Involvement of Slit–Robo signaling in the development of the posterior commissure and concomitant swimming behavior in Xenopus laevis
- Yasuhiko Tosa†1,
- Kiyohito Tsukano†1,
- Tatsuya Itoyama1,
- Mai Fukagawa1,
- Yukako Nii1,
- Ryota Ishikawa1,
- Ken-ichi T. Suzuki3,
- Makiko Fukui1,
- Masahumi Kawaguchi2 and
- Yasunori Murakami1Email author
© Tosa et al. 2015
Received: 23 January 2015
Accepted: 31 August 2015
Published: 5 October 2015
During vertebrate development, the central nervous system (CNS) has stereotyped neuronal tracts (scaffolds) that include longitudinal and commissural axonal bundles, such as the medial longitudinal fascicle or the posterior commissure (PC). As these early tracts appear to guide later-developing neurons, they are thought to provide the basic framework of vertebrate neuronal circuitry. The proper construction of these neuronal circuits is thought to be a crucial step for eliciting coordinated behaviors, as these circuits transmit sensory information to the integrative center, which produces motor commands for the effective apparatus. However, the developmental plan underlying some commissures and the evolutionary transitions they have undergone remain to be elucidated. Little is known about the role of axon guidance molecules in the elicitation of early-hatched larval behavior as well.
Here, we report the developmentally regulated expression pattern of axon-guidance molecules Slit2 ligand and Robo2 receptor in Xenopus laevis and show that treatment of X. laevis larvae with a slit2- or robo2-morpholino resulted in abnormal swimming behavior. We also observed an abnormal morphology of the PC, which is part of the early axonal scaffold.
Our present findings suggest that expression patterns of Slit2 and Robo2 are conserved in tetrapods, and that their signaling contributes to the construction of the PC in Xenopus. Given that the PC also includes several types of neurons stemming from various parts of the CNS, it may represent a candidate prerequisite neuronal tract in the construction of subsequent complex neuronal circuits that trigger coordinated behavior.
External stimuli received by several types of sensory receptors located on the body’s surface are transferred to the peripheral nerves. Subsequently, the nerve afferents enter the brain and send information to relay nuclei, which in turn project to higher centers in which the various sensory inputs are integrated. The motor center then outputs commands to motoneurons located in the hindbrain or in the spinal cord. Thus, the construction of a precise circuit is a crucial step in eliciting appropriate behavioral responses. If such neuronal circuits are disorganized during ontogenesis, early larvae may be unable to perform coordinated body movements. In the developing vertebrate central nervous system (CNS), early-differentiating neurons extend axons toward their target regions, forming stereotyped tracts (scaffolds) consisting of longitudinal and commissural axonal bundles [1–8]. In later development, these early tracts are thought to serve as guideposts for later-developing axons . The basic framework of these tracts is highly conserved in vertebrate evolution [2, 10]. The early tracts consist of longitudinal (extending along the anteroposterior axis) and commissural (connecting to the left and right side of the brain) tracts. The former include the lateral longitudinal fascicle, tracts of the postoptic commissure (TPOC), and the supraoptic tract (SOT); the latter include the anterior, habenular (HC), and posterior (PC) commissures.
Developing vertebrate brains are typically subdivided into series of segments called neuromeres, and those located in the diencephalon are called prosomeres [11–17]. It is known that some commissural bundles are located at a specific region on the neural tube corresponding to prosomeric compartments. The HCs and PCs are formed in prosomere 2 (thalamus) and prosomere 1 (pretectum), respectively, in many vertebrate groups . The highly conserved framework of these commissures implies that a strictly maintained neurodevelopmental program involved in their wiring has been inherited during vertebrate evolution. In fact, some transcription factors and axon-guidance molecules have been shown play an important role in the formation of the network of these early tracts [18–20]. Previous studies have revealed that the interaction between Slit (ligand) and Robo (receptor), which acts as a repulsive guidance signal, plays an essential role in the formation of early scaffolds, e.g., the inhibition of Slit2 or Robo2 results in an abnormal morphology of the TPOC [21–23] and SOT . These molecules are also involved in the formation of commissural tracts in insects [25, 26] and vertebrates (Slit:[27, 28]; Robo:[29, 30]). In zebrafish robo3 mutant, the axons of the Mauthner neuron fail to cross the midline . In mammals, the Slit/Robo interaction is involved in the formation of the corpus callosum, which is a type of commissural system that connects the cerebral hemispheres . The similarity of the Slit-Robo interaction in teleosts and rodents leads to the possibility that the role of slit and robo is conserved in vertebrate evolution. To study such evolutionary conservation in the vertebrate lineage, it is important to study the function of slit and robo in amphibians, as these animals are thought to have diverged between the teleost and amniote lineages. In addition, the development of locomotion patterns in larval stages is well-described in anuran species [33–35]. For these reasons, Xenopus laevis, an anuran species, may be a suitable model for use in phylogenetic studies and behavioral analysis in early hatched larvae. The aim of the present study is to identify the role of Slit-Robo signaling in the formation of early tracts and/or the elicitation of swimming behavior in Xenopus. We also tried to identify the evolutionary transition of Slit-Robo signaling. To this end, we studied the expression pattern of Slit2 and Robo2 in Xenopus embryos, then perturbed their signals using morpholino antisense oligonucleotides (MO) and analyzed the swimming behavior of early larvae. We found that expression domains of Slit2 and Robo2 in Xenopus are similar to those of amniotes, indicating that the axon guidance mechanism that depends on Slit-Robo signaling is evolutionarily conserved in the forebrain of tetrapods. We also found a disorganized swimming behavior and an abnormal morphology of the PC in both slit2- and robo2-MO-injected larvae. These results indicate that interaction between Slit2 and Robo2 is involved in the construction of the PC and the formation of neuronal element(s) that control coordinated body movement in Xenopus larvae.
Materials and methods
Adult Xenopus laevis were purchased from a local farm (Hamamatsu Seibutsu Kyozai Co. Ltd; Shizuoka Prefecture, Japan), and fertilized eggs were obtained in the laboratory via artificial fertilization. Fertilized eggs were then placed in fresh water and incubated at 20 °C. Embryonic stages were determined based on Nieuwkoop and Faber (1967) . The studies were performed according to the Ethical Guidelines for Animal Use of the Animal Care Committee at Ehime University.
Isolation of axon-guidance genes in X. laevis
Xenopus homologues of slit2 and robo2 were isolated by polymerase chain reaction (PCR) using X. laevis embryonic cDNA as a template. The primers of Xlslit2 were designed based on a published sequence (NM_001087668.1). The following primers were used: Xlslit2-F, 5’–TGAATCAGCACCACCAATGG–3’, Xlslit2-R, 5’–CTAGTCTCGATACCTTCTCG–3’; Xlrobo2-F, 5’–TGGATTGTAGAGTGCTGAGG–3’, Xlrobo2-R, 5’–CACGGAGCAATGCTACTTCC–3’.
The PCR products included in the agarose gel were purified using the Wizard SV Gel and PCR Clean-Up System (Promega), and the DNA fragment was cloned into pGEM-T Easy (pGEM-T Easy Vector Systems; Promega).
Injection of morpholinos
To inhibit Slit2 or Robo2 signals specifically in neuronal tissues, a slit2 MO (GCCACCCAAGGAAAGAACCCAACCA; Gene Tools, LLC) and a robo2 MO (AGCCACCAGAAAGCCCATGTTTCCC) were injected at the 8-cell stage into the small blastomere of the animal pole, which differentiates into the CNS . To visualize injected cells, enhanced green fluorescent protein (eGFP) mRNA was co-injected into the blastomere. Morpholinos containing five mismatched nucleotides were used as a control. As controls of slit2 and robo2, GCgAgCCAAcGAAAcAAgCCAACCA and AGCCACgAcAAAcCCgATcTTTCCC were used, respectively (the mismatched nucleotide is shown in lower case). We injected 2.5 pmol of slit2, robo2, and control MOs. Injected embryos were incubated in 3 % ficoll until the blastula stage; subsequently, embryos were replaced in 0.3 × MMR (100 mM NaCl, 5 mM MgCl2, 0.5 mM CaCl2, 5 mM EGTA, 20 mM HEPES-NaOH, pH 7.5) at 20 °C. At stage 28, we checked eGFP illumination under a fluorescence microscope (Lumar V12; Carl Zeiss SMT GmbH, Oberkochen, Germany). Embryos showing neuron-specific localization of eGFP were incubated and used for further analyses.
Paraformaldehyde (PFA)-fixed specimens were observed under a stereomicroscope (Lumar V12; Carl Zeiss). The body length, curvature of the body axis, and surface area of the eyes were measured using the Axio Vision software (release 4.7.2; Carl Zeiss). Ten larvae were examined in each measurement.
Axonal labeling of the posterior commissure
For retrograde labeling of axons using NeuroVue, Xenopus larvae were dissected in phosphate-buffered saline (PBS, pH 7.5). After fixation in 4 % PFA in PBS, a small chip of NeuroVue Red (Funakoshi 24835) was inserted into the primordium of the pretectum. Larvae were then incubated at 37 °C for one week in 2 % PFA in PBS. Labeled specimens were dissected, and the isolated brain was sectioned at 50 μm using a vibratome (EM PRO7; Dosaka EM Co. Ltd, Kyoto, Japan) after embedding in 5 % agar. Sections were then observed under a confocal microscope (LMS 510 META; Carl Zeiss).
Whole-mount in situ hybridization
For whole-mount in situ hybridization, after fixation in 4 % PFA, embryos were dehydrated in a graded series of methanol (30 %, 50 %, 70 %, 90 %, and 100 %) and stored at −25 °C. In situ hybridization was performed as described previously , with minor modifications. After the color development, specimens were dissected, and the isolated brain was observed under a stereomicroscope (Carl Zeiss). Some specimens were cut into 50 μm using a vibratome after embedding in 5 % agar, and were then observed under a microscope (Axio Image A1; Carl Zeiss).
As a primary antibody to visualize developing axons, we used a monoclonal antibody raised against acetylated tubulin (T-6793, diluted 1/1000; Sigma-Aldrich). Developing muscles were stained with MF20, which recognizes myofilaments (obtained from the Developmental Studies Hybridoma Bank, University of Iowa; diluted 1/100). As a secondary antibody, we used Alexa Fluor 555 goat anti-mouse IgG (H + L; A21422; Invitrogen) or Alexa Fluor 488 goat anti-mouse IgG (H + L; A11001; Invitrogen). Nucleus was labeled with DAPI (D9564, 1 mg/mL, Sigma-Aldrich). For whole-mount immunostaining experiments, hatched larvae were prepared as described previously [38, 40]. The stained specimens were observed under a fluorescence microscope (Lumar V12; Carl Zeiss) or a confocal microscope (510 META; Carl Zeiss). Ten larvae were examined in each measurement. Statistical analyses were carried out using Student’s t-test.
In situ hybridization combined with immunohistochemistry
Whole-mount in situ hybridization was performed as described above. Then, the samples were washed several times with Tris-buffered saline with 0.1 % triton-x100 (TBST). The nerve fibers were visualized by an immunohistochemistry by described above. Briefly, samples were incubated for 1 overnight in TBST containing 5 % skim milk (TSTM). They were then treated with the anti-acetylated tubulin antibody. Samples were incubated in the antibody for 2 days at RT in TSTM containing 0.02 % NaN3. The samples were then washed in TBST four times and subsequently incubated in the secondary antibody (Alexa488 anti-mouse IgG, diluted 1:500) for 2 days at RT in TSTM. The samples were then washed four times in TBST. The specimens were observed under a fluorescence stereomicroscope (Lumar V12; Carl Zeiss).
Swimming behavior in MO-treated larvae
To study the molecular functions of Xenopus cognates of slit2 (Xlslit2) and robo2 (Xlrobo2) in the developing nervous system, MO were injected into the blastomere, which differentiates into the CNS. Although many MO-injected individuals showed no abnormal morphology at stage 44, a small number of larvae exhibited head curvature or eye reduction in both the control- and slit2-MO-injected groups. Conversely, the robo2-MO-injected specimens and their control-MO-injected specimens showed a more asymmetrical craniofacial morphology than did slit2-MO- and control-MO-injected specimens. Thus, in the subsequent analysis, we used slit2-MO-injected larvae and their control-MO-injected larvae (with normal body morphology), whereas we used robo2-MO-injected larvae and their control-MO-injected larvae, which have a slightly asymmetrical shape in the head or eye, in addition to larvae with normal body morphology.
External morphology and musculature construction
Expression of Xlslit2 in Xenopus larvae
In Xenopus embryos at late tail-bud stage (stage 44), Xlslit2 transcripts were expressed at high levels in the dorsal part of the diencephalon, mesencephalon, and metencephalon (Fig. 4c). At this stage, Xlslit2 expression regions became broader compared with those of stage 40 embryos, although the Xlslit2 expression domain was still restricted to the specific part of the metencephalon (Fig. 4c). Conversely, Xlslit2 transcripts were weakly expressed in the transitional region between the diencephalon and mesencephalon (arrow in Fig. 4c).
Expression of Xlrobo2 in Xenopus larvae
Next, we observed the expression pattern of the Xenopus ortholog of robo2 (Xlrobo2), which is a putative receptor of Xlslit2. At stage 32, Xlrobo2 was expressed at high levels in the metencephalon (Fig. 4d). Furthermore, the Xlrobo2 transcript was observed in the diencephalon and ventral telencephalon (Fig. 4d). At stage 40, the Xlrobo2 mRNA was detected throughout the dorsal level of the brain (Fig. 4e). In particular, it was expressed in the dorsal sides of the mesencephalon and metencephalon, whereas it was weakly expressed in the ventral neural tube. We did not detect Xlrobo2 transcripts in the spinal cord (data not shown). At stage 44, Xlrobo2 was expressed at higher levels throughout the dorsal part of the brain, as in the previous stage (Fig. 4f).
Morphology of the nervous system in Xenopus larvae
Immunostaining using an anti-acetylated tubulin antibody was performed to investigate the developmental process of the Xenopus nervous system. In the embryonic stage 46, several cranial nerves, including the olfactory (the first cranial nerve, I), the optic (the second cranial nerve, II), the trigeminal (mdV), the anterior lateral line (buAD), and the posterior lateral line (PLLN) nerves, were observed (Fig. 4g, h), as in matured tadpole larvae. We also identified spinal nerves arranged segmentally in the trunk region (data not shown). These nerves showed clear segregation and projected to their correct targets. Next, we studied the axonal organization of the CNS, which receives input from the peripheral nerves. At stage 46, we observed several longitudinal or commissural axonal bundles, in which three distinct commissural tracts were observed in the dorsal side of the neural tube (Fig. 4g, h). Among those, the HC was located in the anterior part of the dorsal diencephalon. The PC was observed in the posterior part of the dorsal diencephalon (apparently corresponding to the pretectum). The commissures in the cerebellum (CC), which may include commissure cerebelli and commissure vestibulo-lateralis , were located on the cerebellum and across the midline on the dorsal side.
Expression of Xlslit2 and Xlrobo2 in relation to the commissural tracts
Next, to identify the positional relationship between the expression domain of Xlrobo2 and the nTPC, stained specimen were cut into coronal sections and were observed at stage 40. In the anterior diencephalon (presumptive thalamus), the expression domain of Xlrobo2 was located in the ventral side, and it was not detected in the dorsal area, including the habenular nucleus (Fig. 6c). In contrast, in the section of the posterior diencephalon (pretectum), the Xlrobo2 transcript was located in the dorsal part, corresponding to the nTPC (Fig. 6d, asterisk).
Morphology of the developing CNS in MO-injected larvae
Morphology of the PC in MO-treated embryos
Overall, the results of this series of experiments suggest that Slit2 and/or Robo2 is specifically involved in the fasciculation of the PC.
In the present study, we focused on the function of Slit2 and Robo2, both of which play crucial roles in the patterning of CNS axons , and identified three novel findings. First, expression patterns of Slit2 and Robo2 are spatiotemporally regulated. Second, slit2-MO- and robo2-MO injected larvae exhibited abnormal swimming behavior. Third, slit2-MO- and robo2-MO-injected larvae showed an abnormal morphology of the PC, a part of the embryonic axonal tract. These results suggest that the Slit2–Robo2 interaction primarily or secondarily affects to the neuronal circuitry that triggers the coordinated swimming pattern.
Involvement of Slit2 and Robo2 in swimming behavior
We found that larvae treated with slit2 and robo2 MO exhibited an abnormal swimming trajectory. In addition, the swimming distance and speed of MO-injected larvae were also reduced, although control-MO-injected larvae showed no apparent defects in swimming behavior. We also found that slit2-MO- and robo2-MO-treated larvae showed a normal body musculature. These data suggest that Slit2 and Robo2 inhibition induced the abnormal swimming pattern observed, without disturbing basic body apparatuses, such as the fins or body muscles. Importantly, as noted above, both Xlrobo2-MO- and control-MO-treated larvae included specimens that exhibited abnormal morphology of the head and eye. Despite the presence of these head abnormalities in both control-MO- and robo2-MO-treated larvae, we observed a significant abnormality in Xlrobo2-MO-treated specimens regarding swimming behavior, whereas no significant problem was detected in the control group. Given that MOs were specifically injected into blastomeres, which differentiate into the nervous system, we speculated that MO-injected larvae exhibited nervous system defects in circuits that control body movement.
Peripheral nerves in MO-treated larvae
We found that larvae treated with slit2 and robo2 MO exhibited an apparent normal morphology in the peripheral nerves including cranial and spinal nerves. Although one previous report indicated that central projection of the optic nerve in zebrafish robo2 mutant was abnormal , we did not find any abnormality of the optic nerve bundle projecting to the brain. However, as we could not follow the trajectory of the optic nerve toward the optic tectum, further experiment by axon labeling will be needed to identify whether MO-treated larvae exhibit abnormalities in the optic tract.
Expression of slit2 and robo2 in developing Xenopus larvae
To clarify whether MO-treated specimens have any problem in the neurodevelopmental process, we studied the expression pattern of the slit2 and robo2 transcripts in relation to the developmental position of the commissural tracts.
Previous studies have showed that the Slit2 and Robo2 mRNAs are expressed in the developing nervous system in many vertebrates [21–24, 27–30]. The present study showed that Xlslit2 was expressed in a specific part of the developing CNS, as in other vertebrates [23, 24, 49, 50]. We found that the expression domains of Xlslit2 and Xlrobo2 corresponded to the region in which the PC is formed; the Xlslit2 expression domain located beneath the PC, and Xlrobo2 was expressed in the nTPC, which may be a source of the PC (see below). Importantly, Xlslit2 domain showed a discontinuous pattern in which PC appears to be extended in Xlslit2-weak region, and the anterior limit of Xlslit2 expression domain corresponds closely to the anterior border of the PC. In addition to the spatial distribution, the temporal timing of Xlslit2/Xlrobo2 expression was though to correspond to the development of the PC. Namely, the early scaffold of Xenopus laevis is formed at stage 32 , a time at which the PC (called TPC in Xenopus) is across the midline of the dorsal diencephalon. Our study showed that the expression of Xlslit2 and Xlrobo2 was observed at stage 32, which corresponds to the onset of PC formation. Xlslit2 and Xlrobo2 were also expressed at stage 40, a time point at which the early tract is being constructed. Subsequently, the Xlslit2 and Xlrobo2 transcripts were observed at stage 44, a stage at which tight bundle of PC could be observed. Thus, Xlslit2 and Xlrobo2 were expressed correspondingly to the developmental time course of the PC.
Interaction between slit2 and robo2 in the formation of the PC
The present study revealed that, although the HC and CC showed normal morphology, the PC exhibited an abnormal morphology in both slit2-MO- and robo2-MO-injected larvae. This may be due to a problem in the fasciculation process; the PC bundle in MO-treated specimens was thinner and wider compared with that of control larvae. Previous studies using insects and vertebrates showed that Slit2 and Robo2 are involved in the formation of axon bundles [22, 23] and commissural tracts [25–30, 32]. Those findings support our present finding that Xenopus cognates of Slit2 and Robo2 seem to be involved in the formation of the PC.
Evolutionary perspective of Slit-Robo signaling in the formation of the PC
Neuronal defects may contribute to abnormal behavior
It is reasonable to speculate that, if Xlslit2-MO- and Xlrobo2-MO-treated larvae exhibit defects in the neuronal circuitry of the CNS, they would show some kind of defect in motor movement, due to the disruption of neuronal wiring. In fact, we found that Xlslit2-MO and Xlrobo2-MO-injected larvae showed an abnormal swimming pattern. As this behavior is thought to be an initial form of free swimming in Xenopus larvae, it may be controlled mainly by a neuronal circuit constructed by an intrinsic neurodevelopmental program. Thus, the Slit–Robo interaction appears to be involved in the establishment of functional brain element(s) that are formed in the embryonic or early larval stage and regulate an initial free swimming. Because these molecules are expressed at early stages, this behavioral defect may originate from the disruption of an early developing neuronal circuit that induces the coordinated swimming pattern. Otherwise, the later-developing axons that follow early-developing tracts constructed by Slit2–Robo2 signaling may contribute to the behavioral problem detected. Previous studies showed that inhibition of Slit2 or Robo2 results in an abnormal morphology of the TPOC in zebrafish and mouse [21–23]. Thus, Xlslit2-MO- and Xlrobo2-MO-injected Xenopus embryos may develop abnormalities in the TPOC itself, as in other vertebrates, which may cause behavioral abnormalities. Otherwise, the neuronal circuits that are formed subsequently on the framework of the TPOC may be related to the behavioral problem observed in the present study, although we were unable to check abnormality in the TPOC using these morphants due to technical difficulties (unavailability of specific antibodies that recognize TPOC). In addition, the commissural interneurons in the spinal cord that produce appropriate muscle contraction and body movements may be affected by Xlslit2-MO- and Xlrobo2 inhibition, although transcripts of Xlslit2 and Xlrobo2 could not be observed in trunk myomeres and the spinal cord, respectively. However, as observed in the present study, abnormal morphology of the PC may induce behavioral changes. Although it is unknown whether there is a direct link between the PC and swimming behavior, it is important to note that axons in the PC stem from several types of neurons. Previous studies have shown that the PC contains axons that stem from several nuclei, including the nTPC [46, 55]. In mammals, the PC is involved in oculomotor movement by transmitting visual information coming from the cerebral cortex (visual area) and superior colliculus [56, 57]. Moreover, in mammals and birds, the PC contains neurons from the medial longitudinal fascicle (MLF), which includes a neuronal circuit involving the coordination of sensory and motor nerve integration. In chicks, neurons consisting of the PC (TPC) were located within the MLF, intermingled with the central and dorsal populations of MLF neurons . These data suggest that the guidance mechanism for visual and/or MLF axons is affected by MO treatment and, hence, fails to form functional neuronal connections. Therefore, we surmise that the malformed sensory circuits in the PC would be a suitable as a candidate that caused the abnormal behavior in the slit2-MO- and robo2-MO-treated larvae. Conversely, it is possible that the other neuronal system that is formed by the interaction between Slit2 and Robo2 results in the behavioral problem. Future neuroanatomical and functional studies are necessary to identify the molecular mechanism via which correct neural circuits and behavioral patterns are elicited.
buccal ramus of anterodorsal lateral line nerve
Commissures in the cerebellum
mandibular division of the trigeminal nerve
maxillary division of the trigeminal nerve
superficial ophthalmic ramus of anterodorsal lateral line nerve
Posterior lateral line nerve
optic nerve. Schlosser and Northcutt (2000) was referred for the morphological identification 
We thank Dr. Mikio Inoue, Tetsuya Kominami, Kei Nakayama and Hiromi Takata for technical support and valuable discussions. We thank all past and present members of the YM laboratory for support and constructive discussions. Work in YM’s laboratory was supported by RIKEN, Kobe, Japan and the Japan Society for the Promotion of Science (JSPS; grant number 24650178 and 26430018 to YM, and grant number 21770238 and 23657149 to MK).
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- Anderson RB, Key B. Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain. Development. 1999;126:1859–68.PubMedGoogle Scholar
- Barreiro-Iglesias A, Villar-Cheda B, Abalo XM, Anadon R, Rodicio MC. The early scaffold of axon tracts in the brain of a primitive vertebrate, the sea lamprey. Brain Res Bull. 2008;75:42–52.View ArticlePubMedGoogle Scholar
- Chitnis AB, Kuwada JY. Axonogenesis in the brain of zebrafish embryos. J Neurosci. 1990;10:1892–905.PubMedGoogle Scholar
- Doldan MJ, Prego B, Holmqvist B, Helvik JV, de Miguel E. Emergence of axonal tracts in the developing brain of the turbot (Psetta maxima). Brain Behav Evol. 2000;56:300–9.View ArticlePubMedGoogle Scholar
- Easter Jr SS, Ross LS, Frankfurter A. Initial tract formation in the mouse brain. J Neurosci. 1993;13:285–99.PubMedGoogle Scholar
- Figdor MC, Stern CD. Segmental organization of embryonic diencephalon. Nature. 1993;363:630–4.View ArticlePubMedGoogle Scholar
- Ishikawa Y, Kage T, Yamamoto N, Yoshimoto M, Yasuda T, Matsumoto A, et al. Axonogenesis in the medaka embryonic brain. J Comp Neurol. 2004;476:240–53.View ArticlePubMedGoogle Scholar
- Ross LS, Parrett T, Easter Jr SS. Axonogenesis and morphogenesis in the embryonic zebrafish brain. J Neurosci. 1992;12:467–82.PubMedGoogle Scholar
- Pike SH, Melancon EF, Eisen JS. Pathfinding by zebrafish motoneurons in theabsence of normal pioneer axons. Developmen. 1992;114:825–31.Google Scholar
- Nieuwenhuys R, TenDonkelaar HJ, Nicholson C, editors. The central nervous system of vertebrates. Heidelberg: Springer-Verlag; 1998.Google Scholar
- Bergquist H, Källén B. On the development of neuromeres to migration areas in the vertebrate cerebral tube. Act Anat. 1953;18:65–73.View ArticleGoogle Scholar
- Lumsden A, Keynes R. Segmental patterns of neuronal development in the chick hindbrain. Nature. 1989;337:424–8.View ArticlePubMedGoogle Scholar
- Orr H. Contribution to the embryology of the lizard. J Morphol. 1887;1:311–72.View ArticleGoogle Scholar
- Puelles L, Rubenstein JL. Forebrain gene expression domains and the evolving prosomeric model. Trends Neurosci. 2003;26:469–76.View ArticlePubMedGoogle Scholar
- Shimamura K, Hartigan DJ, Martinez S, Puelles L, Rubenstein JL. Longitudinal organization of the anterior neural plate and neural tube. Development. 1995;21:3923–33.Google Scholar
- Vaage S. The segmentation of the primitive neural tube in chick embryos (Gallus domesticus). Ergeb Anat Entw Gesch. 1969;4:1–88.Google Scholar
- von Baer K. Über die Entwickelungsgeschichte der Thiere. Königsberg; 1828.
- Cariboni A, Andrews WD, Memi F, Ypsilanti AR, Zelina P, Chedotal A, et al. Slit2 and Robo3 modulate the migration of GnRH-secreting neurons. Development. 2012;139:3326–31.View ArticlePubMed CentralPubMedGoogle Scholar
- Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell. 1994;78:425–35.View ArticlePubMedGoogle Scholar
- Rubenstein JL, Shimamura K, Martinez S, Puelles L. Regionalization of the prosencephalic neural plate. Annu Rev Neurosci. 1998;21:445–77.View ArticlePubMedGoogle Scholar
- Barresi MJ, Hutson LD, Chien CB, Karlstrom RO. Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain. Development. 2005;132:3643–56.View ArticlePubMedGoogle Scholar
- Devine CA, Key B. Robo-Slit interactions regulate longitudinal axon pathfinding in the embryonic vertebrate brain. Dev Biol. 2008;313:371–83.View ArticlePubMedGoogle Scholar
- Ricano-Cornejo I, Altick AL, Garcia-Pena CM, Nural HF, Echevarria D, Miquelajauregui A, et al. Slit-Robo signals regulate pioneer axon pathfinding of the tract of the postoptic commissure in the mammalian forebrain. J Neurosci Res. 2011;89:1531–41.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang C, Gao J, Zhang H, Sun L, Peng G. Robo2--slit and Dcc--netrin1 coordinate neuron axonal pathfinding within the embryonic axon tracts. J Neurosci. 2012;32:12589–602.View ArticlePubMedGoogle Scholar
- Kidd T, Bland KS, Goodman CS. Slit is the midline repellent for the robo receptor in Drosophila. Cell. 1999;96:785–94.View ArticlePubMedGoogle Scholar
- Rothberg JM, Jacobs JR, Goodman CS, Artavanis-Tsakonas S. Slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 1990;4:2169–87.View ArticlePubMedGoogle Scholar
- Plump AS, Erskine L, Sabatier C, Brose K, Epstein CJ, Goodman CS, et al. Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron. 2002;33:219–32.View ArticlePubMedGoogle Scholar
- Shu T, Sundaresan V, McCarthy MM, Richards LJ. Slit2 guides both precrossing and postcrossing callosal axons at the midline in vivo. J Neurosci. 2003;23:8176–84.PubMedGoogle Scholar
- Hocking JC, Hehr CL, Bertolesi GE, Wu JY, McFarlane S. Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells. Mech Dev. 2010;127:36–48.View ArticlePubMedGoogle Scholar
- Lopez-Bendito G, Flames N, Ma L, Fouquet C, Di Meglio T, Chedotal A, et al. Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J Neurosci. 2007;27:3395–407.View ArticlePubMedGoogle Scholar
- Burgess HA, Johnson SL, Granato M. Unidirectional startle responses and disrupted left-right co-ordination of motor behaviors in robo3 mutant zebrafish. Genes Brain Behav. 2009;8:500–11.View ArticlePubMed CentralPubMedGoogle Scholar
- Shu T, Richards LJ. Cortical axon guidance by the glial wedge during the development of the corpus callosum. J Neurosci. 2001;21:2749–58.PubMedGoogle Scholar
- Ten D. Anurans. In: Nieuwenhuys R, TenDonkelaar HJ, Nicholson C, editors. The central nervous system of vertebrates. 2nd ed. Heidelberg: Springer-Verlag; 1998. p. 1151–314.Google Scholar
- van Mier P, ten Donkelaar HJ. Structural and functional properties of reticulospinal neurons in the early-swimming stage Xenopus embryo. J Neurosci. 1989;9:25–37.PubMedGoogle Scholar
- van Mier P, Armstrong J, Roberts A. Development of early swimming in Xenopus laevis embryos: myotomal musculature, its innervation and activation. Neuroscience. 1989;32:113–26.View ArticlePubMedGoogle Scholar
- Nieuwkoop PD, Faber J. Normal Table of Xenopus laevis (Daudin). Amsterdam: North-Holland; 1967.Google Scholar
- Moody SA, Kline MJ. Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres. Anat Embryol (Berl). 1990;182:347–62.View ArticleGoogle Scholar
- Kawaguchi M, Sugahara Y, Watanabe T, Irie K, Ishida M, Kurokawa D, et al. Nervous system disruption and concomitant behavioral abnormality in early hatched pufferfish larvae exposed to heavy oil. Environ Sci Pollut Res Int. 2011;19:2488–97.View ArticlePubMedGoogle Scholar
- Takio Y, Kuraku S, Murakami Y, Pasqualetti M, Rijli FM, Narita Y, et al. Hox gene expression patterns in Lethenteron japonicum embryos--insights into the evolution of the vertebrate Hox code. Dev Biol. 2007;308:606–20.View ArticlePubMedGoogle Scholar
- Kuratani SC, Eichele G. Rhombomere transplantation repatterns the segmental organization of cranial nerves and reveals cell-autonomous expression of a homeodomain protein. Development. 1993;117:105–17.
- Roberts A, Conte D, Hull M, Merrison-Hort R, al Azad AK, Buhl E, et al. Can simple rules control development of a pioneer vertebrate neuronal network generating behavior? J Neurosci. 2014;34:608–21.View ArticlePubMed CentralPubMedGoogle Scholar
- Geisen MJ, Di Meglio T, Pasqualetti M, Ducret S, Brunet JF, Chedotal A, et al. Hox paralog group 2 genes control the migration of mouse pontine neurons through slit-robo signaling. PLoS Biol. 2008;6:e142.View ArticlePubMed CentralPubMedGoogle Scholar
- Nieuwenhuys R. Comparative anatomy of the cerebellum. Prog Brain Res. 1967;25:1–93.View ArticlePubMedGoogle Scholar
- Easter Jr SS, Taylor JS. The development of the Xenopus retinofugal pathway: optic fibers join a pre-existing tract. Development. 1989;107:553–73.PubMedGoogle Scholar
- Key B, Anderson RB. Neuronal pathfinding during development of the rostral brain in Xenopus. Clin Exp Pharmacol Physiol. 1999;26:752–4.View ArticlePubMedGoogle Scholar
- Lázár G, Pál E. Neuronal connections through the posterior commissure in the frog Rana esculenta. J Hirnforsch. 1999;39:369–74.PubMedGoogle Scholar
- Dickson BJ. Molecular mechanisms of axon guidance. Science. 2002;298:1959–64.View ArticlePubMedGoogle Scholar
- Hutson LD, Chien CB. Pathfinding and error correction by retinal axons: the role of astray/robo2. Neuron. 2002;33:205–17.View ArticlePubMedGoogle Scholar
- Holmes G, Niswander L. Expression of slit-2 and slit-3 during chick development. Dev Dyn. 2001;222:301–7.View ArticlePubMedGoogle Scholar
- De Bellard ME, Rao Y, Bronner-Fraser M. Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells. J Cell Biol. 2003;162:269-79.
- Farmer WT, Altick AL, Nural HF, Dugan JP, Kidd T, Charron F, et al. Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals. Development. 2008;135:3643–53.View ArticlePubMed CentralPubMedGoogle Scholar
- Mastick GS, Easter Jr SS. Initial organization of neurons and tracts in the embryonic mouse fore- and midbrain. Dev Biol. 1996;173:79–94.View ArticlePubMedGoogle Scholar
- Miyasaka N, Sato Y, Yeo SY, Hutson LD, Chien CB, Okamoto H, et al. Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system. Development. 2005;132:1283–93.View ArticlePubMedGoogle Scholar
- Lee JS, Ray R, Chien CB. Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration. Dev Dyn. 2001;221:216–30.View ArticlePubMedGoogle Scholar
- Stanic K, Vera A, González M, Recabal A, Astuya A, Torrejón M, et al. Complementary expression of EphA7 and SCO-spondin during posteriorcommissure development. Front Neuroanat. 2014;8:49.View ArticlePubMed CentralPubMedGoogle Scholar
- Bhidayasiri R, Plant GT, Leigh RJ. A hypothetical scheme for the brainstem control of vertical gaze. Neurology. 2000;54:1985–93.View ArticlePubMedGoogle Scholar
- Leichnetz GR, Gonzalo-Ruiz A, DeSalles AA, Hayes RL. The origin of brainstem afferents of the paramedian pontine reticular formation in the cat. Brain Res. 1987;422:389–97.View ArticlePubMedGoogle Scholar
- Ware M, Schubert FR. Development of the early axon scaffold in the rostral brain of the chick embryo. J Anat. 2011;219:203–16.View ArticlePubMed CentralPubMedGoogle Scholar
- Schlosser G, Northcutt RG. Development of neurogenic placodes in Xenopus laevis. J Comp Neurol. 2000;418:121–46.View ArticlePubMedGoogle Scholar