortical pathways, rather than impaired differentiation of midline glial cells 17 , 18 , 20 , 37 . In contrast, we demonstrated that in both mouse and gecko Satb2 mutants, the failure of interhemispheric remodeling is associated with altered distribution of midline glial cells. Furthermore, the analysis of gene expression profiles in mice suggested that Satb2 plays a role in the establishment of astrocytic cell states. Because Satb2 is not expressed in differentiated midline glial components, we hypothesize that Satb2 has a non-cell autonomous effects on altered midline glial distribution. Alternatively, the defect in midline glial cells may be due to the secondary effects of impaired neuronal differentiation, as shown in a previous report of laminar-specific astrocytic differentiation 3 . Interhemispheric fissure is composed mainly of connective leptomeningeal fibroblasts and the extracellular matrix, which act as barriers to the callosal axon projection 12 . It has been reported that in DRAXIN or Msx2-Ctnnb1 KO mutant mice, callosal agenesis is associated with pial membrane hyperplasia 8 , 13 . Several studies have reported that callosal abnormalities are linked with craniofacial abnormalities, suggesting a functional link between cranial mesenchymal differentiation and callosal projection. Intriguingly, Satb2 is expressed in cranial mesenchymal cells of the osteoblast lineage 27 , thus it is possible that the failure of IHF retraction is secondary to cranial abnormalities. Rostral interhemispheric remodeling does not occur in marsupials or monotremes; in these animals, axons from upper cortical neurons are directed to the anterior commissure 2 . Diffusion tensor imaging of human callosal dysgenesis suggested that the failure of midline crossing causes the rerouting of callosal fibers to various white matter regions 38 . A previous study indicated that species-specific routing of intracortical projections is mediated by differential timing of Satb2 expression 22 . Satb2 deficiency in mice has been shown to cause misrouting of callosal projections toward the ventral septum 20 , which partially resemble chick TSM routing. Although mechanisms underlying the developmental and evolutionary plasticity of axonal routing remain to be elucidated, our results suggest that Satb2 plays a key role in the multiple steps of pallial commissure development and evolution, which also provides a better understanding of variable human callosal agenesis. RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tadashi Nomura ( tadnom{at}kit.ac.jp ). Material availability All unique/stable reagents in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and code availability All original code and the data used to generate the figures and in this manuscript are have been deposited at Mendeley Data (DOI: 10.17632/2yx33zfkgn.3): The raw data generated in this study will be shared by the lead contact. AUTHOR CONTRIBUTIONS Conceptualization, R.N. and T.N.; methodology, H.K., M.K., T.A, and Y.N.; investigation, R.N. and T.N.; visualization, R.N. and T.N. funding acquisition, R.N. and T.N. and T.K.; project administration, H.G.,C.O., K.O. and T.Y.; supervision, T.N.; writing – original draft, R.N.& T.N. writing – review & editing, all authors. DECLARATION OF INTERESTS The authors declare that they have no competing interests. STAR METHODS The detailed methods are provided in the online version of this paper and include the following: KEY RESOURCES TABLE View this table: View inline View popup METHODS DETAILS Animals Mouse embryos (CD-1 background) that were originally obtained from Japan SLC were maintained in at the experimental animal facilities of Kyoto Prefectural University of Medicine, Kyoto Institute of Technology in accordance with the relevant guidelines (M2023-199, M2023-202, M2023-205, R2023-5, #5), and the Institutional Animal Care and Use Committee of RIKEN Kobe Branch. Fertilized chicken eggs ( Gallus gallus ) were obtained from a local farm (Yamagishi, Japan) and incubated at 38 °C. The developmental stages of the embryos were assessed using a previously described method 46 . The fertilized Madagascar ground gecko eggs and adults were maintained in Developmental Neurobiology, Kyoto Prefectural University of Medicine and the animal facility of RIKEN BDR at 28 °C-30 °C until the analysis, and the embryonic stages were determined in accordance with previous studies 47 . Fertilized Chinese softshell turtle eggs were obtained from a local breeder (Daiwa Yoshoku) and incubated at 28 °C-30 °C until the time of analysis, and the embryonic stages of the turtles were determined according to a previous study 48 . Paraffin sectioning The embryonic samples were fixed with modified Clarke solution (99.5% ethanol and acetic acid at a ratio of 3:1). After rehydration, decalcification was performed using a 10% EDTA solution (pH 7.4) at room temperature for 1 week, followed by replacement with phosphate-buffered saline (PBS). The tissues were dehydrated using a series of ethanol solutions, and the samples were treated with Neo-Clear. Serial sections (4 µm thickness) were made with a microtome (Yamato Kohki) and mounted onto MAS coated glass slides (Matsunami) and incubated at 50 °C. After deparaffinization, the sections were subjected to hematoxylin and eosin (H&E) staining or immunohistochemical staining. Immunohistochemistry Embryonic samples were fixed overnight at 4 °C in a 4% paraformaldehyde (PFA)/1×PBS solution or a formalin-alcohol solution. After washing with PBS, cryoprotection was achieved with 20% sucrose solution, followed by embedded in OCT compound (Sakura, Japan). The frozen brains were sectioned at a thickness of 10 or 20 µm using via a cryostat. Alternatively, the fixed brains were sliced with a vibrating microtome. For immunohistochemistry, the sections were incubated with primary antibodies (see Key source table). After washing with Tris-buffered saline with Tween 20 (TBST) or PBS with Triton-X (PBST), the sections were incubated with secondary antibodies (see Key source table). Fluorescence images were captured with fluorescence microscopes equipped with a cooled CCD camera and a laser confocal microscope. All the captured images were processed with cellSens standard, FV10-ASW, ImageJ and Adobe Photoshop. Ex vivo slice culture Embryonic chick or turtle brains were dissected in 1×PBS and embedded in 4% low-melting agarose for approximately 10 min in room temperature. To prevent autofluorescence, chicken embryos were perfused with ice-cold PBS before isolation of brains. Embedded brains were sliced with a vibrating microtome with 250 or 300 µm in thickness. Brain slices were placed on a culture filter and cultured in DMEM plus 10% FBS and 1% antibiotics (penicillin/streptomycin). The detection of pan-matrix metalloproteinase (MMP) activity in the developing Chinese softshell turtles was performed according to a previous report 12 with slight modifications. Briefly, brains slices were incubated the culture medium containing 0.4 µM MMPSense 645 Fast fluorescent probe at 30 °C or 37 °C for 24 hours. Axon tracing After the brain was fixed with 4% PFA/1×PBS, small fragments of filters containing lipophilic dye (NeuroVue) were inserted into the specific brain regions. The samples were incubated in 1% PFA/1×PBS at 37 °C for 3–4 weeks, then analyzed following sectioning with a vibrating microtome. Alternatively, the fixed brains were sectioned with a vibrating microtome prior to the axon tracing. For retrograde axon tracing, isolated brains were sliced with a vibrating microtome, and Cholera Toxin Subunit B conjugated with Alexa fluor 488 (CTB488) was injected into the focal region of the slice. After injection, the slices were placed on a culture filter and cultured for 24 hours in 37 °C. Generation of Satb2 -deficient mice Satb2 -deficient mice were generated via CRISPR/Cas12a (Cpf1)-mediated genome editing. A crRNA sequence that targets mouse Satb2 exon 2 was designed by CHOPCHOP. We chose a target sequence with no off targets on the basis of mouse genome information (mm10/GRCm38). Genome-edited mice on the CD-1 background were generated via the i -GONAD method 49 . Briefly, pregnant female mice (E0.75) were anesthetized with 2% isoflurane and the CRISPR/Cas12a solution was injected into the oviduct lumen. Then, square electric pulses were applied to the oviduct via a pulse generator (Nepa gene). Generation of Satb2 -deficient geckoes Satb2 -deficient geckoes were generated via CRISPR/Cas9-mediated genome editing as previously described 50 . The predicted genomic sequences and cDNA of gecko ( Parodedura picta ) Satb2 were identified by the Reptiliomix BLAST search ( https://transcriptome.cdb.riken.jp/reptiliomix/ ). The predicted motif of the gecko Satb2 coding region was identified by MOTIF search ( https://www.genome.jp/tools/motif/ ). gRNA sites were designed via CRISPRdirect ( https://crispr.dbcls.jp ) and specificity was checked based on Ppicta_v2.0. The female geckoes were anesthetized with an isoflurane inhalation solution. Microinjection into the female ovary was performed using an electric microinjector system (IM-400, NARISHIGE) under a stereomicroscope (Leica MZ95). After microinjection, the ovary was returned to the abdominal cavity and the musculature and skin were sutured. Genotyping of Satb2 -deficient mice and geckoes Genomic DNA from embryonic mouse or gecko tissues (skin or tail fragments) was isolated via a DNeasy Blood & Tissue kit. The DNA fragments containing the CRISPR target region were amplified via polymerase chain reaction (PCR). To determine of the edited sequences in F0 embryos, PCR amplicons were subcloned into the pBluescriptSK vector, and six to ten randomly selected clones were examined via Sanger sequencing. Electroporation In ovo electroporation of developing chick embryos was performed according to a previous study 25 . Briefly, a window was opened in the shell of an egg, and then a small amount of DNA solution (less than 0.05 µL) was injected into Hamburger & Hamilton 51 25 chicken embryos with a glass needle. Next, needle-type electrodes (CUY200S, BEX) were placed on the neural tube and square electric pulses (28 V for 50 msec, 3 times) were applied with an electroporator. After electroporation, the extraembryonic cavity was filled with sterilized Hank’s balanced salt solution (HBSS) containing antibiotics (1:1000; penicillin/streptomycin, gentamicin), and the window was sealed with cellophane tape. The electroporated embryos were incubated in an incubator at 38 °C Until the prescribed developmental stage. In utero electroporation was performed according to previously described methods 52 . Briefly, a pregnant mouse was anesthetized with isoflurane, and an incision was made in the abdominal wall to expose the uterine horns. A small amount of DNA mixture (0.3– 0.5 µL) was injected into the lateral ventricle of each embryo, and square pulses (35 V, 50 ms, four times) were applied to the embryos with a tweezer-type electrode (CUY650P3, BEX) connected to a pulse generator (BEX). IHF measurement IHF measurements on H&E-stained samples were performed by using Fiji (ImageJ). IHF lengths were normalized to the entire rostral–caudal length of the terminal brain along the interhemispheric midline as previously described 12 . To determine macroscopic trends, a single regression line was drawn by using the least-squares method, and the direction and degree of remodeling were quasi-investigated by the slope. In this case, a single regression line was obtained and drawn for each developmental stage group of mice and geckos, respectively, via R software (ggplot2). The confidence intervals were set at 95%, and the ranges are shaded in the corresponding figure colors. Spatial transcriptome The brains of the embryos were harvested at chick E14, and directly embedded with O.C.T. compound. Embedded brain stored at −80 °C deep freezer until sectioning. Frozen sections were prepared from each brain at a thickness of 10 μm as manufacture’s protocol. The sections were placed on the Visium Spatial Gene Expression Slide (10x Genomics, USA) and processed with the Visium Spatial Gene Expression Reagent kit (10x Genomics, USA) according to the manufacture’s protocol. H&E brightfield and FL staining images were collected as mosaics using a Keyence BX-800 microscope (Keyence, Japan). The resultant libraries were sequenced at Macrogen inc. by the HiSeqX platform (Illumina, USA) with 150bp paired end sequencing. Data processing of spatial gene expression FASTQ files were processed with Space Ranger 1.3.1 with default parameter settings referring to the gene annotations of the galGal6 genome assembly provided by UCSC (galGal6.ncbiRefSeq.gtf.gz) resulting in a production of the matrices of gene expression profiles of the individual cells. Its FASTQ files were retrieved from the UCSC genome browser( https://hgdownload.soe.ucsc.edu/goldenPath/galGal6/bigZips/galGal6.fa.gz ). The gene expression profiles of the E14 sample were processed with Space Ranger. Read count matrices were normalized, and the spots of the sample were grouped based on their expression profiles by k-means clustering, resulting in 8 clusters. 2D t -SNE plots were visualized with Loupe Browser application. Marker genes, those with significantly higher expression levels in a cluster than the others, were detected with Loupe Browser. The clusters were manually annotated referring to the ‘known’ marker genes that were included in those automatically inferred. RNA-seq data analysis Bulk RNA sequencing data from the cortex of E17.5 wild-type and Satb2 KO mice were downloaded from GSE201561 28 . The tsv files were converted to csv files on R studio. DEGs of genes expressed in parts of the glial sling in the literature described in the text were compared via hierarchical clustering and violin plots in wild-type and Satb2 KO mutant. Single-cell RNA-sequencing data of the E15.5 mouse astrocyte progenitor cells (GFP-positive and RFP-negative cells derived from Nes-GFP/Dcx-mRFP double transgenic reporter mice.) were downloaded from GSE128948. We used the Seurat package (v5.0.3) to analyze the data. Dimensionality reduction was performed via principal component analysis (PCA) and UMAP via the Seurat pipeline with standard parameters. R studio (package: SeuratWrapper, tidyverse, dplyr, SeuratWrappers, ggplot2, ggridges, and Monocle3) were used to draw violin plots and UMAPs, and Monocle 3 was used for psedotrajectory analysis. X-ray computed tomography For micro-CT scanning analysis, samples fixed with 4% PFA in PBS were stained with 1% phosphotungstic acid solution (w/v) in 100% EtOH and scanned with a FLEX-M345 at the Kyoto Institute of Technology. The scanned data were processed with Kyoto CT OD reconstruction or the original version of the software and were converted to a stack of 8-bit tiffs to hyperstack with Fiji (ImageJ). A peak voltage of 40 kV and a current of 100 µA, unfiltered, were used for skeletal staining data, and a peak voltage of 40 kV and a current of 80 µA were used for pan-collagen visualization data. 53 For quantification of collagen in the central and middle of the range of equivalent anatomical positions, data were converted at 8 bits and the ImageJ plug-in (Plot Z axis) was used. To accurately reflect x-ray absorbance, the change in the same region of interest (ROI) over the dorsoventral axis of the embedding material-only region was subtracted and the values were standardized for PCA as described above. For statistical analysis, at least three independent samples from each experimental group were compared. Comparisons between experimental groups were performed using Microsoft Excel (v16.54, Microsoft) and R studio (package: ggplot2). Image processing and quantificational analyses All fluorescence images were captured with a fluorescence microscope equipped with a CCD camera (DP80, Olympus), and a confocal microscope. Images were examined and analyzed by using Fiji ImageJ. All images were processed with ImageJ and Adobe Photoshop 2024. Fluorescent images were displayed in pseudo-colour, which differs from the original colour if necessary. Glial fiber orientation was compared using Fiji/ImageJ software. Regions of a telencephalic hemisphere showing distinct fiber structures were extracted from paraffin or frozen sections of spoonbills and geckos stained with anti-GFAP antibody. The orientation pattern of the glial fibers in each image was calculated as a ‘Fourier component’ using the ‘Directionality plugin’ ( http://fiji.sc/wiki/index.php/Directionality ). The slice images used for analysis were adjusted using the auto threshold to select Max entropy of the Fiji plugin. The resulting csv file was loaded into R Studio and z-score normalization to the number of GFAP-positive fibers in a given direction was performed to examine the differences in the distribution map of GFAP-positive fibers. The standardized values output by the Directionality plugin were approximated using the R library (ggdist, dplyr, patchwork) and Gaussian fitted at the 95% confidence interval, and the ranges are shaded in the corresponding figure colors. Data visualization and statistical analysis To visualize the differential expression of genes, we utilized the ggplot2 package in R. We first imported the gene expression dataset using the read.csv and specified the file path of the data. To focus on key genes involved in interhemispheric remodeling we filtered the data, extracting rows corresponding to these gene symbols for further analysis. Next, we created a custom color-coding system based on the basis of log 2 -fold changes and adjusted p-values . Specifically, data points with a log 2 fold change greater than 1 and an adjusted p-value less than 0.05 were colored red, indicating significant upregulation. Conversely, data points with a log 2 fold change less than −1 and an adjusted p-value less than 0.05 are colored blue, indicating significant downregulation. Data points not meeting these criteria were excluded from the visualization by filtering out missing (NA) values in the color column. A volcano plot was generated using ggplot2, with the log 2 fold change on the x-axis and the negative log 10 value of the adjusted p-value on the y-axis. This plot allowed for the clear identification of significantly upregulated and downregulated genes. We enhanced the visualization by adding a horizontal dashed line to indicate the false discovery rate (FDR) threshold of 0.05, aiding in the interpretation of statistical significance. CTB-labeled soma density was measured on confocal images of the four experimental groups using ImageJ. The data for each group were transformed by multiplying by 10 -4 µm 2 for consistency. For the analysis of neuronal soma density, we used ggplot2, dplyr, and ggpubr in the R environment. Given the presence of missing values in certain groups, tests were performed only on the available data points. On the basis of the results of the normality test, the appropriate statistical test was selected. If both groups passed the normality test ( p-value >0.05), a t -test was performed to compare the means. Otherwise, a nonparametric Wilcoxon rank sum test was performed.10 -4 µm 2 for consistency. This approach was applied for both the comparisons, with corresponding p-values recorded for each test. The mean values and SEM (standard error of the mean) were added for each group via a point-range plot to provide visual context for the overall distribution of the data. Data availability The raw data of spatial transcriptomics have been deposited in the DDBJ data base (DRA BioProject: PRJDB15113). All data generated in this study have been deposited in Mendeley Data (Mendeley Data, V3, DOI: 10.17632/2yx33zfkgn.3). ACKNOWLEDGMENTS The authors thank Ms. Misato Kawami and Mariko Yazaki for their technical assistance, and Anna Yasunaga for providing wild type geckoes. This research was funded in whole, or in part, by Japanese Grant-In-Aid for Scientific Research (KAKENHI, 21H02594 to T.N.), the Takeda Science Foundation (to T.N.), the Ohsumi Frontier Science Foundation (to T.N.), Leading Initiative for Excellent Young Researchers (LEADER, 2020L0019) and JSPS KAKENHI-Grants (20K22665, 22H02638 to T.K.) and the Kyoto Prefectural University of Medicine 2121 Fellowship (to R.N.)., and Leave a Nest Co., 63rd incu·be encourage award (to R.N). Footnotes ↵ 7 X: @TadashiNomura3 ↵ 8 Lead contact https://data.mendeley.com/datasets/2yx33zfkgn/3 REFERENCES 1. ↵ Owen , R . ( 1837 ). On the Structure of the Brain in Marsupial Animals . Philosophical Transactions of the Royal Society of London 127 , 87 – 96 . OpenUrl CrossRef 2. ↵ Suarez , R. , Gobius , I. , and Richards , L.J . ( 2014 ). Evolution and development of interhemispheric connections in the vertebrate forebrain . Front Hum Neurosci 8 , 497 . doi: 10.3389/fnhum.2014.00497 . OpenUrl CrossRef PubMed 3. ↵ Bayraktar , O.A. , Bartels , T. , Holmqvist , S. , Kleshchevnikov , V. , Martirosyan , A. , Polioudakis , D. , Ben Haim , L. , Young , A.M.H. , Batiuk , M.Y. , Prakash , K. , et al. ( 2020 ). Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map . Nat Neurosci 23 , 500 – 509 . doi: 10.1038/s41593-020-0602-1 . OpenUrl CrossRef PubMed 4. ↵ Fenlon , L.R. , Suarez , R. , Lynton , Z. , and Richards , L.J . ( 2021 ). The evolution, formation and connectivity of the anterior commissure . Semin Cell Dev Biol 118 , 50 – 59 . doi: 10.1016/j.semcdb.2021.04.009 . OpenUrl CrossRef PubMed 5. ↵ Rapti , G . ( 2023 ). Regulation of axon pathfinding by astroglia across genetic model organisms . Front Cell Neurosci 17 , 1241957 . doi: 10.3389/fncel.2023.1241957 . OpenUrl CrossRef PubMed 6. Gavrish , M. , Kustova , A. , Celis Suescun , J.C. , Bessa , P. , Mitina , N. , and Tarabykin , V . ( 2023 ). Molecular mechanisms of corpus callosum development: a four-step journey . Front Neuroanat 17 , 1276325 . doi: 10.3389/fnana.2023.1276325 . OpenUrl CrossRef PubMed 7. ↵ De Leon Reyes , N.S. , Bragg-Gonzalo , L. , and Nieto , M. ( 2020 ). Development and plasticity of the corpus callosum . Development 147 . doi: 10.1242/dev.189738 . OpenUrl Abstract / FREE Full Text 8. ↵ Choe , Y. , Siegenthaler , J.A. , and Pleasure , S.J . ( 2012 ). A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation . Neuron 73 , 698 – 712 . doi: 10.1016/j.neuron.2011.11.036 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Silver , J. , Lorenz , S.E. , Wahlsten , D. , and Coughlin , J . ( 1982 ). Axonal guidance during development of the great cerebral commissures: descriptive and experimental studies, in vivo, on the role of preformed glial pathways . J Comp Neurol 210 , 10 – 29 . doi: 10.1002/cne.902100103 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Shu , T. , Puche , A.C. , and Richards , L.J . ( 2003 ). Development of midline glial populations at the corticoseptal boundary . J Neurobiol 57 , 81 – 94 . doi: 10.1002/neu.10252 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Gobius , I. , Suarez , R. , Morcom , L. , Paolino , A. , Edwards , T.J. , Kozulin , P. , and Richards , L.J . ( 2017 ). Astroglial-mediated remodeling of the interhemispheric midline during telencephalic development is exclusive to eutherian mammals . Neural Dev 12 , 9 . doi: 10.1186/s13064-017-0086-1 . OpenUrl CrossRef PubMed 12. ↵ Gobius , I. , Morcom , L. , Suarez , R. , Bunt , J. , Bukshpun , P. , Reardon , W. , Dobyns , W.B. , Rubenstein , J.L. , Barkovich , A.J. , Sherr , E.H. , and Richards , L.J . ( 2016 ). Astroglial-Mediated Remodeling of the Interhemispheric Midline Is Required for the Formation of the Corpus Callosum . Cell Rep 17 , 735 – 747 . doi: 10.1016/j.celrep.2016.09.033 . OpenUrl CrossRef PubMed 13. ↵ Morcom , L. , Edwards , T.J. , Rider , E. , Jones-Davis , D. , Lim , J.W. , Chen , K.S. , Dean , R.J. , Bunt , J. , Ye , Y. , Gobius , I. , et al. ( 2021 ). DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation . Elife 10 . doi: 10.7554/eLife.61618 . OpenUrl CrossRef 14. Morcom , L. , Gobius , I. , Marsh , A.P. , Suarez , R. , Lim , J.W. , Bridges , C. , Ye , Y. , Fenlon , L.R. , Zagar , Y. , Douglass , A.M. , et al. ( 2021 ). DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation . Elife 10 . doi: 10.7554/eLife.61769 . OpenUrl CrossRef 15. ↵ Unni , D.K. , Piper , M. , Moldrich , R.X. , Gobius , I. , Liu , S. , Fothergill , T. , Donahoo , A.L. , Baisden , J.M. , Cooper , H.M. , and Richards , L.J . ( 2012 ). Multiple Slits regulate the development of midline glial populations and the corpus callosum . Dev Biol 365 , 36 – 49 . doi: 10.1016/j.ydbio.2012.02.004 . OpenUrl CrossRef PubMed 16. ↵ Edwards , T.J. , Sherr , E.H. , Barkovich , A.J. , and Richards , L.J . ( 2014 ). Clinical, genetic and imaging findings identify new causes for corpus callosum development syndromes . Brain 137 , 1579 – 1613 . doi: 10.1093/brain/awt358 . OpenUrl CrossRef PubMed 17. ↵ Alcamo , E.A. , Chirivella , L. , Dautzenberg , M. , Dobreva , G. , Farinas , I. , Grosschedl , R. , and McConnell , S.K . ( 2008 ). Satb2 regulates callosal projection neuron identity in the developing cerebral cortex . Neuron 57 , 364 – 377 . doi: 10.1016/j.neuron.2007.12.012 . OpenUrl CrossRef PubMed Web of Science 18. ↵ Britanova , O. , de Juan Romero , C. , Cheung , A. , Kwan , K.Y. , Schwark , M. , Gyorgy , A. , Vogel , T. , Akopov , S. , Mitkovski , M. , Agoston , D. , et al. ( 2008 ). Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex . Neuron 57 , 378 – 392 . doi: 10.1016/j.neuron.2007.12.028 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Srivatsa , S. , Parthasarathy , S. , Britanova , O. , Bormuth , I. , Donahoo , A.L. , Ackerman , S.L. , Richards , L.J. , and Tarabykin , V . ( 2014 ). Unc5C and DCC act downstream of Ctip2 and Satb2 and contribute to corpus callosum formation . Nat Commun 5 , 3708 . doi: 10.1038/ncomms4708 . OpenUrl CrossRef PubMed 20. ↵ Leone , D.P. , Heavner , W.E. , Ferenczi , E.A. , Dobreva , G. , Huguenard , J.R. , Grosschedl , R. , and McConnell , S.K . ( 2015 ). Satb2 Regulates the Differentiation of Both Callosal and Subcerebral Projection Neurons in the Developing Cerebral Cortex . Cereb Cortex 25 , 3406 – 3419 doi: 10.1093/cercor/bhu156 . OpenUrl CrossRef PubMed 21. ↵ McKenna , W.L. , Ortiz-Londono , C.F. , Mathew , T.K. , Hoang , K. , Katzman , S. , and Chen , B . ( 2015 ). Mutual regulation between Satb2 and Fezf2 promotes subcerebral projection neuron identity in the developing cerebral cortex . Proc Natl Acad Sci U S A 112 , 11702 – 11707 . doi: 10.1073/pnas.1504144112 . OpenUrl Abstract / FREE Full Text 22. ↵ Paolino , A. , Fenlon , L.R. , Kozulin , P. , Haines , E. , Lim , J.W.C. , Richards , L.J. , and Suarez , R . ( 2020 ). Differential timing of a conserved transcriptional network underlies divergent cortical projection routes across mammalian brain evolution . Proc Natl Acad Sci U S A 117 , 10554 – 10564 . doi: 10.1073/pnas.1922422117 . OpenUrl Abstract / FREE Full Text 23. ↵ Zhang , Q. , Huang , Y. , Zhang , L. , Ding , Y.Q. , and Song , N.N . ( 2019 ). Loss of Satb2 in the Cortex and Hippocampus Leads to Abnormal Behaviors in Mice . Front Mol Neurosci 12 , 33 . doi: 10.3389/fnmol.2019.00033 . OpenUrl CrossRef PubMed 24. ↵ Kamei , Y. , Inagaki , N. , Nishizawa , M. , Tsutsumi , O. , Taketani , Y. , and Inagaki , M . ( 1998 ). Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin . Glia 23 , 191 – 199 . doi: 10.1002/(sici)1098-1136(199807)23:33.0.co;2-8 . OpenUrl CrossRef PubMed Web of Science 25. ↵ Nomura , T. , Gotoh , H. , and Ono , K . ( 2013 ). Changes in the regulation of cortical neurogenesis contribute to encephalization during amniote brain evolution . Nat Commun 4 , 2206 doi: 10.1038/ncomms3206 . OpenUrl CrossRef PubMed 26. ↵ Nomura , T. , Yamashita , W. , Gotoh , H. , and Ono , K . ( 2018 ). Species-Specific Mechanisms of Neuron Subtype Specification Reveal Evolutionary Plasticity of Amniote Brain Development . Cell Rep 22 , 3142 – 3151 . doi: 10.1016/j.celrep.2018.02.086 . OpenUrl CrossRef PubMed 27. ↵ Dobreva , G. , Chahrour , M. , Dautzenberg , M. , Chirivella , L. , Kanzler , B. , Farinas , I. , Karsenty , G. , and Grosschedl , R . ( 2006 ). SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation . Cell 125 , 971 – 986 . doi: 10.1016/j.cell.2006.05.012 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Guo , Q. , Wang , Y. , Wang , Q. , Qian , Y. , Jiang , Y. , Dong , X. , Chen , H. , Chen , X. , Liu , X. , Yu , S. , et al. ( 2023 ). In the developing cerebral cortex: axonogenesis, synapse formation, and synaptic plasticity are regulated by SATB2 target genes . Pediatr Res 93 , 1519 – 1527 . doi: 10.1038/s41390-022-02260-z . OpenUrl CrossRef PubMed 29. ↵ Mendes , S.W. , Henkemeyer , M. , and Liebl , D.J . ( 2006 ). Multiple Eph receptors and B-class ephrins regulate midline crossing of corpus callosum fibers in the developing mouse forebrain . J Neurosci 26 , 882 – 892 . doi: 10.1523/JNEUROSCI.3162-05.2006 . OpenUrl Abstract / FREE Full Text 30. ↵ Epifanova , E. , Babaev , A. , Newman , A.G. , and Tarabykin , V . ( 2019 ). Role of Zeb2/Sip1 in neuronal development . Brain Res 1705 , 24 – 31 . doi: 10.1016/j.brainres.2018.09.034 . OpenUrl CrossRef PubMed 31. ↵ Seuntjens , E. , Nityanandam , A. , Miquelajauregui , A. , Debruyn , J. , Stryjewska , A. , Goebbels , S. , Nave , K.A. , Huylebroeck , D. , and Tarabykin , V . ( 2009 ). Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors . Nat Neurosci 12 , 1373 – 1380 . doi: 10.1038/nn.2409 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Lopez-Bendito , G. , Flames , N. , Ma , L. , Fouquet , C. , Di Meglio , T. , Chedotal , A. , Tessier-Lavigne , M. , and Marin , O. ( 2007 ). Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain . J Neurosci 27 , 3395 – 3407 . doi: 10.1523/JNEUROSCI.4605-06.2007 . OpenUrl Abstract / FREE Full Text 33. ↵ Liu , J. , Wu , X. , and Lu , Q . ( 2022 ). Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis . Development 149 . doi: 10.1242/dev.199985 . OpenUrl CrossRef 34. ↵ Johnston , J.B . ( 1913 ). The Morphology of the Septum, Hippocampus, and Pallial Commissures in Repliles and Mammals . J Comp Neurol 23 , 371 – 478 . OpenUrl CrossRef 35. ↵ Henrrick , C.J . ( 1910 ). The morphology of the forebrain in amphibia and reptilia . J Comp Neurol Psychol 20 , 413 – 547 . doi: 10.1002/cne.920200502 . OpenUrl CrossRef 36. ↵ Martinez-Garcia , F. , Amiguet , M. , Schwerdtfeger , W.K. , Olucha , F.E. , and Lorente , M.J . ( 1990 ). Interhemispheric connections through the pallial commissures in the brain of Podarcis hispania and Gallotia stehlinii (Reptilia , Lacertidae). J Comp Neurol 205 , 17 – 31 . OpenUrl 37. ↵ Moldrich , R.X. , Gobius , I. , Pollak , T. , Zhang , J. , Ren , T. , Brown , L. , Mori , S. , De Juan Romero , C. , Britanova , O. , Tarabykin , V. , and Richards , L.J. ( 2010 ). Molecular regulation of the developing commissural plate . J Comp Neurol 518 , 3645 – 3661 . doi: 10.1002/cne.22445 . OpenUrl CrossRef PubMed Web of Science 38. ↵ Tovar-Moll , F. , Moll , J. , de Oliveira-Souza , R. , Bramati , I. , Andreiuolo , P.A. , and Lent , R. ( 2007 ). Neuroplasticity in human callosal dysgenesis: a diffusion tensor imaging study . Cereb Cortex 17 , 531 – 541 . doi: 10.1093/cercor/bhj178 . OpenUrl CrossRef PubMed Web of Science 39. Osumi , N. , and Inoue , T . ( 2001 ). Gene transfer into cultured mammalian embryos by electroporation . Methods 24 , 35 – 42 . doi: 10.1006/meth.2001.1154 . OpenUrl CrossRef PubMed Web of Science 40. Butler , A. , Hoffman , P. , Smibert , P. , Papalexi , E. , and Satija , R . ( 2018 ). Integrating single-cell transcriptomic data across different conditions, technologies, and species . Nat Biotechnol 36 , 411 – 420 . doi: 10.1038/nbt.4096 . OpenUrl CrossRef PubMed 41. Stuart , T. , Butler , A. , Hoffman , P. , Hafemeister , C. , Papalexi , E. , Mauck , W.M ., 3rd, Hao , Y. , Stoeckius , M. , Smibert , P. , and Satija , R. ( 2019 ). Comprehensive Integration of Single-Cell Data . Cell 177 , 1888 – 1902 e1821. doi: 10.1016/j.cell.2019.05.031 . OpenUrl CrossRef PubMed 42. Cao , S. , Zhao , X. , Li , Z. , Yu , R. , Li , Y. , Zhou , X. , Yan , W. , Chen , D. , and He , C . ( 2024 ). Comprehensive integration of single-cell transcriptomic data illuminates the regulatory network architecture of plant cell fate specification . Plant Divers 46 , 372 – 385 . doi: 10.1016/j.pld.2024.03.008 . OpenUrl CrossRef PubMed 43. Hara , Y. , Tatsumi , K. , Yoshida , M. , Kajikawa , E. , Kiyonari , H. , and Kuraku , S . ( 2015 ). Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation . BMC Genomics 16 , 977 . doi: 10.1186/s12864-015-2007-1 . OpenUrl CrossRef PubMed 44. Naito , Y. , Hino , K. , Bono , H. , and Ui-Tei , K . ( 2015 ). CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites . Bioinformatics 31 , 1120 – 1123 . doi: 10.1093/bioinformatics/btu743 . OpenUrl CrossRef PubMed 45. Labun , K. , Montague , T.G. , Krause , M. , Torres Cleuren , Y.N. , Tjeldnes , H. , and Valen , E . ( 2019 ). CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing . Nucleic Acids Res 47 , W171 – W174 . doi: 10.1093/nar/gkz365 . OpenUrl CrossRef PubMed 46. ↵ Hamburger , V. , and Hamilton , H.L . ( 1951 ). A series of normal stages in the development of the chick embryo . J Morphol 88 , 49 – 92 . OpenUrl CrossRef PubMed Web of Science 47. ↵ Noro , M. , Uejima , A. , Abe , G. , Manabe , M. , and Tamura , K . ( 2009 ). Normal developmental stages of the Madagascar ground gecko Paroedura pictus with special reference to limb morphogenesis . Dev Dyn 238 , 100 – 109 . doi: 10.1002/dvdy.21828 . OpenUrl CrossRef PubMed 48. ↵ Tokita , M. , and Kuratani , S . ( 2001 ). Normal Embryonic Stages of the Chinese Softshelled Turtle Pelodiscus Sinensis (Trionychidae) . Zool Sci 18 , 705 – 715 . OpenUrl CrossRef Web of Science 49. ↵ Gurumurthy , C.B. , Sato , M. , Nakamura , A. , Inui , M. , Kawano , N. , Islam , M.A. , Ogiwara , S. , Takabayashi , S. , Matsuyama , M. , Nakagawa , S. , et al. ( 2019 ). Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD . Nat Protoc 14 , 2452 – 2482 . doi: 10.1038/s41596-019-0187-x . OpenUrl CrossRef PubMed 50. ↵ Abe , T. , Kaneko , M. , and Kiyonari , H . ( 2023 ). A reverse genetic approach in geckos with the CRISPR/Cas9 system by oocyte microinjection . Dev Biol 497 , 26 – 32 . doi: 10.1016/j.ydbio.2023.02.005 . OpenUrl CrossRef PubMed 51. ↵ Asgari , S. , Luo , Y. , Akbari , A. , Belbin , G.M. , Li , X. , Harris , D.N. , Selig , M. , Bartell , E. , Calderon , R. , Slowikowski , K. , et al. ( 2020 ). A positively selected FBN1 missense variant reduces height in Peruvian individuals . Nature 582 , 234 – 239 . doi: 10.1038/s41586-020-2302-0 . OpenUrl CrossRef PubMed 52. ↵ Tabata , H. , and Nakajima , K . ( 2001 ). Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex . Neuroscience 103 , 865 – 872 . doi: 10.1016/s0306-4522(01)00016-1 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Hanly , A.R.D. , Johnston , C. , Lemass , A. , Jose , B.T. , and Lally , C . Phosphotungstic acid (PTA) preferentially binds to collagen-rich regions of porcine carotid arteries and human atherosclerotic plaques observed using contrast enhanced micro-computed tomography (CE-ΜCT) . Front Physiol 14 , 1057394 . 54. ↵ Fish , J.L. , Villmoare , B. , Kobernick , K. , Compagnucci , C. , Britanova , O. , Tarabykin , V. , and Depew , M.J . ( 2011 ). Satb2, modularity, and the evolvability of the vertebrate jaw . Evol Dev 13 , 549 – 564 . doi: 10.1111/j.1525-142X.2011.00511.x . OpenUrl CrossRef PubMed Back to top Previous Next Posted December 04, 2024. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum Ryota Noji , Mari Kaneko , Takaya Abe , Hiroshi Kiyonari , Yukihiro Nishikawa , Takuma Kumamoto , Hitoshi Gotoh , Chiaki Ohtaka-Maruyama , Katsuhiko Ono , Tatsuya Yoshizawa , Tadashi Nomura bioRxiv 2024.12.03.625459; doi: https://doi.org/10.1101/2024.12.03.625459 Share This Article: Copy Citation Tools Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum Ryota Noji , Mari Kaneko , Takaya Abe , Hiroshi Kiyonari , Yukihiro Nishikawa , Takuma Kumamoto , Hitoshi Gotoh , Chiaki Ohtaka-Maruyama , Katsuhiko Ono , Tatsuya Yoshizawa , Tadashi Nomura bioRxiv 2024.12.03.625459; doi: https://doi.org/10.1101/2024.12.03.625459 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 (8013) Biochemistry (18739) Bioengineering (14888) Bioinformatics (44418) Biophysics (22599) Cancer Biology (19723) Cell Biology (26899) Clinical Trials (138) Developmental Biology (13965) Ecology (21005) Epidemiology (2067) Evolutionary Biology (25455) Genetics (16166) Genomics (23507) Immunology (18705) Microbiology (42503) Molecular Biology (18059) Neuroscience (93451) Paleontology (700) Pathology (2977) Pharmacology and Toxicology (5095) Physiology (8114) Plant Biology (15999) Scientific Communication and Education (2095) Synthetic Biology (4560) Systems Biology (10235) Zoology (2391) window.__CF$cv$params={r:'a3e0d69b5f61dde1',t:'MTc4OTkwNzM5Mw==',u:'01a0becb652d763c8a3f3fd50af5742c',ut:'9WqlAlj7YWZW1ZhoGYbctFQRtrJxvpVvtCvas.mEHD8-1789907395-1.2.1.1-85sei_v.T4F8TnkVcrgkwhGkxcdMioRK.MTI2_lITvJt7tbh4tGY8YkKHDf3fD7IxJeNWo6.bX01WpyoxRsgfO4rMmezhyor4.y4TSVqEeU',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);})();