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, 12 (5), 957-75

Embryonic Neurons of the Developing Optic Chiasm Express L1 and CD44, Cell Surface Molecules With Opposing Effects on Retinal Axon Growth

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Embryonic Neurons of the Developing Optic Chiasm Express L1 and CD44, Cell Surface Molecules With Opposing Effects on Retinal Axon Growth

D W Sretavan et al. Neuron.

Abstract

The first retinal ganglion cell axons arriving at the embryonic mouse ventral diencephalon encounter an inverted V-shaped neuronal array defining the midline and posterior boundaries of the future optic chiasm. These neurons express L1, an immunoglobulin superfamily molecule known to promote retinal axon outgrowth, and CD44, a cell surface molecule that we find inhibits embryonic retinal axon growth in vitro. Incoming retinal axons do not penetrate this L1/CD44 neuron array, but turn to establish the characteristic X-shaped optic chiasm along the anterior border of this array. These results suggest that L1/CD44 neurons may serve as an anatomical template for retinal axon pathways at the embryonic mouse ventral diencephalon.

Figures

Figure 1
Figure 1. Schematic Diagrams of the Ventral Surface of the Embryonic Mouse Diencephalon at E11, E12–E13, and E15–E16, Showing the Relationship between the Retinas, Optic Nerves, and Optic Chiasm
RET, retina; ON, optic nerve; OC, optic chiasm. The shaded regions represent the approximate extent of the retinal axon projections into the mammalian brain at different ages. Concomitant with the formation of the X-shaped pattern of axon pathways on the ventral diencephalon, axons from nasal retina cross the midline to grow into the contralateral optic tract, whereas axons from a group of ganglion cells in ventral–temporal retina turn away from the midline to grow into the ipsilateral optic tract. The rectangular box in the E11 diagram outlines the approximate region shown in Figures 2A and 2B.
Figure 2
Figure 2. Sections through the Future Optic Chiasm Region in E12 Mouse Embryos following Immunostaining with Neuron-Specific Markers or Labeling with Dil
(A) A horizontal section showing the pattern of immunoreactivity in the ventral diencephalon following staining with an anti-MAP2 antibody. Bar, 100 μm. (B) A horizontal section showing the pattern of immunoreactivity following staining with an anti–β III tubulin antibody. Bar, 100 μm. Note that at E12, prior to arrival of retinal axons, MAP2-and β III tubulin-immunopositive cells are distributed in an inverted V-shaped array on the ventral diencephalon surface straddling the midline, with the tip of the V pointing anteriorly and the legs of the V extending posteriorly (arrows in [A] and [B] indicate position of the anterior tip of the inverted V neuronal array). (C) A coronal section through the diencephalon following placement of a Dil crystal at the ventral midline to label the axon projections from embryonic chiasm neurons extending along the lateral walls of the diencephalon. Dorsal is toward the top; ventral is below; the midline is at the center. During their course within the diencephalon, the axons of the chiasm neurons run just underneath the pial surface. Bar, 250 μm. (D–F) Coronal sections of the ventral diencephalon showing embryonic chiasm neurons in an E12.5 embryo that have been backfilled from their axons with Dil. Retrogradely labeled cells are found in the ventral diencephalon below the pial surface (dotted white lines) and are aligned with their axons running in the medial-lateral direction. These cells have long axons and multiple dendrite-like short processes emanating from their cell bodies. The midline is not present in (D)–(F). Bar, 30 μm.
Figure 3
Figure 3. Whole Mounts of the Ventral Embryonic Diencephalon following Immunostaining with a Polyclonal Antibody Directed against the Cell Surface Molecule L1
Anterior is toward the top; the midline is at the center. OS, optic stalk (called the optic nerve after retinal axons have entered). The two eyes normally present at the ends of the optic stalks were not included in these preparations. (A) An immunolabeled whole mount from an E11 embryo. At this stage, retinal axons have yet to enter the diencephalon region. However, immunostaining with anti-L1 antibody reveals that L1-positive axons are already present in the future region of the optic chiasm arranged in an inverted V shape. Bar, 300 μm. (B) Ventral diencephalon whole mount from an E12 embryo. At this age, retinal axons (arrowheads), which arealso immunoreactive for L1, have begun to invade the ventral diencephalon but have yet to reach the chiasm neurons. Bar, 200 μm. (C) Whole mount from an E13 embryo. By this stage, a large number of retinal axons have grown into the diencephalon and are intermixed with the cell bodies and processes of the embryonic chiasm neurons. Note that retinal axons leaving the optic nerve to enter the ventral diencephalon do not grow straight, but on average all turn about 45° posteriorly (see arrows). As a result of this turn, axons head toward the inverted V-shaped array of chiasm neurons and their processes. Bar, 200 μm.
Figure 4
Figure 4. Horizontal Sections through the Ventral Diencephalon of E13 Embryos Showing the Relationship between Incoming Retinal Axons and Embryonic Optic Chiasm Neurons and Their Processes
The retinal axons from one eye have been anterogradely labeled from the retina with Dil, and in these photographs appear yellow-orange; chiasm neurons and their processes have been retrogradely labeled from their axons with DiO placed on both sides of the diencephalon and appear green. The dotted white lines represent the borders of the V-shaped array of embryonic optic chiasm neurons. Retinal axons, upon entering the ventral diencephalon, grow into and overlap with the anterior one-third of the inverted V-shaped array of chiasm neurons. Retinal axons, however, do not penetrate further into this array, but instead turn and either cross the midline to head to the other side (A) or grow away from the midline to project ipsilaterally (B). The midline in (A) is in the center. In (B), the midline is toward the left edge. Bars, 50 μm (A); 25 μm (B).
Figure 5
Figure 5. Sections through the Ventral Diencephalon of E12 Embryos Showing Immunostaining of Embryonic Optic Chiasm Neurons with Anti-CD44 MAb and Anti–β III Tubulin Antibody
(A–C) Horizontal section through the future optic chiasm region showing the pattern of immunoreactivity obtained with each of the anti-CD44 MAbs: (A) KM201, (B) IM7, (C) 18C8. The staining pattern is similar with all three antibodies and appears as an inverted V shape, corresponding to the distribution of the embryonic chiasm neuron cell bodies as revealed by anti-MAP2 and anti–β III tubulin staining. (The plane of section in [C] is at a slight angle compared with the others.) The dotted line in (A) represents the plane of section for (D). Bars, 100 μm. (D) A coronal section through the ventral diencephalon region showing embryonic chiasm neurons stained with MAb KM201. Glial and neuroepithelial cells are not stained. The dotted white line indicates the ventral pial surface. Bar, 40 μm. (E and F) Coronal section through the ventral diencephalon of an E12 mouse embryo showing embryonic chiasm neurons following immunostaining with an anti–β III tubulin antibody(E) and anti-CD44 MAb KM201 (F). Bars, 20 μm. (C and H) Coronal section through the ventral diencephalon of an E14 mouse embryo showing embryonic chiasm neurons following immunostaining with an anti–β III tubulin antibody (G) and anti-CD44 MAb KM201 (H). Bars, 20 μm. The colocalization of β III tubulin and CD44 immunoreactivity on the same cells demonstrates the neuronal identity of CD44+ cells. At E14, β III tubulin is present in a large number of neurons, only some of which are CD44+.
Figure 6
Figure 6. CD44 Immunoblots
(A) Membrane fractions from the ventral diencephalon of E12 C57/B16 mouse embryos and membrane fractions from peripheral circulating lymphocytes of adult C57/B16 mice. Tissues from the ventral diencephalon contain a single diffuse immunoreactive band at 85K, a finding consistent with the presence of the low molecular weight isoform of CD44. Adult circulating lymphocytes have two immunoreactive bands, a major band at 95K and a minor band at 115K. (B) CD44 immunoblots using membrane fractions from the ventral diencephalon of E12–P0 mice. A single immunoreactive band at 85K is seen during these stages of embryonic development. Higher molecular weight forms were not detected. (C) CD44 immunoblots following enzymatic digestions. Lane 1, undigested membrane protein fraction. (CD44 immunoreactivity following incubation under conditions for digestions appear as a doublet.) Lane 2, neuraminidase digestion; lane 3, chondroitinase ABC digestion. The shift in the molecular weight of CD44 from 85K to 70K after neuraminidase treatment demonstrates the presence of sialic acid. Chondroitinase treatment did not result in a mobility shift of the CD44-immunoreactive band, indicating that CD44 in the embryonic diencephalon is not modified with glycosaminoglycan side chains, a finding consistent with the existence of a low molecular weight form of CD44 in the ventral diencephalon.
Figure 7
Figure 7. Reverse Transcription PCR Analysis of CD44 Isoforms in the Embryonic Ventral Diencephalon
(A) Diagram illustrating the domain structure and posttranslational modifications on both the long and short forms of CD44. Open circles, N-linked glycosylations; closed circles, O-linked glycosylations; closed triangles, glycosaminoglycan side chains; open triangles, cysteine; EX, extracellular domain; TM, transmembrane domain; CYTO, cytoplasmic domain. Alternative splicing of a 132–165 amino acid segment into the extracellular domain creates a long form of CD44 that promotes cellular metastasis (Gunthert et al., 1991). (6) Diagram showing the location of the PCR primers used in this study (see Experimental Procedures for nucleotide sequences of PCR primers). Primer 1 is located in exon 5, primer 2 in exon 15, primer 3 in exon 17 (transmembrane domain), and primer 4 in exon 19 (cytoplasmic tail). Primers 5 and 6 are located in the alternatively spliced extracellular region of the molecule and are located in exon 8 (also known as v4) and exon 10 (also known as v6), respectively. (Exon nomenclature is from Screaton et al., 1993, and Screaton et al., 1992.) (C) PCR products obtained using the primers identified in (B). In each lane, the PCR product was obtained using the reverse primer (RP) together with the upstream primer indicated by the number at the top of the lane. Results from this PCR analysis demonstrate the presence of mRNA encoding the short form of CD44 at the embryonic ventral diencephalon. There was no evidence for mRNA transcripts encoding longer forms of CD44 containing alternatively spliced inserts in the extracellular segment. Numbers at the left of (C) denote size in bases.
Figure 8
Figure 8. E14 Ventral Diencephalon Whole Mount following Immunostaining with Anti-CD44 Mab
Previous studies have shown that contralaterally projecting retinal axons arriving at the ventral diencephalon at E14 cross the midline over to the opposite side wheras ipsilaterally projecting axons do not cross, but instead turn away from the midline to grow into the ipsilateral optic tract (Colello and Guillery, 1990; Godement et al., 1990; Sretavan, 1990; Sretavan and Reichardt, 1993). At this age, CD44 immunoreactivity is present at the midline (arrow) and forms the posterior border of the optic chiasm (arrowhead). ON, optic nerve. Bar, 200 μm.
Figure 9
Figure 9. CD44 Cell Surface Labeling and Immunoblots from CD44+ and CD44 Parental Cell Lines
(A and B) CD44+ cells. (A) Staining of nuclei with 4,6-diamidino-2-phenylindole (DAPI) (B) Anti-CD44 immunostaining. (C and D) CD44 parental cells. (C) DAPI staining. (D) Anti-CD44 immunostaining. CD44 is expressed on the surface of transfected cells, whereas untransfected cells demonstrate no immunoreactivity. Bar, 20 μm. (E) CD44 immunoblots. Lane 1, membrane fractions from E12 mouseventral diencephalon; lane 2, CD44 parental (K562) cells; lane 3, CD44+ cell line (high expressor); lane 4, CD44+ cell line (low expressor); lanes 5 and 6, transfected cell lines with no detectable expression of CD44. CD44 expressed on transfected cells appears as a diffuse 85K band, similar in size to CD44 present in the embryonic ventral diencephalon.
Figure 10
Figure 10. Montage Showing E13 Mouse Retinal Explants Growing in Collagen Gels under Different Conditions
(A) Retinal explants in the presence of membrane fragments from untransfected parental K562 cells. (B) Retinal explants growing in the presenceof membranefragments from a CD44+ cell line. (C) Retinal explants in the presence of CD44+ membranes and anti-CD44 rat MAb KM201. (D) Retinal explants in the presence of CD44+ membranes and anti-CD44 rat MAb IM7. (E) Retinal explants in the presence of CD44+ membranes and anti-CD44 rat MAb IRAW 14.4. Bar, 500 μm. Retinal explants in collagen gels seeded with parental K562 membranes extended fascicles of axons in a radial fashion from the explant. In explants growing in the presence of CD44+ membranes, axon fascicles of similar thicknesses extended in a radial fashion but were on the average much shorter. This decrease in axon fascicle growth was not reversed by anti-CD44 MAb KM201 or IM7. It was, however, partially reversed after the addition of anti-CD44 MAb IRAW 14.4.
Figure 11
Figure 11. Total Length of Axon Fascicles from E13 Retinal Explants Grown in Collagen Gels Seeded with CD44+ Membranes in the Presence of AntiCD44Antibody IRAW and Hyaluronidase or Hyaluronic Acid
Each black dot represents the total fascicle length from one retinal explant. The numbers of explants (n), mean fascicle length (x̄), and SD are given at the bottom. (A) Compared with explants in the presence of membranes from untransfected parental cells, explants in the presence of CD44+ membranes showed on average a 60% decrease in fascicle length (p < .001). The addition of hyaluronidase (HA ase) did not effect this inhibitory effect of CD44. (B) Fascicle length of retinal explants grown under the following conditions were compared and determined to be significantly different when analyzed using the two-tailed t test. CD44+ + IRAW versus CD44+ (p < .005). CD44++ HA ase + IRAW versus CD44+ (p < .005). Thus addition of hyaluronidase did not affect the ability of IRAW to reverse partially the inhibitory effects of CD44. The same statistical analysis revealed no significant difference in fascicle lengths between CD44+ versus CD44+ + hyaluronic acid (HA) or CD44+ versus CD44+ + HAase, indicating that the effects of CD44 on retinal axon growth are likely not to be mediated through its N-terminal hyaluronate-binding region.
Figure 12
Figure 12. Total Axon Fascicle Length from Retinal Explants Growing under Different Conditions
Fascicle length of retinal explants grown under the following conditions was compared and determined to be significant when analyzed using the two-tailed t test. CD44 versus CD44+ (p < .001), CD44+ versus CD44++ IRAW (p < .01), CD44+ versus CD44+ + antibody (Ab) mix (p < 035). The same statistical analysis revealed no significant difference in fascicle length between retinal explants under the conditions of CD44+ versus CD44+ + KM201 MAb or CD44+ versus CD44+ + IM7 MAb.
Figure 13
Figure 13. Summary Diagram of Findings
Top: at E11, a group of early generated CD44+ neurons is present as an inverted V-shaped array at the future region of the optic chiasm prior to the arrival of retinal axons. Middle: at E12–E13, L1-expressing retinal axons have begun to arrive and become closely associated with L1/CD44 embryonic chiasm neurons and their processes. Bottom: by E15/E16, retinal axons have established an X-shaped optic chiasm within which ipsilaterally and contralaterally projecting axons are specifically routed into the correct optic tracts. Throughout the period when initial retinal pathways are laid down at the ventral diencephalon, CD44+ neurons are present about the midline and define the posterior boundary of the developing optic chiasm. The X-shaped optic chiasm is established anteriorly, capping the inverted V array of chiasm neurons.

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