The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.     

The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons

XIHbox 6 is an early spatially restricted marker for molecular studies of neural induction. The sequence of the full-length XIHbox 6 protein is reported. An antibody raised against a beta-galactosidase/XIHbox 6 fusion protein was used to analyze the expression of XIHbox 6 proteins during frog embryogenesis. The anterior border of XIHbox 6 expression lies just posterior of the hindbrain/spinal cord junction. Immunostaining extends the entire length of the spinal cord. A much weaker transient expression with a similar anterior border is observed in mesoderm. Almost all nuclei in the newly closed spinal cord contain XIHbox 6. The number of positive nuclei decreases over the next stages of development, until in later embryos XIHbox 6 is restricted to nuclei of the dividing neuroepithelium, and not the mantle or marginal zones of the spinal cord. When the limb buds begin to grow, there is a second burst of XIHbox 6 expression in proliferating neurons of the cervical and lumbar enlargements, where nerves arise that supply the limbs. The data suggest that XIHbox 6 expression is spatially and temporally restricted to immature neurons of the spinal cord, before their differentiation into mature neurons.     

Rig-1 a new member of Robo family genes exhibits distinct pattern of expression during mouse development

The Robo genes encode a family of proteins that are the receptors for the midline repellent Slits and play a role in axon guidance. In addition to Robo1 and Robo2, Rig-1 has been recently identified in mouse as a novel member of the Robo family of proteins. As a first step in elucidating the role of Rig-1 during vertebrate development, we characterised the expression of Rig-1 by in situ hybridisation together with Robo1 and Robo2 in the spinal cord and other tissues of the mouse embryo. Our results show that Rig-1 has a dynamic pattern of expression in the developing CNS. In the spinal cord Rig-1 shows overlapping but distinct pattern of expression with Robo1 and Robo2.     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

Colloquium 14: New Functions for Retinoic Acid in the Developing CNS. The role of retinoic acid in the patterning of the developing nervous system

Retinoic acid (RA) is metabolised from its precursor, retinol (vitamin A). In mammalian embryos, retinol is provided by the mother via the placenta and in birds retinol comes from the yolk. We have studied the role of RA in CNS development in quail embryos by depriving adult quails of retinol in the diet which results in them laying eggs which have no retinol stores. The resulting embryos are therefore retinol and RA deficient. The CNS of these embryos is abnormal in three regards; patterning, neural crest production and neurite outgrowth. With regard to patterning, at an early stage of development prior to somitogenesis, hindbrain patterning genes are not induced which leads to the respecification of the posterior hindbrain territory. This region is not lost from the embryo but instead becomes transformed into an enlarged anterior hindbrain. Another aspect of patterning that is abnormal in these RA deficient embryos is the dorsoventral gene expression domains in the anterior spinal cord. These domains are required for the proper specification of motor neurons, sensory neurons and various classes of interneurons. Consequently these neuronal classes are mis‐specified in the RA deficient embryos. With regard to the neural crest, these cells often fail to migrate correctly and then die in the absence of RA. With regard to neurite outgrowth, very little outgrowth seems to take place in these deficient embryos which suggests that RA is involved in neurite outgrowth. Taking these experiments into the adult to examine the role of RA in neurite regeneration, we have had success in inducing neurite outgrowth in vitro from adult mouse spinal cord by manipulating the retinoic acid receptors which transduce the RA signal at the level of the nucleus.     

gsh1 demarcates hypothalamus and intermediate spinal cord in zebrafish

We have cloned and characterized the expression of zebrafish genetic screen homeobox 1 (gsh1). Early expression is confined to hindbrain rhombomeres; by mid-somitogenesis gsh1 is expressed in precise domains within the mesencephalon and diencephalon, as well as in intermediate spinal cord. Double-label experiments revealed that the diencephalic domain is coincident with hypothalamus and that spinal cord expression is in a region that generates interneurons. These data suggest gsh1 may play a role in patterning cell types generated in these domains.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.     

Bcl-2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.     

Cloning and embryonic expression of zebrafish neuropilin genes

Neuropilin (Nrp), a cell surface receptor for class 3 semaphorins and for certain heparin forms of vascular endothelial growth factors, functions in many biological processes including axon guidance, neural cell migration and angiogenesis in the development of the nervous system and the cardiovascular system. To understand the role of neuropilins in zebrafish embryogenesis, we have cloned three zebrafish neuropilin homologues, nrp1b, nrp2a and nrp2b. Based on synteny, zebrafish nrp1b and the previously cloned nrp1a are orthologous to human nrp1, and zebrafish nrp2a and 2b orthologous to human nrp2. We have characterized the expression patterns of these four zebrafish neuropilin genes in wild type embryos from the beginning of somitogenesis to 48 h post-fertilization. Zebrafish nrp1a is expressed in the neural tube including telencephalon, epithalamus, cells along the axonal trajectory of the posterior commissure and the medial longitudinal fascicle, hindbrain neurons, vagus motor neurons and spinal motoneurons. Zebrafish nrp1b is expressed in the nose, the cranial neural crest cell (NCC) derived tissue underlying the hypothalamus, endothelial precursors and the trunk and tail vasculature. Zebrafish nrp2a is expressed in telencephalon, anterior pituitary, oculomotor and trochlear motor neurons, cells along the supra-optic and posterior commissures, hindbrain rhombomere 1, hindbrain neurons, cranial NCCs and sclerotome. Zebrafish nrp2b is expressed in telencephalon, thalamus, hypothalamus, epiphysis, cells along the anterior and posterior commissures, post-optic and supra-optic commissures and the olfactory axonal trajectory, hindbrain neurons, cranial NCCs, somites and spinal cord neurons.     

Xenopus brevican is expressed in the notochord and the brain during early embryogenesis

A complete cDNA encoding the Xenopus laevis homologue of the aggrecan/versican family member, brevican (Xbcan) was cloned from an embryonic stage 42 cDNA library. In the deduced amino acid sequence, 1152 in length, similarity to the hyaluronan-binding (link) domains of brevicans from other species were present in the N-terminal region as well as EGF-, lectin- and complement regulatory protein-like domains in the C-terminal part, the latter three being characteristic for brevican found within the extracellular matrix (J. Biol. Chem. 269 (1994) 10119). Indeed, Xbcan was secreted into the extracellular space as a soluble protein when expressed in oocytes. No cDNAs encoding a GPI-anchored bcan variant could be isolated from that cDNA library. During embryonic development, the expression of this gene was first observed in the notochord of neurula stage embryos. In addition to this, in tailbuds, Xbcan was also found to be expressed within the fifth and sixth rhombomere of the hindbrain. In tadpole stage embryos, expression was furthermore observed in periventricular regions of the developing brain and the rostral part of the spinal cord.     

Developmental expression of the axonal glycoprotein TAG-1: differential regulation by central and peripheral neurons in vitro.

TAG-1 is a 135,000 Mr axonal glycoprotein of the immunoglobulin superfamily that promotes axon extension in vitro. One distinguishing feature of TAG-1 is its transient expression on subsets of axons in the developing nervous system. To examine the mechanisms that regulate TAG-1, we have monitored the expression of this protein by developing central and peripheral neurons in vitro. TAG-1 was detected on the surface of a subset of E11 to E13 spinal cord neurons in vitro and was also released by these neurons. Expressions of TAG-1 on the cell surface was transient but it was possible to detect a released form of TAG-1 at all times in vitro. Spinal cord neurons isolated from older embryos did not express surface TAG-1 when they regenerated axons in vitro. Changes in the environment of spinal cord neurons did not alter the time course of TAG-1 expression, suggesting that regulation of the protein is cell autonomous. In contrast to these results with spinal cord neurons, surface expression of TAG-1 by DRG neurons persisted in vitro and adult DRG neurons re-expressed TAG-1 when grown in vitro. The cell surface and released forms of TAG-1 therefore appear to be regulated differently by central and peripheral neurons.     

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals¹. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact^2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)^5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.