chiasmatic neurons in the ventral diencephalon of mouse embryos—changes in arrangement and...

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Developmental Brain Resea

Research Report

Chiasmatic neurons in the ventral diencephalon of mouse embryos—

Changes in arrangement and heterogeneity in surface antigen expression

Ling Lin, Anny W.S. Cheung, Sun-On Chan*

Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China

Accepted 7 May 2005

Available online 13 June 2005

Abstract

We have investigated the changes in arrangement of the SSEA-1 immunoreactive chiasmatic neurons in the mouse ventral diencephalon

from embryonic day (E) 9 to the end of gestation. A regionally specific staining of SSEA-1 was first detected in the ventricular layer of the

caudal diencephalon at E10 and later at E11 on the cells in the subventricular layer. At E12, these cells formed the characteristic V-shaped

configuration caudal to the optic axons in the chiasm. At E13–E15, this neuronal array changes gradually to a configuration that facilitates

contact with the optic axons only at the midline and the initial segment of the optic tract. Colocalization studies showed that CD44 was

localized strongly on the neurons in the central but not lateral domains of the array, suggesting existence of heterogeneity in these neurons in

terms of surface antigen presentation. This difference between the central and lateral domains raises the possibility that the chiasmatic

neurons may regulate the patterning of axon orders at the midline and the optic tract through presentation of distinct combination of guidance

cues at these strategic positions in the optic pathway. Furthermore, exogenous Lewis-x/SSEA-1 inhibited neurite outgrowth from the E14

retinal explants; this inhibition was observed in neurites from both ventral temporal and dorsal nasal retina. These findings suggest an action

of this surface carbohydrate on the control of axon growth and guidance in the mouse optic pathway.

D 2005 Elsevier B.V. All rights reserved.

Theme: Development and regeneration

Topic: Axon guidance mechanism and pathways

Keywords: Chiasm; Axon guidance; Midline; Optic tract; SSEA-1

1. Introduction

In the mouse embryos, the optic chiasm and the

proximal segment of the optic tract are two critical regions

where significant sorting and reordering of axons occur in

the retinofugal pathway [18,28]. A number of studies have

indicated the contribution of a population of early-

generated neurons in the ventral diencephalon to these

axon sorting processes. These cells express surface mole-

cules including CD44 and stage-specific embryonic anti-

gen-1 (SSEA-1) [26,31]. The functional significance of

0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2005.05.001

* Corresponding author. Fax: +852 26096898.

E-mail address: [email protected] (S.-O. Chan).

these chiasmatic neurons is first demonstrated in the study

which shows a failure of axon entry into the chiasm after

elimination of the CD44 immunoreactive neurons [32]. A

more recent report has demonstrated that CD44 is the

molecule on these neurons that serves the permissive role

for axon crossing at the midline [24]. Furthermore,

chondroitin sulfate proteoglycans, which are localized on

and around these neurons, have been shown to affect the

development of axon divergence at the midline and

formation of chronotopic axon arrangement in the optic

tract [7,8,21]. These findings indicate a multiple role of the

chiasmatic neurons on axon growth and patterning in the

mouse optic pathway.

SSEA-1 is another surface molecule that is expressed

prominently on the chiasmatic neurons. This molecule is

rch 158 (2005) 1 – 12

L. Lin et al. / Developmental Brain Research 158 (2005) 1–122

originally defined by a monoclonal antibody against murine

teratocarcinoma cell line F9 [30]. It is a carbohydrate

moiety carried by glycoproteins or glycolipids and has a

structure as Gal-h-(1-4)-[Fuc-a-(1-3)]-GlcNAc (lacto-N-

fucopentaose, LNFIII or FAL) [17]. It is also known as

CD15 (leukocyte cluster of differentiation 15) and Lewis-x,

a member of the Lewis blood group antigens in human [2].

Expression of SSEA-1 has been shown in mouse embryos

at late eight-cell stage [30] and is expressed abundantly on

the surface of trophectoderm, primitive ectoderm and

endoderm and primordial germ cells during migration

[13,16]. In the mouse diencephalon, SSEA-1 immunoreac-

tivity is observed on the chiasmatic neurons, which exist in

patterns closely related to the growth of the earliest

generated axons [26] and the turning of the uncrossed

axons in the chiasm [27]. Although SSEA-1 is one of the

first identified molecules on the chiasmatic neurons [26],

whether it is involved in the regulation of retinal axon

growth and patterning in the chiasm and the optic tract is

undetermined. In this study, we investigated the changes in

arrangement of these neurons in the mouse ventral

diencephalon and related these changes to fiber order

changes at the chiasmatic midline and the optic tract.

Furthermore, we determined the possible contribution of the

SSEA-1 to axon growth in the chiasm by examining effects

of exogenous SSEA-1/Lewis-x carbohydrate on neurite

outgrowth from mouse retinal explants.

2. Materials and methods

2.1. Animals

Experimental procedures in the present study were

approved by the University Animal Ethic Committee.

Time-mated pregnant pigmented C57 mice were obtained

from the Laboratory Animals Services Center of the

Chinese University of Hong Kong. The day on which

the vaginal plug was found was considered as embryonic

day 0 (E0).

2.2. Immunohistochemistry

Pregnant mice were killed by cervical dislocation, and

embryos at the ages of E9–E18 were removed by Cesarean

section and killed by decapitation. The heads of the

embryos were fixed in 4% paraformaldehyde in 0.1 M

phosphate buffer (pH = 7.4) overnight at 4 -C. The heads

were embedded in a gelatin–albumen mixture and sec-

tioned on a vibratome at the thickness of 100 Am. Frontal

or horizontal sections containing the retinofugal pathway

from the eyes to the proximal parts of the optic tract were

collected in 0.1 M phosphate buffer saline (PBS, pH 7.4).

Localization of SSEA-1 in these brain sections was

examined using immunocytochemistry, which followed

the procedures described in our early studies [7,9]. Sections

were incubated with a mouse monoclonal antibody against

SSEA-1 (IgM, from Developmental Studies Hybridoma

Bank, Iowa, USA, under the contract N01-HD-6-2915

from NICHD) [30] at dilution of 1:5 overnight at 4 -C.Sections were then incubated in FITC-conjugated goat anti-

mouse IgM (1:100, Jackson Laboratories, Maine, USA) for

90 min at room temperature. Control sections were

processed with the same procedures but in the absence of

the primary antibody. For double-label studies, sections

were probed with antibodies against SSEA-1 (1:5) and

CD44 (IM7; 1:50, rat IgG, PharMingen, USA) simulta-

neously overnight followed by incubation with goat anti-

mouse IgM conjugated to FITC and donkey anti-rat IgG

labeled with Cy3 (both at 1:100, Jackson Laboratories,

Maine, USA). This IM7 antibody recognizes an extrac-

ellular epitope outside the hyaluronic acid binding site of

the CD44 molecule [35]. It was used in this study because

it gives a more distinct label of the chiasmatic neurons than

the other CD44 antibody, Hermes-1 [24]. Sections were

coverslipped with 50% glycerol in PBS and examined by

using a confocal imaging system. In all control prepara-

tions, there was no obvious staining in the brain sections.

In this part of the study, at least 4 litters of embryos were

examined in each age group.

2.3. Preparation of retinal explants and treatment with

Lewis-x

Retinal explants were prepared from E14 mouse embryos

according to the procedure described in an earlier study [5].

The eyes were collected in cold DMEM/F-12 medium, and

the retinas were dissected free of the lens, vitreous and

pigmented epithelium. Retinal explants were isolated from

peripheral regions of either ventral temporal or dorsal nasal

quadrant, which give rise to mostly uncrossed and crossed

axons, respectively, in the chiasm at this developmental stage

[4,10,15]. Explants were placed evenly on polylysine–

laminin coated coverslips and cultured in DMEM/F-12

supplemented with 1% bovine serum albumin, 0.4%

methylcellulose, 0.5% insulin, 0.5% transferrin and 0.1%

sodium selenite (all purchased from Sigma, USA). The

trisaccharide Lewis-x [Galh1,4(Fuca1,3)GlcNAc, Cat. No.434630, from CalBiochem, USA] at various concentrations

was added into the medium at the start of the culture. After

18 h in culture, explants were fixed with 4% paraformalde-

hyde in 0.1 M phosphate buffer for 1 h. Control for this part

of the study consisted of preparations without addition of the

Lewis-x carbohydrate, with added lactose, or with addition

of another carbohydrate Lewis-y [Fuca1,2Galh1,4(Fuca1,3)GlcNAc; Cat. No. 434634; CalBiochem, USA].

Neurite outgrowth from the whole explant was imaged

sequentially using the Neurolucida Image Analysis System

(MicroBrightField, Inc., USA) under phase contrast optics

(20�, Plan-Neofluar, NA 0.5, from Zeiss, Germany). The

images were then reassembled into a montage with clear

visualization of the whole explant and its neurites. Using the

L. Lin et al. / Developmental Brain Research 158 (2005) 1–12 3

image analysis software MetaMorph (Universal Imaging

Corp, USA), retinal neurites were highlighted by adjusting

the threshold, and the pixel area covered by the retinal

neurites was measured. The data in controls and the Lewis-x

treated groups were compared using the Kruskal–Wallis

nonparametric ANOVA test of the InStat software (Graph-

Pad Inc., USA).

2.4. Confocal imaging

Sections stained for SSEA-1 or double-labeled for

SSEA-1 and CD44 were examined using a confocal

imaging system (MRC600, Bio-Rad, Hertford, UK) con-

nected to a Zeiss Axiophot photomicroscope (Zeiss,

Germany). The excitation filter set BHS (488 nm excitation

and 515 nm emission long-pass) was used for imaging

FITC; another filter set GHS (514 nm excitation and 550 nm

emission long-pass) was used for Cy3 observation. Digital

images were processed by the Confocal Assistant software

(Bio-Rad, USA).

3. Results

3.1. SSEA-1 expression in the ventral diencephalon of

E9–E12 embryos

We had examined expression of SSEA-1 in the ventral

diencephalon of mouse embryos, aged from E9 to E12,

when the eye primordia and the optic stalk are being

formed. In E9 embryos, the earliest age that we examined,

immunoreactivity for SSEA-1 was predominantly found on

the ventricular lining of the diencephalon and the lumen of

the optic stalk (Fig. 1A). Intense staining of SSEA-1 was

also observed in the lumen of the Rathke’s pouch, but not

in the infundibular stalk (Fig. 1A). At E10, when the optic

cup first appears, intense label of SSEA-1 was restricted to

the ventricular layer in the caudal parts of the ventral

diencephalon (Fig. 1B), whereas weak label was observed

in other diencephalic regions, the Rathke’s pouch and the

intraretinal space. SSEA-1 positive cells started to appear in

the subventricular zone of the diencephalon at E11

(Fig. 1C). These cells were located in the caudal regions

of the ventral diencephalon and appeared to link with the

strong label on the ventricular surface (Fig. 1D). Intense

label was also found at the junction between the dience-

phalon and the telencephalon (Fig. 1C). The characteristic

V-shaped configuration of the chiasmatic neurons was first

detected in the caudal regions of the diencephalon at E12

(Figs. 1E–H), with the tip extending rostrally toward the

midline of the ventral diencephalon where the future chiasm

develops (Fig. 1G). At this stage, retinal axons start to enter

the ventral diencephalon [14,26]. Examinations of these

SSEA-1 positive neurons showed that they were multipolar

in shape, with a clear soma and a number of thin processes

(Figs. 1F and H).

3.2. SSEA-1 expression in the E13–E15 ventral

diencephalon

During E13 to E15, many retinal ganglion cell axons

arrive at the chiasm and the optic tract and undergo the

major axon sorting processes [3,11,15]. These axon order

changes are accompanied by a change in the configuration

of the chiasmatic neurons. In E13 embryos, immunostain-

ing of SSEA-1 revealed a V-shaped array of the

chiasmatic neurons in horizontal sections of the ventral

diencephalon at the level 300 Am above the ventral pial

surface (Fig. 2A). However, this cellular array differs from

that in E12 diencephalon, in which there is a clear

segregation of cells in the lateral regions into a rostral and

a caudal division (Fig. 2B). This bifurcated array was less

obvious in sections taken close to the pial surface, which

contains the chiasm (Fig. 2C). The SSEA-1 positive

neurons at this level were packed in the characteristic V-

shaped configuration.

Similar arrangement of the SSEA-1 positive neurons was

found in the ventral diencephalon of E14 and E15 embryos

(Figs. 2D–E). This neuronal array straddled the midline at

the level of the chiasm (Fig. 2D) and extended to the initial

segment of the optic tract (Figs. 2D and E; see also Fig. 4).

Processes of these cells formed a dense plexus along the

deep border of the optic tract; some were found penetrating

into the optic fiber layer (Fig. 2F), providing probably a

cellular substrate that may interact with axons at this region

of the optic pathway.

3.3. SSEA-1 expression at E16–E18 diencephalon

At later stages of gestation, i.e. from E16–E18, most

axons have already passed the intermediate targets in the

ventral diencephalon and arrived at the subcortical targets

in the thalamus and the midbrain. At E16, immunostain-

ing for SSEA-1, instead of revealing the V-shaped array,

was largely confined to two cellular domains on each

side of the ventral diencephalon (Figs. 3A, D, F). The

extensions to the midline and the initial segment of the

tracts were maintained (Figs. 3B, C, E). At E18, the

latest age that we have examined, SSEA-1 immunor-

eactivity was restricted clearly in two distinct domains on

each side of the diencephalon (Fig. 3G). The larger

domain was found in the rostral region, lying close to the

midline (Fig. 3H); the smaller one was located at a more

caudal and lateral position. The identity and fate of these

SSEA-1 expressing domains were not determined in this

study.

3.4. Heterogeneity in the chiasmatic neurons

Another question that we had addressed was whether all

SSEA-1 positive chiasmatic neurons are immunoreactive for

the CD44 antibody. We double-labeled horizontal (n = 4)

and frontal sections (n = 4) of the ventral diencephalon of

Fig. 1. Confocal micrographs showing SSEA-1 immunoreactivity in horizontal sections of the ventral diencephalon of E9–E12 mouse embryos. Anterior is up.

The vertical arrows in panels (E) and (G) show the midline. (A) At E9, immunoreactivity for SSEA-1 was detected along the ventricular surface (arrows) of the

third ventricle (V) and in the lumen of the optic stalk (OS). Strong label was also observed in the lumen of the Rathke’s pouch (RP) but not in the infundibular

stalk (IN). (B) At E10, when the optic cup (OC) appears, staining was most intense at the ventricular layer (empty arrow) of the caudal parts of the

diencephalon. (C) At E11, when the retina (R) is formed, SSEA-1 staining was localized largely in the ventricular layer in the caudal diencephalon (empty

arrow) and at the junction of the diencephalon and telencephalon (solid arrow). (D) Higher power view of the box in panel (C) showing the intense SSEA-1

staining on the cells (black arrows) within the subventricular zone of the diencephalon. (E) At E12, the SSEA-1 positive cells were localized in two

symmetrical domains in the caudal regions of the diencephalon. (F) Higher magnification of the boxed area in panel (G) showing the appearance of the labeled

cell bodies and processes of the immunopositive chiasmatic neurons (empty arrows). (G) In another section, 100 Am ventral to panel (E), these immunoreactive

cells were arranged in an inverted V-shaped array (outlined by the broken lines). (H) Higher magnification of the boxed area shows the labeled chiasmatic

neurons at the central or midline domain of this neuronal array. Scale bar: A, B, D = 100 Am; C, E, G = 200 Am; F, H = 50 Am.


E14 embryos with monoclonal antibodies against SSEA-1

and CD44. The SSEA-1 antibody revealed the characteristic

pattern of chiasmatic neurons in the ventral diencephalon

(Fig. 4B), as described above. The anti-CD44 staining,

however, did not label the whole population of chiasmatic

neurons on the same section but showed a more restricted

Fig. 2. Confocal micrographs showing SSEA-1 expression in the ventral diencephalon of E13–E15 mouse embryos. Images were taken from horizontal (A–E)

and frontal (F) sections of the ventral diencephalon. Rostral is up in panels (A–E) and dorsal is up in panel (F). The solid vertical arrows indicate the midline,

and the white solid line indicated the pial surface. (A) Immunopositive cells were located in the caudal region of the diencephalon. (B) Higher magnification of

the boxed area in panel (A) shows the bifurcation (marked by the broken lines) at the lateral region of the neuronal array. (C) At a more ventral section, the

chiasmatic neurons were packed as a characteristic V-shaped array (broken lines). (D) Similar arrangement of the SSEA-1 positive neurons (broken lines) was

observed in the E14 ventral diencephalon. (E) At E15, the chiasmatic neurons (asterisk) extended rostrally to appose the initial segment of the optic tract. (F)

These lateral extensions from the chiasmatic neurons (asterisks) consist of both cells and their processes, which form a dense plexus (empty arrows) at the deep

border of the optic tract (marked by the broken line). Some processes (horizontal arrows) intruded into the optic axon layer. OE: olfactory epithelium; OS: optic

stalk; OT: optic tract. Scale bar: A, E = 200 Am; B, C, D, F = 100 Am.


pattern that was confined to the central but not lateral

regions of this array (Figs. 4A and C). Within the central

region, including the raphe structure at the chiasmatic

midline, the neurons were largely double-labeled for

SSEA-1 and CD44 (Figs. 4C, E and F). Lateral to this

region, the cells and their processes were predominately

immunoreactive to SSEA-1 (Figs. 4D and E). These

processes, as shown in earlier parts of the result, formed a

Fig. 3. SSEA-1 expression in the ventral diencephalon in E16–E18 mouse embryos. Panels (A–C) and (G–H) are horizontal sections; rostral is towards the

top. Panels (D–F) are frontal sections; dorsal is to the top. The large arrows indicate the midline. The deep border of the optic tract is marked by the broken

line. (A) In E16 ventral diencephalon, immunostaining was largely confined to two cellular domains (asterisks) and to the deep border of the optic tract.

(B) Higher power view of the boxed area in panel (A), showing the restricted labeling (bound by the two arrows) at the initial segment of the optic tract.

(C) Another high power view showing the SSEA-1 immunoreactivity on the chiasmatic neurons (small arrows) adjacent to the optic tract. (D) In frontal

sections of E16 ventral diencephalon, the staining was found in a group of cells adjacent to the midline and along the deep border of the optic tract (small

arrows). (E) Higher power view of the optic tract in another E16 embryo, showing the localization of SSEA-1 positive cells and processes (small arrows).

(F) High magnification view of the boxed area in panel (D), showing the SSEA-1 immunopositive cells adjacent to the midline. (G) At E18, just before birth,

immunoreactivity for SSEA-1 was localized in two distinct domains (asterisks) and along the ventricular regions. Relationship of these cells to the optic axons

was not obvious at this stage. (H) Higher magnification showing the SSEA-1 positive domains (asterisks) next to the midline. OS: optic stalk; OT: optic tract.

Scale bar: A = 200 Am (applied to G); B = 50 Am (applied to C, E, F, H); D = 100 Am.


plexus along the deep border of the initial segment of the

optic tract (Fig. 4G). There were a number of neurons, lying

along the caudal border of the array that appeared

predominately immunoreactive to the CD44 antibody (Figs.

4C and E). These observations suggested that the chiasmatic

neurons were not a homogenous cell population; they could

be segregated into a central and two lateral domains based

on their surface antigen presentations.

Fig. 4. Colocalization of SSEA-1 and CD44 in the ventral diencephalon of E14 mouse embryos. Rostral is up in the horizontal sections (A–E), and dorsal is up

in the frontal sections (F–G). Vertical arrows in panels (A) and (F) indicate the midline. The pial surface is marked by solid lines, while the deep border of the

optic axon layers is demarcated by the broken lines. (A–C) Immunostaining for CD44 was largely localized in the central domain (bound by the two arrows in

panel C) of the SSEA-1 positive chiasmatic neurons, while the lateral regions of the array were predominately SSEA-1 positive. (D) Higher magnification of

the merged image of the chiasmatic neurons, showing colocalization of CD44 and SSEA-1 immunoreactivity at the central domain, which extends to the

chiasmatic midline, and the predominate localization of SSEA-1 (white arrows) in the lateral region that apposes the optic tract. (E) Higher power view of the

chiasmatic neurons at the central domain of the array (indicated by the boxed area in C), showing cells that are labeled preferentially with antibody against

SSEA-1 (white arrows), CD44 (empty arrows) or both (white triangles). (F) At rostral level of the chiasm, chiasmatic neurons in the central domain that are rich

in both CD44 and SSEA-1 extended processes into the fiber layer at the midline. (G) At the caudal level of the chiasm, where axons enter the optic tract, the

chiasmatic neurons extended processes (white arrows) which formed a dense plexus along the deep border of the tract. Note the predominant localization of

SSEA-1 but not CD44 in these processes. OS: optic stalk; OT: optic tract. Scale bar: A = 200 Am (applied to panels B and C); D = 100 Am (applied to F and G);

E = 50 Am.


3.5. Lewis-x is inhibitory to retinal neurite outgrowth

In order to determine whether the carbohydrate molecule

SSEA-1/Lewis-x affects the growth of retinal axons, we

investigated the outgrowth of neurites from E14 retinal

explants in the presence of various concentrations of Lewis-x.

After 18 h in culture, in controls treated without addition of

Lewis-x, substantial neurite outgrowth was seen in explants

of both dorsal nasal and ventral temporal retina (Figs. 5A

and B). A low concentration (10–100 Ag/ml) of Lewis-x

did not induce obvious reduction in neurite outgrowth

(Figs. 5C and D). However, when explants were treated

with 400 Ag/ml Lewis-x, an obvious reduction in neurite

outgrowth was observed (Figs. 5E and F). Quantitative

analyses showed that neurite outgrowth from both ventral

temporal and dorsal nasal explants was significantly


reduced only in the presence of 400 Ag/ml Lewis-x (P <

0.01) when compared with corresponding data in controls

that were cultured without Lewis-x (Fig. 5G). Such

inhibition was neither observed in explants treated with

lactose nor in those treated with lower concentration of

Lewis-x (P > 0.05). Moreover, treatment with the explants

with Lewis-y (400 Ag/ml), another molecule of the

carbohydrate antigen family, elicited a response in neurite

growth which is distinct from that observed with a

comparable concentration of Lewis-x. Lewis-y, instead of

reducing neurite growth from both retinal regions, selec-

tively inhibited outgrowth from dorsal nasal (P < 0.001) but

not ventral temporal explants (P > 0.05), when compared

with the control without addition of the carbohydrate

molecule (Fig. 5G).

4. Discussion

In the present study, we have investigated the

developmental changes in configuration of the chiasmatic

neurons, which express a surface carbohydrate SSEA-1,

in the ventral diencephalon of mouse embryos. The major

findings are: (1) regionally specific staining of SSEA-1

can be detected along the ventricular surface in the caudal

regions of the diencephalon at E10 and on the chiasmatic

neurons at E11, long before arrival of retinal axons; (2)

during the time of axon growth in the ventral dience-

phalon, these neurons undergo a change in arrangement:

from a simple V-shaped domain at E12 to a more

elaborated arrangement at later (from E13–E16) stages of

development, which apposes optic axons at the chiasmatic

midline and the initial segment of the optic tract; (3) at

the late gestational stages (E16–E18), when most optic

axons have arrived at their subcortical targets, SSEA-1

expression is confined to two distinct domains on each

side of the ventral diencephalon; (4) colocalization studies

of SSEA-1 and CD44 expression reveal a heterogeneity

in these neurons, segregating neurons in the midline

domain from those at the lateral regions; (5) exogenous

SSEA-1/Lewis-x carbohydrate inhibits neurite outgrowth

from explants of both ventral temporal and dorsal nasal

retina, suggesting a negative influence of this surface

epitope on axon growth at the optic chiasm and the optic

tract.

Fig. 5. Effects of exogenous Lewis-x/SSEA-1 on the outgrowth of neurites from d

The micrographs in panels A–F were processed by the ‘‘Find Edge’’ filter in th

neurites. (A–B) After 18 h in culture, the control explants (without addition of L

from both retinal regions was not obviously affected by the presence of 10 Ag/ml L

reduction in neurite outgrowth was seen in explants from both retinal regions. No

(A–D). (G) A plot of the results on the explant culture study, showing the signific

outgrowth from E14 mouse retina, when compared with control preparations that

were inhibited. Such inhibition was not observed in explants treated with lactose

explants with Lewis-y caused a reduction in outgrowth of DN but not VT neu

different from the pattern observed in the groups treated with similar concentration

panels (B–D); the one in panel (E) applies to panel (F).

4.1. Dynamic changes in configuration of the chiasmatic

neurons

We have shown in this study that intense SSEA-1

immunostaining can be detected in the ventricular layer of

the caudal regions of the diencephalon at as early as E10,

which possibly be one of the sites that gives rise to the

SSEA-1 positive cells in the ventral diencephalon at later

stages of development [4,26,31]. As shown in the current

study, these cells are arranged in a V-shaped configuration

and attain a typical neuronal morphology. The neuronal

identity of these cells has been proven in past reports by

their expression of neuronal markers such as h-III tubulinand microtubule associated protein-2 [27,31]. However, it

should be noted that there are some morphological studies

reporting a possible glial nature of the CD15/SSEA-1

positive cells in the ventral diencephalon of developing

mouse embryos and newborn wallaby [6,25], which may

serve to compartmentalize the prosencephalic regions in the

brain. This possibility of glial specific expression of SSEA-

1 deserves further investigation to determine if the CD15

staining colocalizes with that of the markers expressed

specifically in the radial glia [27].

Previous studies have shown that array of the chiasmatic

neurons has direct contact with the length of the earliest

ingrowing optic axons in the chiasm, particularly along the

rostral border of the array. It is thus argued that the

chiasmatic neurons may provide a template to shape the

paths of the first generated axons in the diencephalon

[26,29,31], which arise exclusively from the central retina

[10,14]. On the contrary, at later developmental stages, the

optic axons that are generated from peripheral parts of the

retina encounter a different influence from the chiasmatic

neurons. As shown in the current study, these axons are in

contact with the chiasmatic neurons only at a narrow raphe

structure at the midline and at the threshold of the tract.

Such pattern of changes confirms earlier findings that there

is a major change in configuration of these neurons during

the development of the mouse optic chiasm, which appears

to relate to the axon divergence process at the midline that

occurs during this period of development [26,27]. We

propose further that these changes may enable the

chiasmatic neurons to affect axon growth and patterning

not only at the midline but also at the initial segment of the

optic tract. This spatial rearrangement probably presents

orsal nasal (DN) and ventral temporal (VT) retina of E14 mouse embryos

e Photoshop (v. 6.0, Adobe, USA) in order to enhance the contour of the

ewis-x) showed extensive neurite outgrowth. (C–D) The neurite outgrowth

ewis-x. (E–F) However, in the presence of 400 Ag/ml Lewis-x, substantia

te that these micrographs are at a higher magnification than those in panels

ant inhibitory effect (asterisk, P < 0.01) of Lewis-x at 400 Ag/ml on neurite

did not receive added carbohydrate. Neurites from both DN and VT retina

nor in explants treated with lower concentrations of Lewis-x. Treatment o

rites, when compared with control without added carbohydrate, which is

of Lewis-x. Scale bar: A, E = 500 Am; the one in panel (A) also applies to

.

l

f


guidance signals on these neurons at strategic positions to

control axon patterning in the optic pathway. One example

is chondroitin sulfate glycosaminoglycans, which are

localized on the chiasmatic neurons [7,22] and have been

shown to contribute to the turning of ipsilaterally projecting

axons at the chiasmatic midline and to the formation of age-

related fiber order in the initial segment of the optic tract

[8,21].

Mechanisms that drive the change in configuration of the

chiasmatic neurons are not clear. There may be an active

migration of this neuronal population towards the caudal

parts of the diencephalon; or there is an addition of cells that

interpose between the chiasmatic neurons and the optic

axons in the chiasm. Recent study has shown that there is a

population of neurons, which are immunoreactive to

sialylated form of neural cell adhesion molecule, that appear

in regions located between the chiasm and the CD44

positive neurons during this period of development [9].

This finding raises the possibility that generation of these

neurons may serve as a force to drive the configurational

change of the chiasmatic neurons.

By the end of gestation, most retinal axons have already

grown through the chiasm and reached the subcortical

targets. At this stage, expression of SSEA-1 is clustered in

two distinct domains on each side of the ventral diencepha-

lon. Contacts of these domains with the optic pathway are

eventually lost, suggesting that the influence of the SSEA-1

positive neurons to optic axon growth is no longer operating

when most axons have arrived at the targets. Unlike the

pioneering subplate neurons in the developing cortex [1],

the chiasmatic neurons may remain after contributing to the

optic pathway development and differentiate into one of the

nuclear groups in the hypothalamus. However, it is possible

that these SSEA-1 rich domains are not derived from the

chiasmatic neurons but from some other neurons that begin

to synthesize SSEA-1 at the late gestational stages. More-

over, the fates of these SSEA-1 positive domains are not

known and need to be defined in future investigations using

Fig. 6. Schematic drawing of the configuration of the chiasmatic neurons in relation

developmental stage, retinal axons from the central retina enter the optic stalk (OS

arrow). These neurons at the midline/central domain (area marked by red vertical

chiasm, which includes turning of the uncrossed axons (red) and crossing of the cro

of the tract (OT) (marked by the empty arrow), they are sorted according to their ti

repulsive cues at the deep parts of the optic tract, where the SSEA-1 positive nerve

receptors on the optic axons (light blue) (see [21]).

markers of specific molecules expressed by the hypothala-

mic nuclei.

4.2. Heterogeneity in the chiasmatic neurons and axon

growth in the chiasm and optic tract

Further to the spatial changes in the chiasmatic neuronal

array, we have shown in the colocalization studies that

CD44 is expressed on the SSEA-1 positive neurons only at

the midline region but not in the lateral domains, indicating

a regionally specific difference in expression of these cell

surface antigens on the chiasmatic neurons. The functional

significance of this difference to axon growth and fiber

order patterning is unknown; neither do we know whether

there is preferential expression of other guidance molecules

in one part but not the other of the population of the

chiasmatic neurons. Nevertheless, this difference raises an

interesting idea on the contribution of this neuronal

population to the axon patterning in the mouse optic

pathway. At E13–E16, when most optic axons are growing

in the pathway, the specific spatial relationship of the

chiasmatic neurons to the optic axons suggests a dual

influence of these cells on axon growth and guidance (see

Fig. 6). The chiasmatic neurons may present cues, including

CD44, at the midline that control axon crossing [24,32] and

segregation of crossed from uncrossed axons [8,15,24,27].

At the threshold of the optic tract, these neurons probably

contribute signals that guide the formation of the age-related

fiber order [3,7,21]. These guidance signals may be

indifferent from those at the midline, which then would

have to be associated with a change in surface receptors

expression and/or signaling mechanisms within the optic

growth cones when they travel from the chiasm to the optic

tract [21]. One example of these signals is the chondroitin

sulfate proteoglycan, which is localized at both the midline

and the optic tract [7,8,21,22]. Alternatively, the set of

guidance signals at the optic tract may be different from

those at the midline, so that axons read a combination of

to the axon routing processes at the E14 mouse retinofugal pathway. At this

) and the chiasm and encounter the chiasmatic neurons at the midline (solid

bars) probably present cues that control axon routing at the midline of the

ssed axons (deep blue). When the crossed axons arrive at the initial segment

me of arrival. These processes probably rely on an interaction of axons with

plexus (green area) is located, and on a regionally specific change in surface


cues at the midline that generates axon divergence and

another set of cues at the optic tract that instructs the

formation of the age-related order. The existence of

heterogeneity in the chiasmatic neurons, as demonstrated

in the current colocalization studies, appears to support this

argument. The difference in expression of surface antigens,

probably together with other axon growth regulating

molecules, may contribute to the distinct influences of the

chiasmatic neurons to retinal axon growth and patterning at

the midline and the optic tract.

4.3. SSEA-1 may have an influence on retinal axon growth

We have shown in this study that exogenous Lewis-x/

SSEA-1 has a significant inhibitory action on neurite

outgrowth from both ventral temporal and dorsal nasal

retina isolated from E14 mouse embryos, suggesting that

this carbohydrate molecule may be involved in regulating

axon growth and guidance in the optic pathway. Such

inhibition is not observed in explants treated with lactose

nor in those treated with Lewis-y (which affects selectively

outgrowth of dorsal nasal neurites), indicating the specific

influence of Lewis-x/SSEA-1 to the retinal neurite growth.

Previous investigations of the functions of Lewis-x have

demonstrated its adhesive properties through a homophilic

interaction since incubation with free LNFIII (SSEA-1)

inhibits compaction of mouse morulae that express SSEA-1

[2,30] and blocks adhesion of SSEA-1 positive embryonic

carcinoma cells in a calcium-dependent manner [12,19]. In

the frog, antibodies against Lewis-x produce a suppression

of neurite outgrowth from explant culture of tadpole brain

tissue where Lewis-x is normally expressed [34]. In the

developing mouse brain, such homophilic interactions may

be responsible for binding and self-adhesion of the

chiasmatic neurons. However, the negative effect of

Lewis-x on neurite outgrowth from the retina is not likely

explained by such homophilic binding since retinal axons

are not immunoreactive to SSEA-1 at all stages studied.

This inhibitory influence likely depends on interactions

with other guidance molecules that are present on the

retinal axons. In the immune system, Lewis-x has been

demonstrated as a ligand of selectins, a family of trans-

membrane glycoproteins [33]. This ligand–receptor bind-

ing has been implicated in the adhesion of leukocytes to

activated endothelium, which eventually leads to extrava-

gation of leukocytes into lymphoid tissues and site of

inflammation [23]. Recently, a molecule homologous to

selectin has been identified in Drosophila and has been

shown to participate in the development of the eye and the

mechanosensory bristles [20]. It remains to be determined

whether selectins are expressed on the retinal axons during

the period of optic pathway development; and if it exists on

the axons, whether such interactions may form a novel

regulatory mechanism for the development of axon order

changes in the chiasm and the optic tract of mouse

retinofugal pathway.

It is not clear whether Lewis-x exists in the mouse

ventral diencephalon at concentration comparable to that

in the in vitro condition and whether Lewis-y is expressed

in the ventral diencephalon to exert its influence to the

optic axons. Nevertheless, the current results show a

lack of difference in the inhibitory function of Lewis-x

to neurite outgrowth from both ventral temporal and

dorsal nasal retina, suggesting that this carbohydrate

alone is not likely involved in the generation of the axon

divergence at the midline of the chiasm. It is, however,

possible that Lewis-x may function together with other

guidance molecules to deviate newly arrived growth

cones, which arise from all retinal quadrants, away from

the deep parts of the optic tract, thus producing the age-

related fiber arrangement. This has to be addressed in a

future study by investigating the fiber order changes in

the mouse optic tract after perturbation of the Lewis-x

functions.

Acknowledgments

This project was partially supported by grant from the

Research Grant Council of the Hong Kong Special

Administrative Region (Project No. CUHK4417/03M) and

a direct grant from The Chinese University of Hong Kong

(Ref. No. 2004.1.051).

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