Joel C. Glover

Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo

We have used recently developed fluorescein- and rhodamine-conjugated dextran-amines as axonal tracers in in vitro preparations of the nervous system of the chicken embryo. These substances are efficiently taken up by injured axons and transported rapidly to the cell bodies. They are also, albeit to a lesser degree, taken up by intact nerve terminals in skeletal muscle. They reveal the dendritic and axonal structure of labelled neurons, and are well suited for use in double-labelling experiments.

Projection patterns of commissural interneurons in the lumbar spinal cord of the neonatal rat

We have studied the axonal projection patterns of commissural interneurons (CINs) in the neonatal rat spinal cord. Some CINs are integral components of the neuronal networks in the vertebrate spinal cord that generate locomotor activity. By using differential retrograde labeling protocols with fluorescent dextran amines, we show that CINs with ascending axons (ascending CINs, or aCINs) and CINs with descending axons (descending CINs, or dCINs) constitute largely different populations. We show that aCINs and dCINs occupy partially overlapping domains in the transverse plane. The aCINs are located at the dorsal margin, within the dorsal horn, centrally within the intermediate zone, and in the medial region of the ventral horn, whereas the dCINs are located predominantly among the ventral and central aCINs and in smaller numbers within the dorsal horn. The labeled aCINs and dCINs project for at least one and a half segment rostrally or caudally and are present in roughly equal numbers. We also demonstrate the presence of a third, smaller population of CINs whose axons bifurcate to project for at least one and a half segment both rostrally and caudally (adCINs). The adCINs are located predominantly among the central and ventral groups of aCINs and dCINs. Finally, we demonstrate the presence of CINs with axons projecting for fewer than one and a half segment in either direction. These “short‐range CINs” are intermingled with the aCINs, dCINs, and adCINs. Our results provide an anatomical framework for further electrophysiological studies aimed at identifying the CINs that participate in the mammalian locomotor central pattern generator. J. Comp. Neurol. 447:349–359, 2002.

Characterization of commissural interneurons in the lumbar region of the neonatal rat spinal cord

Neurons with axons that extend to the contralateral side of the spinal cord—commissural interneurons (CINs)—coordinate left/right alternation during locomotion. Little is known about the organization of CINs in the mammalian spinal cord. To determine the numbers, distribution, dendritic morphologies, axonal trajectories, and termination patterns of CINs located in the lumbar spinal cord of the neonatal rat, several different retrograde and anterograde axonal tracing paradigms were performed with fluorescent dextran amines and the lipophilic tracer 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI). CINs with ascending (aCINs) and descending (dCINs) axons were labeled independently. The aCINs and dCINs occupied different but overlapping domains within the transverse plane. The aCINs were clustered into four recognizable groups, and the dCINs were clustered into two recognizable groups. All dCINs and most aCINs were located within the gray matter, with somata ranging from 10–30 μm in diameter and with large, multipolar dendritic trees. One group of aCINs was located outside the gray matter along the dorsal and dorsolateral margin and had dendrites that were nearly confined to the dorsolateral surface. All CIN axons traversed the ventral commissure at right angles to the midline. CIN axons coursed up to six or seven segments rostrally and/or caudally in the ventral and ventrolateral white matter and gave off collaterals over a shorter range, predominantly to the ventral gray matter. These findings show that the lumbar spinal cord of the neonatal rat contains substantial numbers of CINs with axon projections and collateral ranges spanning several segments and that CINs projecting rostrally vs. caudally have different distributions in the transverse plane. The study provides an anatomical framework for future electrophysiological studies of the spinal neuronal circuits underlying locomotion in mammals. J. Comp. Neurol. 403:332–345, 1999.

Development of projection-specific interneurons and projection neurons in the embryonic mouse and rat spinal cord

Interneurons and projection neurons in the lumbar spinal cord of mouse and rat embryos were labeled retrogradely with fluorescent dextran amines from a distance of one segment from the segment of origin [lumbar segment (L) 2]. Six classes with specific axonal projections (ipsilateral ascending, descending, and bifurcating, and commissural ascending, descending, and bifurcating) were identified by differential labeling in both species and followed from embryonic day (E)12 to birth in the mouse. Neurons with shorter projections (intrasegmental interneurons) were not studied. We show that the four nonbifurcating neuron classes occupy characteristic, partially overlapping domains in the transverse plane, indicating a systematic pattern of migration and settlement related to axon trajectories. The number of neurons in each of the nonbifurcating classes increased steadily during development. Bifurcating neurons represented a minor fraction of the total throughout development and had relatively scattered positions within the ipsilateral and commissural neuron domains. Combination of retrograde tracing and immunohistochemistry for the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) showed that none of the spinal neurons in the six projection‐specific classes was GABA positive, suggesting that all GABA‐positive spinal neurons, including previously described GABA‐positive commissural neurons, are unlikely to have projections exceeding one or two segments in either direction. J. Comp. Neurol. 483:30–47, 2005.

Fate-Mapping the Mammalian Hindbrain: Segmental Origins of Vestibular Projection Neurons Assessed Using Rhombomere-Specific Hoxa2 Enhancer Elements in the Mouse Embryo

As a step toward generating a fate map of identified neuron populations in the mammalian hindbrain, we assessed the contributions of individual rhombomeres to the vestibular nuclear complex, a major sensorimotor area that spans the entire rhombencephalon. Transgenic mice harboring either the lacZ or the enhanced green fluorescent protein reporter genes under the transcriptional control of rhombomere-specific Hoxa2 enhancer elements were used to visualize rhombomere-derived domains. We labeled functionally identifiable vestibular projection neuron groups retrogradely with conjugated dextran-amines at successive embryonic stages and obtained developmental fate maps through direct comparison with the rhombomere-derived domains in the same embryos. The fate maps show that each vestibular neuron group derives from a unique rostrocaudal domain that is relatively stable developmentally, suggesting that anteroposterior migration is not a major contributor to the rostrocaudal patterning of the vestibular system. Most of the groups are multisegmental in origin, and each rhombomere is fated to give rise to two or more vestibular projection neuron types, in a complex pattern that is not segmentally iterated. Comparison with studies in the chicken embryo shows that the rostrocaudal patterning of identified vestibular projection neuron groups is generally well conserved between avians and mammalians but that significant species-specific differences exist in the rostrocaudal limits of particular groups. This mammalian hindbrain fate map can be used as the basis for targeting genetic manipulation to specific subpopulations of vestibular projection neurons.

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9-cis retinoic acid only detected after all-trans-retinoic acid levels are super-elevated experimentally.

Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [ 1998 ] Nature 395:398–402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [ 1998 ] J. Chromatogr. A 828:451–460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all‐trans retinoic acid (at‐RA), the ligand for RARs, and relate this to the expression of several retinoid‐synthesizing enzymes. However, we do not detect 9‐cis‐retinoic acid (9‐cis‐RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at‐RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9‐cis‐RA potentially is present and demonstrates that at‐RA predominates over 9‐cis‐RA by at least 30‐ to 180‐fold in different spinal cord regions.

Regional patterning of reticulospinal and vestibulospinal neurons in the hindbrain of mouse and rat embryos

The dispositions and axonal trajectories of bulbospinal neurons in the pons and medulla of mouse and rat embryos is described from the earliest times these projections can be labelled retrogradely from the cervical spinal cord. Reticulospinal and vestibulospinal neurons are clustered into identifiable groups, each with a characteristic combination of spatial domain and axon trajectory. The various groups can be labelled retrogradely in a specific developmental sequence. The position of some groups shifts from medial to lateral with development, apparently through cell migration. These observations show that the basic regional organization of the reticulospinal and vestibulospinal projections is similar in mouse and rat and is already established during early stages of axon outgrowth. J. Comp. Neurol. 411:288–300, 1999.

Correlated patterns of neuron differentiation and Hox gene expression in the hindbrain: a comparative analysis

Hindbrain neurons are organized into coherent subpopulations with characteristic projection patterns and functions. Many of these serve vital functions that have been conserved throughout the vertebrate radiation, but diversification to modified or highly specialized functions has also occurred. The differentiation of identifiable neuron groups in specific spatial domains must involve the regional expression of determinants within the hindbrain neuroepithelium. The Hox genes are involved in longitudinal regionalization of the neural tube, and their expression patterns in the hindbrain are closely related to the rhombomeres which partition the hindbrain into morphogenetic units. Hox gene expression also exhibits conserved patterning as well as phylogenetic variation. One plausible mechanism that may have contributed to evolutionary diversification in hindbrain neuron populations is therefore the emergence of species-specific differences in Hox gene expression. This article presents a comparative overview of the regional patterning of selected Hox genes and hindbrain neuron populations in several embryologically important species. Although tantalizing correlations exist, the relationship between Hox genes and neuronal patterning is complex, and complicated by dynamic features in each. Much more comparative and developmental data must be obtained before the link between Hox gene expression and hindbrain neuron patterning can be elucidated satisfactorily in an evolutionary context.

Retinoid X receptor γ gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors

We have examined the distribution of the retinoid X receptor γ (RXRγ) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRγ transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRγ‐positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRγ‐positive cells at these stages is unknown, due to the lack of cell type‐specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRγ‐positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRγ2 isoform. Between d16 and early posthatch stages the level of RXRγ2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRγ1 isoform were substantially lower than mRNA levels of the RXRγ2 isoform at all time points examined. These results demonstrate that RXRγ mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRγ as the earliest marker of photoreceptor differentiation documented to date. J. Comp. Neurol. 391:204–213, 1998.

Developmental dynamics of functionally specific primary sensory afferent projections in the chicken embryo

Abstract The central projections of specific subpopulations of lumbar primary afferents were selectively labeled with the lipophilic tracer DiI in fixed preparations of the chicken embryo. Muscle or cutaneous afferents were selectively labeled by applying DiI to identified peripheral nerves. Medial or lateral afferent populations were selectively labeled by partially lesioning the dorsal root. Muscle and cutaneous afferent populations each contribute to both the medial and the lateral afferent populations. Medial muscle afferents terminate in the intermediate zone and lateral motor column proximally, but only in the intermediate zone distally. Lateral muscle afferents terminate in a ventrolateral region of the dorsal horn both proximally and distally. Medial cutaneous afferents terminate predominantly in lamina III, but a few terminate in the medial region of the intermediate zone. Lateral cutaneous afferents terminate in lamina II and in a ventrolateral region of the dorsal horn. On the basis of the principle termination patterns, specific termination fields were defined and related to the classical cytoarchitectonics of the spinal gray matter. Differential retrograde tracing from the spinal cord with fluorescent dextran-amines demonstrated that the medial afferents originate from the earlier-generated ventrolateral population of large sensory cell bodies, while the lateral afferents originate from the later-generated dorsomedial population of small sensory cell bodies. The medial afferents establish their central projections earlier than the lateral afferents, but for each subpopulation the initial pattern of termination prefigures the mature pattern, throughout the segmental range of the collaterals. Birthdating with 3H-thymidine showed that potential target neurons in the different terminal fields within the dorsal horn are born at different times. In particular, interneurons in lamina II are born after those in lamina III, paralleling the early and late termination of cutaneous afferents in these laminae. Our observations support the notion that primary afferents recognize specific cues in the spinal cord, but also implicate the relative timing of afferent and target differentiation as an important determinant of primary afferent termination patterns.