ontogeny of gnrh and olfactory neuronal systems in man: novel insights from the investigation of...

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Frontiers in Neuroendocrinology 25 (2004) 108–130

Frontiers inNeuroendocrinology

Ontogeny of GnRH and olfactory neuronal systems in man:novel insights from the investigation of inherited forms

of Kallmann�s syndrome

David Gonzalez-Martınez*, Youli Hu, Pierre Marc Bouloux

Department of Medicine, Neuroendocrinology Unit, Upper Third Floor, Royal Free and University College Medical School,

University College of London, NW3 2PF, London, UK

Available online 24 August 2004

Abstract

GnRH embryonic neuronal fate is determined by discreet spatio-temporal expression patterns and interactions of axonal guid-

ance and cell adhesion molecules and extracellular matrix proteins. Expression of several transcription factors, locally derived

growth factors and neurotransmitters influence GnRH ontogeny and rostral forebrain specification. In man, disrupted GnRH neu-

ronal ontogeny can be caused by several monogenic disorders leading to isolated hypogonadotrophic hypogonadism (IHH); these

include mutations within KAL-1, GnRH-R, and FGFR1. Mutations in KAL-1 and its encoded protein anosmin-1, causes X-linked

Kallmann�s syndrome (XKS) characterized by IHH, anosmia, synkinesis, and unilateral renal agenesis. Anosmin-1 has an obligate

functional interaction with membrane associated heparan sulphate proteoglycans (HSPG) and FGFR-1 (KAL-2) whose mutations

lead to the autosomal dominant form of KS (AKS). FGFR1 and anosmin-1 may interact via a HSPG dependent mechanism raising

the possibility of interaction between two single gene defects cause similar phenotypic abnormalities.

� 2004 Elsevier Inc. All rights reserved.

Keywords: GnRH; Anosmin-1; FGFR-1; Heparan sulphates; Rostral forebrain; Olfactory bulb; Kallmann�s syndrome; Axonal guidance molecules;

Development; Ontogeny

1. Introduction

The neuropeptide gonadotrophin releasing hormone(GnRH) serves both as hormone and a neurotransmitter

exerting multiple actions on reproductive physiology

and behaviour. In many vertebrate species including hu-

mans, the population of GnRH-I neurones regulating

pituitary LH and FSH (luteinizing and follicle stimulat-

ing hormone) secretion originate in the peripheral olfac-

tory system [150]. During development, these GnRH cell

bodies undergo a migratory process from their medialolfactory placodal origin across the terminal nerve

(TN) to their final destination in the septo-preoptic

hypothalamus [165,210], from where about 2000 neuro-

0091-3022/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yfrne.2004.06.001

* Corresponding author. Fax: +442074332871.

E-mail address: [email protected] (D. Gonzalez-Martınez).

nes extend axonal processes to establish contact with the

median eminence portal capillary loops. This TN-septo-

preoptic GnRH-I system serves as the principal regula-tor of gonadotrophin release in vertebrates. In mouse

and chick and presumably the rest of vertebrates, a reci-

procal relationship exists between the appearance and

(apparent) disappearance of GnRH neurones in the

olfactory epithelium (OE) and their translocation and

subsequent appearance into the CNS during embryo-

genesis [125,192,213,214]. As gestation progresses, great-

er numbers of GnRH cells become demonstrable in thesepto-preoptic hypothalamic area, while a lower number

remain in the olfactory pit and nasal septum [168]. In the

OE, GnRH cells never exceed a few hundred in number,

even though greater numbers subsequently appear

entwined in the cranial nerve 1 complex, suggesting ra-

pid transition to acquisition of peptide following initial

mailto:[email protected]

Fig. 1. Schematic drawings showing GnRH-I and anosmin-1 immuno-

reactivity in the olfactory system and rostral forebrain during human

embryogenesis. GnRH neurones: Anosmin-1, 4. (A) 4 weeks (CS13),

8/9mm. (B) 5.5 weeks (CS17), 11/12mm. (C) 6 weeks (CS18), 13/15mm.

OP, olfactory placode; PON, primary olfactory neurones; bv, blood

vessel; NM, nasal mesenchyma; CP, cribriform plate; F, forebrain;

ORN, olfactory receptor neurones; OE, olfactory epithelium; OB,

olfactory bulb; ONT, olfactory nerve tract; TN, terminal nerve; NTg,

terminal nerve ganglion cells. Refs. [46,72,140,204].

D. Gonzalez-Martınez et al. / Frontiers in Neuroendocrinology 25 (2004) 108–130 109

rostral migration away from the OE. An alternative pos-

sibility however, remains that GnRH-immunoreactive

cells, or their progenitors, may be capable of dividing

outside the OE during migration.

The migration of GnRH neurones away from the

medial OE/TN is highly organized and targeted, pro-gressing rostrally along the nasal septum, prior to their

entrance into the forebrain [211]. This journey not only

ends in the dispersal of GnRH cell bodies in the neuropil

spanning a broad region of the anterior hypothalamus,

but also enables the integration of chemical information

from close spatial relationships with other peptidergic

neurones, several of these physiological modulators of

GnRH release [49].The ontogeny of the TN-septo-preoptic system re-

flects its evolutionary origin as a peripheral endocrine

organ associated with the olfactory system. Very early

during human embryonic development, GnRH-I cell

ontogeny commences in the medial olfactory placode.

In the terminal nerve region, GnRH cells are first evi-

dent in the ventral telencephalon, initially at approxi-

mately 6 weeks (Carniege Stage 17, CS17, 15mm)gestation [167], although they appear to have been �born�in the olfactory placode somewhat earlier than this, as

evidenced by studies in a 5.5 weeks old embryo (CS16,

12mm) in which Verney et al. [204] demonstrated

GnRH-I-ir neurones medial to the olfactory placodes

within the vomeronasal organ (VNO). At this stage of

human embryogenesis, migrating olfactory nerve fibres

have already established contact with the ventral telen-cephalic vesicle, at the site of the presumptive olfactory

bulb (OB) anlage [204]. OB morphogenesis becomes dis-

tinct at week 7 (CS19) [140] (see Figs. 1 and 2).

Comparable results have been obtained in the rhesus

monkey [150] where two GnRH populations emerge

from the olfactory placode and migrate towards the ros-

tral forebrain; a first group of pioneer neurones establish

the migratory pathway early on, while the later-risinggroup may form direct/indirect connection with the first

group in order to reach the brain. After migrating from

the olfactory placode, ‘‘early’’ GnRH-I cells can be visu-

alized in the rostral and ventral edge of the ventral wall

of the forebrain (embryonic day 30, equivalent to CS14)

prior to OB development (embryonic day 34–40) [150],

confirming that a proportion of GnRH neuronal migra-

tion occurs prior to OB histogenesis.The comparative endocrinology of GnRH neuronal

development has contributed much to unravelling the

complexity of the brain–pituitary–gonadal axis. Among

vertebrates, GnRH neurones have been extensively stud-

ied in teleosts using morphological, electrophysiological,

behavioural, and molecular approaches [21,25,44,79,

135,174,175,218]. Recent observations on the ontogeny

of three different GnRH neuronal systems in perciformteleosts have shown that the GnRH forebrain systems

(GnRH-I and GnRH-III) share a common embryonic

origin in the olfactory placode with an inverse gradient

in rostro-caudal GnRH cells distribution within the ven-

tral telencephalon and diencephalon, while GnRH-II

emerges from the synencephalic region where its expres-

sion is restricted [64–66]. These data share a remarkable

similarity with higher vertebrates and validate teleosts as

Fig. 2. Summary of GnRH-I and anosmin-1 immunoreactivities in the human embryonic CNS. OB, olfactory bulb; mOP, medial olfactory placode;

PON, primary olfactory neurones; SC, spinal cord; Th, Thalamus; vrF, ventro-rostral forebrain; vTel, ventral telencephalon. CS, Carniege stage; ED,

embryonic day. �, Ref. [167]; m, Ref. [204]; %, Ref. [72]; }, Ref. [140]; +, Ref. [46].

110 D. Gonzalez-Martınez et al. / Frontiers in Neuroendocrinology 25 (2004) 108–130

potential models for investigating GnRH ontogeny and

in general neurodevelopmental studies.The vertebrate GnRH neuronal migratory complex

requires several key cellular elements. Upon leaving

the OE, in mice, GnRH neurones retain intimate appo-

sition with each other and with peripherin-positive ter-

minalis axon fibres [56]. Cells of glial appearance are

present in the GnRH–axonal fascicle complex, corre-

sponding to ensheathing cells which migrate from the

olfactory placode along with the olfactory axons. Later,these form processes that ensheath GnRH cells within

the external surface of the axonal fascicles. These cells

seems to restrict the axonal fascicles and their migratory

cells to the nasal mesenchyme, but upon rostral fore-

brain entry, the nerves defasciculate and apposition

ceases and a relationship with a mixed population of

neural and glial cells is established [106] as cell bodies

disperse within the anterior hypothalamic neuropil.Recent studies have shed light on the mechanisms

of GnRH migration from the nasal region to the

brain, identifying mechanical and chemical forces that

guide these neurones to their final destination. This re-

view initially describes recent developments in GnRH

neuronal ontogeny, focussing on molecular mecha-

nisms affecting both the early and later events in this

ontogeny. This is followed by a detailed review of themolecular pathogenesis of Kallmann�s syndrome, the

prototypic human disorder of failed GnRH neuronal

migration.

2. Molecular selection of GnRH cells and placodal

development

The nasal placodes are ectodermal thickenings on the

frontal and ventrolateral aspects of either side of the

head. The epithelia from the nasal placode contain both

non-sensory and sensory olfactory epithelia, as demon-

strated by specific phenotypic markers [34]. In contrast

to non-sensory cells, much is known about the sensory

OE. This produces both the main olfactory as well as

the VNO epithelia, the former a chemosensory, the lat-ter a pheromone receptive system. Neurones within the

developing pheromone receptor system not only express

GnRH [168], but also other factor including neuropep-

tides and catecolaminergic enzymes and neurotransmit-

ters [75,83,124,197,204].Several transcription factors orchestrate the induc-

tion and differentiation of the placode (see Table 1). In

mice, several genes are invloved in placodal differentia-

tion and identity including Eya1 and Eya2 [215], Pax6

[5], Otx1 and Otx2 [19,178,179]. Other factors such as

Gata-4, Ap-2, Olf-1, have been shown to be related to

GnRH and odorant signal transduction functions dur-

ing early development. The above factors are likely toprepare the placode and the rostral forebrain substrates

enabling GnRH cell migration, as well as activating

molecular pathways essential for cell survival, growth

and migratory differentiation.

3. Factors influencing GnRH and rostral forebrain

development

After placodal differentiation, synaptic contact be-

tween olfactory fila and the telencephalic vesicle plays

a pivotal role not only in the later stages of GnRH ros-

tral forebrain migration, (distinct from the ‘‘early’’

GnRH wave migration described in primates), but also

in events leading to OB histogenesis induction. In the

mouse, at E11.5 (human equivalence: CS 16), olfactoryaxon outgrowths already form a structural platform

on which future GnRH neurones migrate in association

with peripherin-positive axons [56]. However, these

tracts do not provide any chemical guidance for GnRH

neurones; axonal guidance molecules, extracellular ma-

trix proteins, growth factors, and neurotransmitters

are required to complete this function (see Table 1 and

Table 2). These factors, exerted in short range cuesand expressed in this short passage, are direct/indirectly

involved on olfactory axon routing and GnRH neuronal

migration.

3.1. Axonal guidance molecules

Axonal guidance molecules are involved in the con-

trol of olfactory system development and mediated byboth attractive and repulsive interactions. Failure of this

function disrupts targeting of axons, and the induction

of vomeronasal organ, accessory olfactory bulb, lateral

Table 1

Extracellular matrix proteins (ECM), neurotransmitters (NT), and transcription factors (TF) involved in GnRH system, placodal, and brain

development

Group Family Knock-out phenotype References Comments

ECM ECM

Anosmin-1 x� Kallmann�s syndrome [110,111,

151,152]

CSP 6B4 PG [131] 6B4PG surrounds

GnRH cells in the Olf.

Nerve in chick embryos

HSPG Knock-outs Nes-EXT1-null mice in

CNS presented same phenotypic

defects presented

in Fgf8—mutant mice

[54,78] Nes-EXT1-null forebrains

primary cultures show

no proliferative response

to FGF treatments

Laminin No significant observation in

forebrain or GnRH development

[184,209]

NT NT

GABA(c-aminobutyric acid) GABA-A-R signalling manipulation inhibit

GnRH cells biosynthetic capacity and migration.

GAD-67 transgenic mice show abnormal

positioning of GnRH cells in the forebrain.

Muscimol inhibited GnRH cells migration

[18,57,58,

181,212]

Axons from GABA

olfactory cells terminate

at the cribriform plate

and their expression

correlates with GnRH

neuronal migration from

the nasal region

AMPA (a-amino-3- hydroxy-5-methyl-

4-isoxazole propionic acid)

[180] Affect tangential

migration and positioning

between nose and forebrain

NMDA (N-methyl-DD-aspartate) [180] Affect positioning in

diagonal band of Broca

and preoptic area

TF TF

Eya1, Eya2 Affects the specification of the placode [215] Drosophila homologous

sine oculi gene family.

Regulatory hierarchy

downstream of Pax

Pax6 ·x Olfactory pathway disruption/anosmia [5]

Otx1, Otx2 Failure of rostral forebrain, temporal

perirhinal areas, and cortex development

[178]

Gata-4 Gata-4 �/� die before any GnRH or

forebrain differentiation

[6,96,100,

121,128]

Zinc finger family.

Expressed in the OE.

Bind to GnRH promoter

Olf-1 Mutation does not alter spatiotemporal

pattern of gene expression in

olfactory sensory neurons

[35,61,95] Helix–loop–helix family.

Expressed in sensory

receptors. Regulates

odorant signal

transduction cascade

Ap-2a � Ap-2a �/� showed cranio-abdominoschisis,

severe dismorphogenesis and failure in the

closure of the cranial neural tube.

Ectsopic expression of GnRH in the

respiratory epithelium.

No association with abnormal

olfactory morphogenesis

[26,90,

164,225]

Gene mutation does not

affect olfactory/respiratory

epithelium separation and

GnRH migration. This

TF does not bind directly

to GnRH or peripherin.

Expressed in the

respiratory epithelium

Pax6 ·>/Gsh2 Failure of Tel development,

olfactory hypoplasia, specification

of progenitor cells in

cerebral cortex (Gsh2) and striatum

(Pax6). Loss of PAX6 in human

produces defects

in the eye (aniridia), forebrain

(OB hypoplasia and anosmia),

cerebellum and spinal cord

[5,182,

191,199]

Pax6 and Gsh2 regulate

genetically opposing

programs that seem to

control each others

expression (i.e., Gsh2

represses Pax6 expression

in the lateral ganglionic

eminence and lateral

cortex). Pax6 controls

dorsoventral telencephalon

and diencephalon patterning

(continued on next page)


Table 1 (continued)


Gli-3 (Gli-Kruppel

family protein 3)

Xt/Xt mutant mice affects normal

development of Tel, LOT cell

distribution, and cerebral

cortex. Craniofacial abnormalities

and polydactily

[55,97,104,

205,206]

Zinc finger family. Gli-3 is involved in the

Sonic hedgehog- Patched-Gli (Shh-Pthc-Gli)

pathway and is expressed in the neocortical

ventricular zone

Vax1 > In Xenopus and mice, Vax1 mutants

showed defects in basal Tel, dysgenesis

of optic nerve, coloboma, and lobar

holoprosencephaly

[11,15,

69,70,123]

These observations also suggested that Vax1

may interfere negatively with the expression

of Pax6 and in the Rx vertebrate homebox

Eya3 Affects specification of the CNS [215]

Ebf Ebf1 targeted disruption affect molecular

specification of SVZ and mantle cells

and affects genetics hierarchies

for neural differentiation. Ebf2 disrupts

GnRH migration into de hypothalamus.

Ebf2-null mice show disrupted GnRH

neuronal migration into the hypothalamus

with no gross alterations of the migration

substrate including the OE, VNO,

and nerves, the OB and the ventral forebrain

[43,61,

94,30]

Helix–loop–helix family. Ebf1 plays an

essential role in the acquisition of mantle

cell molecular identity in developing

striatum and provides information on the

genetic hierarchies governing ventral

telencephalic neuronal differentiation

·, factor also detected in the olfactory system; x, factor also detected in the forebrain; >, factor also detected in the diencephalon; �, factorrestricted to the respiratory epithelium; italics, receptors; TF, transcription factors; NT, neurotransmitters; CSP, condroitin sulphates; HSPG,

heparan sulphates proteoglycans.


olfactory tract, and glomeruli development in the

olfactory bulb, all essential for GnRH migration (see

Table 2).

3.1.1. DCC and netrins

Deleted in colorectal cancer (DCC) is a vertebrate

receptor for the guidance molecule netrin-1 [171]; these

molecules determine govern axon pathway growth, for-mation as well as neuronal positioning of GnRH cells.

Netrins can function not only as chemoattractants

through interaction with Dcc, but also as chemorepel-

lents by activating receptors of the Unc5 subfamily of

the immunoglobulin superfamily [194]. Failure of

expression of any of these genes would be expected to

disturb GnRH neuronal ontogeny because of the associ-

ated disruption of structural or physiological barriers.

3.1.2. Ephrins

The Eph are the largest subfamily of tyrosine kinase

receptors and their ligands, the ephrins, are expressed

on olfactory axons in a complex spatio-temporal pattern

[51,81]. Eph-ephrin, repulsive interactions are involved

in axon guidance, cell migration and sorting, cell adhe-

sion within nerve bundles, and neurite outgrowth [82].More specifically, ephrin-A5 is found to be expressed

more strongly on apical than basal VNO axons, while

the EphA6 receptor is preferentially expressed in the

mouse anterior accessory olfactory bulb (AOB) [87].

Thus, axons with higher levels of ephrin-A5 project onto

a region of the AOB with higher EphA expression. This

has led to a model whereby the EphA-ephrin-A can be

envisaged as chemoactive molecules guiding the projec-

tion of apical axons to the anterior AOB. ‘‘Knock-out’’

models support this concept: in ephrin-A5 mutant mice,

apical axons terminate in both anterior and (topograph-

ically inappropriate) posterior AOB [86,87]. In the rat,

down-regulation of EphA4 may facilitate interaction of

ephrin-A5 with its receptor/s on target cells in the bulb

[187]. The dynamic spatio-temporal expression patterns

of ephrin-B1, ephrin-B2, and EphB2 coincide with majorperiods of axon growth, axon sorting and glomerular

formation, suggesting that these molecules have distinct

roles in different regions and at different times during the

development of the primary olfactory system develop-

ment [188].

3.1.3. Semaphorins and slits

Semaphorins and slits (as well as their receptors: neu-ropilins and Robo, respectively) have been identified in

the olfactory system [129,139] and appear to play a role

in the establishment and maintenance of olfactory net-

works. Neuropilin-2 (Npn-2) is expressed on apical but

not basal VNO axons and Npn-2 mutant mice also

showed a disruption of the zonal projection of apical

VNO axons [29,207]. Slit has been proposed as a factor

involved in the septal division between the lateral olfac-tory tract (LOT) the septum and olfactory cortex, based

on its expression in embryonic septum and its ability to

repel and collapse rodent olfactory axons [107,146].

3.1.4. NELF

A nasal embryonic GnRH factor (Nelf) has been

isolated in mice by single cell RNA extraction [91]. Nelf

appears to exert a role as the common guidance mecha-

Table 2

Cell adhesion molecules (CAM), axonal guidance molecules (AGM), and growth factor (GF) present in the olfactory system and involved in GnRH

system development


CAM N-CAM x Genetic deletion, enzymatic or topical

application of antibody only partially

affect GnRH migration with minor changes

in fasciculation in olfactory/VNO pathways

[31,170,222] NCAM, TAG-1, and L1 are

part of the Ig superfamily having a

role as transient axonal surface molecules

Ark(AXL) x Inhibition of the Racfi ERK signalling

pathway avoid the Ark activity

[2] The extracellular domain contains a

combination of FNIII

Galectin-1 Galectin-1 null mice showed an aberrant

topography of olfactory axons

[148]

TAG-1 [59,223]

CC2-

glycoconjugates

[198] Chemical guidance for GnRH

migratory cells

AGM Netrin-1(Dccx

and Unc5h3)

Dcc/Net1 �/� mice present nerve hypoplasia

and misrouting of VNO axons affecting the

visual and neuroendocrine systems

[37,38,171] Ig superfamily. Expressed in the OE

and VNO, and in GnRH cells migrating

within E11 to E14

Ephrins (EphA5-

ephrin-A, EphA4,

ephrin-A5, ephrin-B1,

ephrin-B2, and EphB2)

Inappropriate targeting of apical axons

in the AOB

[86,87] Spatiotemporal expression pattern

during development

Semaphorins

(Semaphorin

Class 3/Neuropilin-2)

npn-2 mutant mice it is disrupted the zonal

projection of apical VNO axons

[27,29,

63,207]

Neuropilin-2 (NPN-2) has been described

inmouse and rat as part of the receptor

complex for some class 3 Semaphorins

Slit/Robo Slit1- and/or Slit2-deficient micee show a

dose-dependent decrease of their repulsive activity.

Slit1/Slit2 double-deficient embryos had a

complete disorganization of the LOT

[129]

NELF No mice mutant model. Heterozygous

missense mutation (1438A > G, T480A) which

may be associated with IHH

[91,120] Candidate for GnRH neural migration

disruption. Expressed in olfactory sensory

cells and GnRH cells during embryonic

development

GF FGF/FGFR Fgf8�/� die around the time of gastrulation.

Fgf8 null allele over a hypomorphic allele presents

small Tel and no OB. Fgfr1-deficient mouse presents

no OB and is essential for Tel morphogenesis.

FGFR1 gene is now considered as KAL-2 gene.

Loss of function mutation preduces autosomal

recessive Kallmann�s syndrome

[41,42,74,113,

118,153,177]

Fgf2-deficient mice regulates cortical

neurogenesis and promoting neural

progenitor cell proliferation. Fgf3, Fgf15,

Fgf17, and Fgf18 are all expressed

at the anterior end of the

developing telencephalon

HGF(SF)-Met Knock-out models of either HGF(SF) or

Met die early in embryogenesis

[62,200] Mouse immortalized GnRH GN11

cell line presented a increased tropism

using different HGF(SF) treatments.

HGF(SF)-Met complex is expressed

in the olfactory system

x, factors also detected in the forebrain; italics in the second column represent the receptors.


nism for olfactory axon projections in early GnRH-I

neuronal migration [91] acting either directly or indi-

rectly. The human homolog, NELF, is a candidate gene

for Kallmann�s syndrome. The recent characterization

of NELF and mutational analysis of a patient with

IHH showing a novel heterozygous missense mutation

(1438A > G, T480A) within NELF has highlighted the

potential importance of this protein in GnRH migration[120].

3.2. Cell adhesion molecules

Within the nasal regions, cell adhesion molecules play

a secondary role in GnRH neuronal migration, guiding

olfactory axons from OE to OB; such chemoactive mol-

ecules include diffusible chemorepellents that exert ac-

tions on axonal/neuronal guidance. They may provide

a chemical corridor required for neuronal migration into

the rostral forebrain (see Table 2).

3.2.1. N-CAM

The role of N-CAM (neural cell adhesion molecule)and its polysialylated form has been investigated in the

GnRH neural ontogeny of several vertebrates. N-

CAM is present during these initial stages and GnRH

neurones and the olfactory nerve have been found to ex-

press the highly polysialylated form of N-CAM (PSA-

N-CAM or N-CAM-H; [125,172,222]). PSA-N-CAM


with its long a-2,8-linked sialic acid polymer (PSA), has

been shown to serve as an overall negative regulator of

cell interactions [161], probably through the ability of

this large carbohydrate to interfere with intermolecular

and/or membrane–membrane contacts [190,220,221].

3.2.2. Ark(AXL)

Ark (adhesion-related kinase) is the mouse homolog

of the human receptor tyrosine kinase AXL (UFO)

[133] and is expressed along the GnRH-I neuronal

migratory route in the cribriform plate, promoting

GnRH-I neuronal migration via a Ras GTPase mecha-

nism [50]. Ark also suppresses GnRH-I gene expression

via the co-ordinated activation of a Rac/ERK (extracel-lular signal-regulated kinase) signalling pathway and a

distinct myocyte enhancer factor-2 (MEF2) mechanism.

Gas6 (product of the Growth arrest specific gene 6)

functions as a common ligand for Ark(AXL) receptors

[126,203]. The Gas6–Ark signalling also promotes ac-

tin-cytoskeletal reorganization and migration of

GnRH-I neurones via one of the Rho GTPases (vide in-

fra) [2–4]. Therefore, while promoting GnRH neuronalsurvival, migration and level of expression, Ark acts to

control the GnRH cell phenotype until the final hypo-

thalamic destination is reached.

4. Factors influencing GnRH migration and cerebral

specification

The passage from the nasal mesenchyme (NM) into

the rostral forebrain constitutes the second stage of

GnRH neuronal ontogeny. New factors are involved

in the brain migration route, switching on GnRH gene

expression and determining final cell positioning in the

brain; disruption of these pathways may also have a po-

tential role in IHH (see Table 1). The mechanisms

directing the GnRH pathway to this area are unclear,but are likely to require both chemoattractive and

chemorepulsive stimuli. These transcription factors in-

clude Pax6 (paired box gene 6) and Gsh2, have been

incriminated in mouse olfactory system development

and govern cortical and striatal progenitor cell develop-

ment respectively. The loss of PAX6 in human produces

multiple CNS defects in the eye (aniridia), forebrain (OB

hypoplasia and anosmia in humans), and other severedefects in the cerebellum and spinal cord [182,191]. Cor-

rect expression of the transcription factor Gli-3 in mouse

forebrain is a prerequisite for normal telencephalic and

later cerebral cortex development [97]. In the human,

dysregulation of the SHH–PTCH–GLI pathway leads

to several diseases involving telencephalic patterning

leading to several birth defects (for review [205]).

Moreover, the Vax1 gene [70], clearly related tothe Emx and Not genes, is required for the formation

of several brain structures in early embryogenesis

[114,142,149,193,224]. Additionally, Ebf (early B-cell

factors) transcriptional factors are implicated in numer-

ous developmental processes and neural functions

[30,43]. Human orthologues of Ebf2 would constitute

plausible candidates for genetic studies of hypogonado-

trophic hypogonadism (HH); moreover, the EBF2 genemaps to within 450kb of the GnRH in human chromo-

somal band 8p21 [30].

5. Cellular biology of GnRH cells

5.1. Migratory mechanisms of GnRH cells

Activation of Gas6-Ark(AXL) and HGF(SF)-Met

activate the molecular pathways involved in cellular

migration. Both have been described in tissues where

migratory GnRH systems are present. Gas6 treatment

stimulates migration of mouse immortalized GnRH

NLT cells, an effect resulting from reorganization of

the actin filaments producing lamellipodia and cell

membrane ruffles. This effect is blocked by treatmentwith the AXL extracellular domain and an Ark anti-

body [1].

Cytoskeletal reorganization is triggered via activation

of signal pathways linking membrane receptors to mem-

brane surface ruffles, lamellipodia, and filopodia ulti-

mately associated with cell migration. Mechanisms

involve the Rho GTPase family, including Rho, Rac,

and Cdc42 (for review [68]) and have been extensivelyinvestigated in NLT cells, in which Gas6 activation

showed a rapid effect, inducing GTP-bound Rac, an ef-

fect blunted in an adenoviral-mediated expression of

dominant negative N17Rac. The migratory and cyto-

skeleton phenotype changes were confirmed by a GFP

construct which responded to Gas6 but not to the dom-

inant negative N17Rac [1]. Downstream of Rac, p38

mitogen-activated protein kinase (p38 MAPK) is alsoresponsive to Gas6; this migratory phenotype was also

blocked by chemical inhibitors and adenoviral infection

of a dominant negative MAPK (T180A and Y182F) [1].

Finally, downstream of Gas6-Ark(AXL), Rac, and p38

MAPK, additional stimulation of MAPK-activated pro-

tein kinase-2 induced phosphorylation of HSP25 also in-

volved in cortical actin remodeling regulation [1].

Involvement of the HGF(SF)–Met complex onmouse immortalized GnRH GN11 cell migration has al-

ready been discussed. In other cell systems, activation of

this system is followed by diverse intracellular signalling

pathways, including PI3K and Ras/p38 MAPK, which

mediate the HGF(SF) induced effect. (In this regard it

should be noted that Ras and the Rho family have a re-

ciprocal relationship, Ras as well as Cdc42 being capa-

ble of activating Rac which in turn can activate Rho[132,155].) It is known that the PI3K pathway is coupled

to Met through the interaction of the p85 subunit with

Fig. 3. Confocal laser scanning micrograph (650·) demonstrating

GnRH-I immunoreactivity in the nasal epithelia of an early trimester

human fetus.


the multi-docking sites of Met, while the Ras/p38

MAPK pathway is bridged by the adaptor protein

Grb2 linking Met to SOS, a Ras guanine nucleotide ex-

change factor [147]. HGF(SF) induces scattering and

branching of Madin-Darby canine kidney (MDCK) epi-

thelial cells [122,189]. Treatment of MDCK cells withPI3K inhibitors or Wortmannin abolishes HGF/SF in-

duced scattering, suggesting that PI3K pathway is essen-

tial for cell motility [158]. When treated with a p38

MAPK pathway inhibitor, MDCK cells also show loss

of motility and fail to form branching tubule structures

in response to HGF(SF) [84]. HGF(SF) via activated

ERK, also induces phosphorylation and activation of

paxillin and focal adhesion kinase (FAK) [105]. Thisactivation is abrogated with an ERK inhibitor which

also inhibits HGF/SF-induced cell spreading and adhe-

sion [105]. These data suggest that both Gas6-Ar-

k(AXL) via Rho GTPases and HGF(SF)-Met via

PI3K and Ras activated p38 MAPK pathways are essen-

tial for cell migration, actions relevant to GnRH cell

migration.

Human GnRH cellular models have also been used tostudy the biology of GnRH systems. Primary long term

cell cultures from human embryonic olfactory neuroep-

ithelium have been isolated, established, cloned, and

propagated in vitro [202]. These cells originate from

the ‘‘stem cell’’ compartment that gives rise to mature

olfactory receptor neurones. Named FNC-B4, these

cells synthesize neuronal proteins and olfactory-specific

markers as well as olfactory neurotransmitters. FNC-B4 cells also express the GnRH gene and protein and

combined HPLC and RIA studies indicated that they re-

lease GnRH in media in a time-dependent manner and

this is modulated by sex steroids and odorants [12].

These GnRH cells were also shown to express activin

A, a GnRH-secretion modulator [52] and endothelin-1

(ET-1), a peptide with different functions in reproduc-

tive functions (steroidogenesis and vascular activity)and central control of the sexual activity. Moreover,

they also express the ET-1 converting enzyme ECE-1

and two classes of binding sites, corresponding to the

ETA and the ETB receptors. Functional studies indicate

distinct functions; the ETA receptor subtype mediates

an increase in intracellular calcium and GnRH secretion

while the ETB subtype induced DNA synthesis and

mitogen-activated protein kinase p44ERK1 expressionbut without stimulating GnRH secretion [112]. The lat-

ter data derived from FNC-B4 cells suggests that there is

an autocrine GnRH loop underlying neuronal migra-

tion, where GnRH can modulate the differentiation

and migration of GnRH-secreting neurones, by recep-

tor-mediated mechanisms [157]. The presence of human

GnRH cells in the olfactory epithelium of adults includ-

ing normal subjects, XKS and HH patients support thenotion that these cells continue to proliferate during life

(Fig. 3), although their physiological function post na-

tally is currently speculative [152]. Nevertheless, FNC-

B4 represents an in vitro model for studying neurogene-

sis, cell differentiation, GnRH pulsatility, and migratory

phenotypic functions.

5.2. GPR54: puberty and the onset of GnRH activity

Although the GnRH pulse generator is already func-

tional by 0.3 gestation (20 weeks gestation) intrinsic

CNS inhibition and sex steroid negative feedback damp

down GnRH release, except for a short period in the

postnatal male. This is followed by a period of dampen-

ing of oscillatory activity in late infancy, followed byquiescence during childhood (juvenile pause). During

late childhood, gradual disinhibition and reactivation

of the pulse generator occurs mainly at night, when in-

creased amplitude and frequency of GnRH release oc-

curs, culminating in reproductive maturation in all

vertebrates. Transcriptional regulation of the GnRH

gene is not a major factor governing onset of mamma-

lian puberty. GnRH content does not change in associ-ation with the pubertal acceleration induced by onset of

pulsatile GnRH release [47], and a modest increase in

GnRH mRNA levels at this stage of development has,

to date, been observed only in the agonadal paradigm

[47]. Thus, it appears that during pubertal development,

the limiting factor in the onset of puberty must lie up-

stream to the GnRH neuron.

An explanation for this pubertal enigma has recentlybeen uncovered. A genetic factor named, GPR54, has

been described to have a potential role in the onset of

the puberty in mice and humans. Homozygosity whole

genome mapping of a consanguineous pedigree with five

siblings affected by isolated hypogonadotrophic hypogo-

nadism has revealed a homozygous 155 nucleotide dele-

tion in GPR54 encompassing a splicing acceptor site of

intron4–exon5 junction and part of exon 5 [36]. GPR54encodes a rhodopsin family G protein-coupled receptor

[36,173] and binds to a natural ligand, kisspeptin-1 a

Fig. 4. Structure of KAL-1 protein. SP, signal peptide; Cys box,

cysteine rich box; WAP, whey acidic protein domain; FNIII,

fibronectin domains; H, histidine rich region.


54 aminoacid peptide, encoded by KISS1 [89], which

may also have an important role in this pubertal phe-

nomenon. Seminara et al. have investigated an IHH

pedigree with an L148S mutation of GPR54, and cre-

ated a deficient Gpr54 �/� mouse, showing the same

human phenotype with IHH and normal response toboth exogenous GnRH and gonadotrophins [173].

These recent data suggest that GPR54 is an essential

factor for normal GnRH release, and underline the

importance of this receptor for normal GnRH secre-

tion and induction of puberty.

5.3. GnRH apoptosis: Ark(AXL)

The final number of GnRH neurones populating the

adult brain, after the ontogenic event and the pubertal

onset, represents the balance beween mitosis and apop-

tosis. Studies have estimated that a high percentage of

the neurones expressed during embryogenesis die before

maturation of the organism [169]. The GnRH neuronal

population is unique in that, unlike other neurones that

express hypothalamic releasing factors, the neuronesmust migrate from the olfactory placode into the fore-

brain. In addition, cell numbers are small, with only

800–2000 neurones in higher vertebrates. Given the

importance of appropriately targeting this subpopula-

tion of neurones into the hypothalamus for reproductive

competence, it might be anticipated that complex mech-

anisms modulate programmed cell death during

migration.In 1995, Bellosta et al. reported that Gas6-Ark(AXL)

signalling protected fibroblasts from apoptosis induced

by tumor necrosis factor-a and c-Myc, determining the

rate of programmed cell death [13]. Unlike non-neural

systems, in GnRH cells, Gas6/Ark(AXL) signalling is

not associated with a mitogenic response, not unex-

pected since neuronal cells are not subject to significant

proliferative stimuli. Ark is expressed in GN10 cells, amigratory GnRH cell line but not in the hypothalamic

GT1-7 GnRH cells [4]. Moreover, Ark GN10 GnRH

cells survived longer in serum withdrawal-induced apop-

tosis than GT1-7 (Ark negative). Gas6, augmented this

effect when added to the cell culture; therefore, Gas6–

Ark molecules appears to stimulate the extracellular

signal-regulated kinase, ERK, and the serine-threonine

kinase, Akt, a downstream component of the phospho-inositide 3-kinase (PI3-K) pathway [4]. In the presence

of Gas6, GN10 cell apoptosis is attenuated; this action

was blocked by ERK and PI3-K signalling cascades

inhibitors such as PD98059 or Wortmannin but with

equivocal effects being obtained in the presence of rapa-

mycin [4,67]. These results point to the critical role of the

ERK pathway in transmitting the Gas6 signal from the

membrane to the intracellular targets to rescue theseneuronal cells from growth factor withdrawal-induced

apoptosis [4].

However, Gas6 is not expressed in these neuronal

cells, so one could hypothesize that adjacent glia or neu-

ronal cells synthesize the Ark(AXL) ligand during the

migratory process, consistent with an increasing litera-

ture supporting the role of glial elements in neuronal

migration and survival [154]. It thus appears that inGnRH migratory cells, Gas6–Ark(AXL) signalling via

ERK and PI3-K (via Akt) pathways, modulate sensitiv-

ity to trophic factor withdrawal and protection from

programmed cell death.

6. Novel insights gleaned from the pathogenesis of

Kallmann�s syndrome (KS)

6.1. Molecular genetics of XKS

KAL-1, the gene for XKS, was cloned 13 years ago

using positional cloning approaches. It spans 210kb of

genomic DNA on Xp22.3 and has 14 exons, with a

cDNA of 2043bp giving a conceptual 680 amino acid

residue protein �anosmin-1,� a component of variousembryonic extracellular matrices (vide infra). This mod-

ular extracellular matrix protein comprises an N-termi-

nal cysteine-rich domain, followed by a WAP (whey

acidic protein) domain, four FNIII (fibronectin type

III) domains and a C-terminal histidine-rich region

(Fig. 4). The combination of WAP and FNIII domains

is unique to anosmin-1; however, the 4-disulphide core

WAP domain is similar to that present in a number ofsmall protease inhibitors (http://smart.embl-heidel-

berg.de/), while the FNIII domains are structurally anal-

ogous to members of the N-CAM protein family,

including membrane-bound proteins such as TAG-1

and L1 [156].

A large number of loss of function mutations have

been described within this locus associated with XKS

(Fig. 5). These include various point mutations (mostcases), intragenic deletions [16,115], complete gene dele-

tion [73], and even larger deletion of the Xp22.3 region

associated with a contiguous gene syndrome (KS and

ichthyosis [10]).

6.2. Spatio-temporal distribution of KAL-1

Initial studies focused on the spatio-temporal expres-sion of KAL-1 at the transcript and protein level. In the

early 90s, studies of normal human embryos and

foetal brain showed KAL-1 expression at 6 [46] and 19

weeks of development [108]. Using RT-PCR and in situ

http://smart.embl-heidelberg.de/

http://smart.embl-heidelberg.de/

Fig. 5. Ribbon representations of the WAP domain and the four homology models for the FNIII domains of anosmin-1. For the WAP domain:

C163Y, blue and C172R, indigo indicate aminoacid substitutions which disrupt the four cysteine disulfide core motif of the WAP domain. For the

FNIII domains: The N- and the C-termini of each domain is denoted by N and C. The seven conserved b-strands are represented as blue ribbons,

with the ABE face shown on the left and the GFCC0 face shown on the right in each model. The loop regions are represented as orange ribbons. The

a-carbon atoms of the three missense mutations are denoted as brown spheres (N267K, E514Km, and F517L). The a-carbon atom of Cys residues

are denoted as yellow spheres (C638, C-496-C523, and C-467). The predicted heparan sulphate proteoglycans binding site is indicated as six green

spheres on the b-strand G of FNIII-1 (residues SKHFRS).


hybridization techniques, Duke and collaborators dem-

onstrated low level gene expression in 45-day-old human

embryos in the spinal cord and the excretory system. In

1999, anosmin-1 immunoreactivity was confirmed in theolfactory system from week 5 and onwards (Figs. 1 and

2), and was transiently detected in the respiratory epi-

thelium of the nasal cavity and in the adjacent mesen-

chyme at the end of week 6 and during week 7 [72].

Within the developing human brain, anosmin-1 immu-

noreactivity is detectable within the forebrain from early

week 5, as well as in the telencephalon in the presump-

tive OB area. Immunoreactivity is also demonstrableat the end of week 6 in the OB anlage, as well as the con-

tact areas where olfactory, vomeronasal and terminal

nerve axons and GnRH make contact within the fore-

brain as well as within the medial wall of the primitive

cerebral hemisphere; however, a complete analysis of

the brain during embryogenesis has not been reported

due to technical difficulties (Fig. 2) [72]. There is signif-

icant variation in anosmin-1 expression across mamma-lian species. However, in the Asian musk shrew,

anosmin-1 immunoreactivity is discernible within the

peripheral olfactory system [39]. How then does aber-

rant expression of anosmin-1 predispose to disruption

of both olfactory and GnRH ontogeny?

6.3. Extracellular matrix: understanding the molecular

mechanism of KS

Recently, anosmin-1 has been added to the list of

extracellular matrix factors essential for GnRH neuro-

nal ontogeny and olfactory bulb development. The

extracellular matrix (ECM) has a complex molecular

composition; while this matrix is made and oriented

by cells within it, matrix itself can affect the function

of proteins released into it. There are two main classesof proteins forming the ECM: the unbranched polysac-

charide chains named glycosaminoglycans (GAGs) and

fibrous proteins including collagen, laminin, fibronectin,

and elastin which have both structural and adhesive

functions. Furthermore, most GAGs are covalently at-

tached to protein forming proteoglycans (PGs). Proteo-glycans include the heparan sulphate proteoglycans such

as perlecan and N-syndecan. These molecules have a

major role in chemical signalling between cells, having

inhibitory or active interactions with several molecules

including FGFs and TGFb or even anosmin-1. Such

interactions include protein immobilization, stearic

blockade of protein, creation of a protein reservoir, pro-

tection from proteolytic degradation or concentratingthe protein to enable specific effects.

Heparan sulphate PGs and laminins are present in

the ECM in the same spatio-temporal distribution as

migratory GnRH cells and expression of anosmin-1.

However, no effects on rostral forebrain or GnRH

development are seen in the laminin knock-out mouse

[54,184]. Knock-outs on HS (heparan sulphates) poly-

merizing enzymes severely disrupts a variety of distinctsignaling pathways in different species [78,143]. Specific

studies on brain morphogenesis have shown that selec-

tive disruption of Ext1 (Nes-EXT1-null mice) in the

central nervous system of mice which express non-glyc-

anated syndecan-3 (indicating disrupted HS synthesis),

demonstrated similar phenotypic defects present in

Fgf8-mutant mice (vide infra). Interestingly, primary

culture from Nes-EXT1-null mice forebrains showedno proliferative response to FGF2 and FGF8 treat-

ments [78].

6.4. KAL-1 function in OB development and GnRH-I

migration.

Anosmin-1 is already expressed in human embryos by

day 35 (CS 15–CS16) as well as in the forebrain wall, theregion where olfactory nerves make initial contact with

the rostro-ventral forebrain (Fig. 1) [72]. This temporal

Fig. 6. T1-weighted MRI demonstrating heterogeneity of appearance

of olfactory bulbs and sulci in KS. (A,B) Regions of reduced grey

matter volume are projected in yellow onto a template brain image,

demonstrating bilaterally reduced volume of the entorhinal cortex in

XKS: (A) coronal view; (B) axial view. (c) KS: coronal view showing

olfactory bulbs (white arrows) with abnormally angulated olfactory

sulci (red arrows). (D) Unilateral aplasia of the OB of a KS patient. (E)

Normal subject: coronal view of olfactory bulbs and sulci. (F) KS:

coronal view showing absent olfactory bulbs with shallow sulci. Voxel-

based morphometric analysis of pooled grey matter MRI data of nine

XKS patients versus matched normal controls. (G) and (H) Increased

white matter density in relation to the course of the corticospinal tracts

of patients with XKS demonstrating mirror movements. Regions of

increased white matter density (p < 0.01) are superimposed in yellow

on the Montreal Neurological Institute Reference Brain. Reproduced,

with permission.


association of expression reinforces the notion that

anosmin-1 is a key determinant of early synaptogenesis

between these pioneer olfactory axons and rostral fore-

brain, possibly acting as a chemoattractant and having

a role in the neural plasticity [72]. Thus, in the absence

of functional anosmin-1 expression, early olfactory axo-nal contact with the telencephalic vesicles does not occur

in humans, and axonal terminals of olfactory, termi-

nalis, and vomeronasal nerves become trapped below

the meninges ending in neuronal tangles on either side

of the midline [166].

In the central nervous system, connections of a single

neuron with multiple target cells are achieved by collat-

eral branching. These collaterals form either by bifurca-tion of the leading growth cone or, more frequently, by

the development of interstitial collateral branches from

the primary axon shaft [134]. In this case, neurones first

send out their (pioneer) axons toward the primary tar-

gets, and after a protracted period, collaterals bud from

primary axons and project toward their final targets (re-

view in [80]). The projections of OB output neurones,

the mitral and tufted cells, develop according to this de-layed axonal branching model [76]. Anosmin-1 appears

to influence the development of both primary and sec-

ondary olfactory processing regions, both of which are

impaired in XKS. A recent study in rodents has also

shown that anosmin-1 demonstrated two separate bio-

logical activities: Firstly, the protein stimulates axon

outgrowth from the mitral/tufted cell layer of the rat

OB and secondly, it induces collateral branching fromaxons at the time when these project into the olfactory

cortex [185]. An alternative proposal is that anosmin-1

may be involved in OB histogenesis independent of

incoming olfactory axons. Thus mesenchymal/epithelial

interactions appear to influence initial OB formation in

mouse [98]; during development, the frontonasal mesen-

chyme is apposed to the ventrolateral forebrain where at

least one mesenchymal signal, RA, activates gene expres-sion in the ventrolateral forebrain [5,99,208]. Consonant

with this notion, olfactory axons are not necessary for

bulb morphogenesis in embryologically manipulated

frogs or Emx-2 mutant mice [24,224]. Although a plau-

sible hypothesis, the temporal expression of anosmin-1

and GnRH-I migration through this pre-olfactory re-

gion of the telencephalic vesicle appear more than mere

coincidence, and a direct effect of anosmin-1 on the laterphases of GnRH-I neuronal cell migration cells remains

possible. The spatio-temporal distribution of anosmin-1

and GnRH-I neurones are similar and the adhesive nat-

ure of this protein makes an anosmin-1-GnRH-I neuro-

nal interaction plausible. It is well known that the

interaction of anosmin-1 with heparan sulphate proteo-

glycans (HSPG) at the cell surface/ECM is essential for

its biological activity [186]. A further pointer to an anos-min-1-GnRH-I interaction is the fact that in rats, N-

syndecan, a membrane-bound HSPG, is confined to

migrating cells and around the migrating cell cluster

which contain both calbindin and GnRH-I immunore-

activity well before the OB becomes apparent. It is

highly likely that N-syndecan molecules are produced

in migrating neurones and expressed on their cell mem-

brane [196].Detailed immunohistochemical and morphological

analysis of a solitary 19-week-old XKS foetus affected


by complete deletion of KALIG-1 (Kallmann�s syn-

drome interval gene-1) has failed to shed further light

on the mechanism of anosmin-1 action. An absence of

GnRH-I neurones occurred in the hypothalamus, and

thick GnRH-I-ir positive nerve bundles terminated at

dorsal surface of the cribriform plate beneath the menin-ges, with total failure of bulb development [166,196].

Absence of the olfactory bulb is not an invariable con-

comitant of KS however. Magnetic resonance imaging

(MRI) has shown considerable morphological heteroge-

neity of OB and olfactory sulci appearance in XKS,

ranging from totally absent to normal, though with con-

sistent reduction in volume of the olfactory cortex

[111,151] (Figs. 6A–F). These important observationssuggest that anosmin-1 is involved in the early events

of bulb development at a time when GnRH-I neuronal

migration is most active. One can conclude that other

factors also play a role in OB development, although

normal functional organization of the OB necessitates

anosmin-1 activity.

6.5. Evolutionary conservation of anosmin-1

The existence of a KAL-1 gene in humans is not un-

ique, and a number of its orthologues have been char-

acterized in cross-species analysis. Kal-1 genes are

present in a variety of vertebrates and invertebrates,

ranging from model organisms such as Caenorhabiditis

elegans and Drosophila melanogaster, through to Fugu

rubripes, Brachyodanio rerio, Coturnix coturnix, andGallus gallus. In some of these species, both mRNA

and protein expression have been confirmed during

development [7,23,101,102,160]. Moreover, the func-

tional activity of these �anosmins� resides in a multido-

main module consisting of a highly conserved

N-terminal WAP domain, followed by a variable num-

ber of FNIII domains. A detailed genetic analysis of

anosmin-1 function and its interacting factors has notbeen possible in rodents, as mouse and rat Kal-1 ortho-

logues have not yet been definitely cloned [185]. The

first systematic genetic analysis of anosmin function

was carried out in C. elegans [23,160], where CeKal-1

influenced both axonal outgrowth and neurite branch-

ing, both in loss of function mutants and when CeKal-

1 was overexpressed in a number of neuronal subtypes

[23,160]. CeKal-1 is also required for ventral enclosureduring embryogenesis and visualization of epidermal

cell boundaries in comma-stage embryos demonstrates

abnormal positioning between cells, both in loss of

function mutants and in worms overexpressing Ce-

Kal-1, suggesting that these cells migrate to surround

the embryo during morphogenesis. However, they fail

to form reciprocal adherent contacts in loss of function

mutants [160], resulting in striking phenotypes in malemutants with ventral enclosure and ray (tail) abnormal-

ities [160].

6.6. Factors influencing anosmin-1 function

To date, the only detailed genetic analysis of factors

modifying Kal-1 related phenotypes have been carried

out in C. elegans transgenic lines which permit study

of gain-of-function phenotypes [23]. The most penetrantaxon branching phenotype observed in worms with neu-

ron specific CeKal-1 over-expression is unaffected by

Eph-R, semaphorin [28], netrin, Robo, FGFR, integrin,

and other ECM mutant [23] deficient backgrounds.

Ephrin/EphR and semaphorin are central to neurite

branching and for axon guidance, in addition to the cell

movements and interactions that occur during morpho-

genesis. By contrast, expression in mutant backgroundsof two b-spectrins showed a higher suppression of phe-

notype (29–36%) explained by their proposed role in

localizing cell adhesion/signalling proteins [23,71]. Fur-

thermore, a modifier screen to isolate new mutations

in genes required for CeKal-1 to exert its function

demonstrated almost complete suppression of CeKal-1-

induced branching of AIY neurones in a heparan-6O-sul-

photransferase deficient background [23]. This enzymecatalyzes the transfer of a sulphate moiety on position 6

of the glucosamine residue, part of the disaccharide repeat

unit of heparan sulphates.

These and previous observations show that the inter-

action of anosmin-1 with HSPG at the cell surface/ECM

is essential for its biological activity [23]. HSPGs have

markedly heterogeneous structures in which distinct pat-

terns of sulphation determine the binding specificityfor ligand proteins. These ‘‘fine structures’’ of heparan

sulphate are mainly produced by the regulated introduc-

tion of sulphate groups at the N-, 2O-, 6O-, and 3O-po-

sitions. Recent studies have demonstrated that these fine

structures mediate distinct molecular recognition events

that regulate a variety of cellular functions. HPSG is in-

volved in neurogenetic events such as neurite outgrowth,

neuron migration and tissue morphogenesis throughregulating uptake, degradation, diffusion, and biological

function of a wide range of ligands, such as, Slit/Robo,

BMP/Noggin, fibroblast growth factors (FGF) and

high-affinity receptors [77,138]. For example, the impor-

tance of 6-O-sulphated glucosamine residues have been

recognized for the binding affinity of HS to antithrom-

bin, FGF, FGFR, ApoE, and lipoprotein lipase,

respectively [9,103,109,117]. Mice mutated for the HS2-O-sulphotransferase (Hs2st) gene demonstrate bilat-

eral renal agenesis secondary to defective FGF signal

transduction [22]. When Hs2st activity is disrupted, no

detectable abnormalities were observed in the mutant

embryos until late gestation, potentially explained by a

compensatory increases in N- and 6-O-sulphation, with

no overall change in charge distribution [127]. The 6-

O-sulphate group is also responsible for the cellular re-sponse to FGF [141]. Moreover, the role of HSPG

expression permitting the biological function of FGF/


FGFR and anosmin-1 seems to be tissue- and temporal-

ly-specific. How then do these putative biological ac-

tions of anosmin-1 relate to the clinical manifestations

and pleiotropic effects of the condition?

6.7. Phenotypic manifestations of KS

KS is characterized by isolated hypogonadotrophic

hypogonadism (IHH) and anosmia (due to absent or

abnormal OB development and failure of GnRH neuro-

nal migration from the medial nasal placode into the

hypothalamus) [151]. OB development may be com-

pletely or partially absent (Figs. 6C–F), and in some pa-

tients dysplastic. Although a genetically heterogeneouscondition, with X-linked, autosomal dominant and

recessive modes of inheritance [41,136,152] recognized,

most clinical presentations are sporadic. The incidence

of KS is about 1 in 8000 males and 1 in 40,000 females.

Failed pubertal development is generally the first mani-

festation of the disease in both sexes; in males, micrope-

nis, cryptorchidism as well as delayed or arrested

puberty are generally present. Males with XKS haveadditional phenotypic characteristics: 85% demonstrate

the phenomenon of bimanual synkinesis (upper body

Fig. 7. (A and B) Surface EMGs recorded simultaneously from the left and r

right index finger abduction. (A) Normal control subject: three bursts of EM

abduction of the right index finger; there is no EMG activity on the left side.

voluntary EMG activity recorded from R1DI during right index abduction, b

the L1DI. (C) Focal magnetic brain stimulation, using a 70mm figure-of-eigh

UK), of the left motor cortex was used to study the laterality of corticospina

homologous muscle pairs. (D) Cross-correlogram of a XKS patient with m

correlograms constructed from multi-unit surface EMGs recorded during vol

was constructed from �5000 trigger spikes from the left 1DI and 5000 even

correspond to spikes in the right 1DI preceding (and following) spikes in th

mirror movements) (Figs. 6G and H) [92,93,116] with

urogenital defects including renal agenesis present in

up to 33% of cases [46,85].

Although previously recognized as an absolute phe-

notypic marker of XKS, bimanual synkinesis may, how-

ever also occur in AKS (vide infra). Could disturbedanosmin-1 action perturb corticospinal development?

We initially sought to investigate this phenotypic abnor-

mality in affected patients using combined neurophysio-

logical [116] and positron emission tomographic (PET)

approaches [93]. Electromyographic (EMG) recordings

taken from the first dorsal interosseous (DI) muscle dur-

ing voluntary self-paced abduction of one index finger in

XKS patients evoke simultaneous EMG activity in thecontralateral DI [116], with no significant difference be-

tween time of onset of the bursts of voluntary and invol-

untary mirroring EMG (Fig. 7C). Focal magnetic

stimulation of the hand area of the motor cortex re-

vealed the presence of fast conducting bilateral corti-

cospinal projections from the motor cortex in all

subjects (Fig. 7D). Overall, these studies concluded that

XKS patients have a novel ipsilateral corticospinal tractand that activity in this tract is responsible, at least in

part, for pathological mirroring [116].

ight first dorsal interossei muscles (L1DI and R1DI) during self-paced

G activity are present on the right side, each burst represents a single

(B) Patient with XKS and mirror movements: similarly, three bursts of

ut simultaneous involuntary bursts of EMG activity can also be seen in

t coil with a Magstim 200 stimulator (The Magstim Company, Dyfed,

l projections. EMGs were recorded simultaneously from left and right

irror movements: there is a large short duration central peak. Cross-

untary sustained left and right index finger abduction. The correlogram

t spikes from the right 1DI, so that negative (and positive) time lags

e left 1DI, respectively.


Measurement of regional cerebral blood flow in XKS

patients using H215O-PET performed during voluntary

hand movement in XKS patients showed a strong pri-

mary motor cortex (M1) activation contralateral to the

voluntarily moved hand, but there was also a significant

degree of M1 activation ipsilateral to the voluntarilymoved hand, i.e., contralateral to the mirroring hand.

Further, study suggested that the small but significant

activation of the ipsilateral M1 in XKS might be due

to sensory feedback from the involuntarily mirroring

hand [93], although transcallosal activation could not

be excluded (Fig. 8).

Statistical analysis of pooled white matter data

from structural MRI scans were used to further inves-tigate the aetiology of mirror movements in such XKS

Fig. 8. PET activation in six individuals with XKS and bimanual synkines

sections have been cut through M1 at the height of the focus in M1with

individual�s MRI scan. For each scan subjects received a 20s intravenous bolu

scans were collected at 10min intervals.

patients [92]. The T1-weighted brain scans of XKS pa-

tients were compared with two non-mirroring groups

of AKS and normal men. This study demonstrated

hypertrophy of the corpus callosum in both KS

groups: the anterior and midsection in XKS, and the

genu and posterior section in AKS. However, bilateralhypertrophy of the corticospinal tract was found only

in the group of XKS patients exhibiting mirror move-

ments (Figs. 7A and B) suggesting that mirror move-

ments in XKS result from abnormal development of

the ipsilateral corticospinal [92] tract fibres consistent

with previous electrophysiological evidence [116]. Ta-

ken together, it appears that anosmin-1 constituted

not only a major determinant of olfactory bulb andGnRH neuronal ontogeny, but was also implicated

is during voluntary movements of their left or right hand. Transverse

the highest Z-score. Areas of activation are co-registered on to the

s of H215O through a cubital fosa vein of the left arm. Consecutive PET


in a more fundamental way in human corticospinal

tract development.

6.8. Other manifestations of KS

While 33% affected males with XKS have unilateralrenal agenesis, renal impairment in the solitary remain-

ing kidney may also occur, associated with renal dyspla-

sia leading to chronic renal impairment [45]. The precise

role of anosmin-1 in nephrogenesis is presently

unknown.

Autosomal forms of KS (which form the majority of

familial KS cases) have no additional phenotype, other

than IHH and anosmia. A variety of associated defectsare, however, seen in a minority of cases, including, mid-

line developmental defects, including cleft lip and palate,

other craniofacial anomalies, coloboma, and sensori-

neural deafness [136,152]. Although a number of genes

influence the development of both olfactory and

GnRH-I neuronal systems in humans [111,136,152],

other genes causing AKS have proved hard to identify

by conventional molecular genetic approaches, since pa-tients are invariably infertile (unless treated with

gonadotrophins or pulsatile GnRH therapy) and pedi-

grees are thus invariably small.

7. FGFR1 and KS

7.1. FGF systems

Members of the fibroblast growth factor (FGF) fam-

ily have multiple roles during central nervous system

development. At least 23 different members of the

FGF family exist, all sharing a conserved 120 amino

acid core region. FGF family members exert diverse

functions, being potent modulators of cell proliferation,

migration, differentiation, and survival (for review [53]).There are five FGF receptor genes, FGFR-1-5, and,

within these, alternative splicing create receptor iso-

forms with distinct specificities for particular FGFs

[183]. There have been multiple studies on the expression

patterns of FGF ligands and receptors during CNS

development that indicate sites of activity. There have

also been many functional in vitro and in vivo assays

that, together, emphasize the critical role of FGFs inthe initial generation of neural tissue at the stage of neu-

ral induction. This activity is also present in the rostral

forebrain, directly affecting olfactory bulb development.

The hepatocyte growth factor (HGF), was originally

identified as a mitogen for hepatocytes and identical to

the SF (scattering factor) [17]. HGF/SF is secreted as

pro-HGF(SF), which is activated by proteolytic cleav-

age of a single polypeptide precursor at arginine 494,yielding heavy (consisting of an N-terminal domain

and four disulfide-linked kringle domains) and light

chains (containing a protease-like domain that lacks

enzymatic activity) [17,159]. HGF(SF) binds to a high

affinity tyrosine kinase c-Met (Met) receptor [20]. Met

is synthesized as a polyprotein and is proteolytically

cleaved into subunits as it matures on the cell surface.

HGF(SF)-Met signalling is essential for some develop-mental processes including promoting migration of mus-

cle precursor cells and motoneurones [17]. This complex

also appears to play a role in nervous system develop-

ment and function [219] where HGF(SF)-Met is present

in olfactory regions such as OB, OE, and the olfactory

nerve layer [195] (Table 2).

7.1.1. FGF

Fgf2 has also been implicated in telencephalic devel-

opment. In particular, analysis of Fgf2-deficient mice

demonstrates a role for this gene in regulating cortical

neurogenesis and promoting neural progenitor cell pro-

liferation [42,137,153], although no patterning defects

were observed in Fgf2-deficient mice. In human, FGF2

has been shown to support human olfactory neurogene-

sis in vitro; this basic FGF had a dose-dependentgrowth-promoting activity which was accompanied by

morphological changes and differential expression of

NF (Neurofilament gene) in GnRH expressing human

primary olfactory FNC-B4 neuroblasts [48].

Fgf8 may play a key role in rostral telencephalic pat-

terning. Fgf8 is expressed just anterior to the neural

plate in the anterior neural ridge as early as E8.5 in

mouse, and in the anterior forebrain from E9 to at leastE12.5 [32]. Mice completely deficient in Fgf8 die around

the time of gastrulation [119]. However, partial loss of

function of Fgf8 in E18.5 embryos carrying an Fgf8 null

allele over a hypomorphic allele can result in a small

telencephalon lacking olfactory bulbs and a normal mid-

line [119]. Results from experiments in which Fgf8-

coated beads were ectopically applied to forebrain tissue

[33,176] and in which Fgf8 was overexpressed in thetelencephalon [60] also suggest that Fgf8 induces the

formation of anterior telencephalic structures.

Fgf genes other than Fgf8 may also be expressed and

play a role in the developing telencephalon. For exam-

ple, Fgf3, Fgf15, Fgf17, and Fgf18 are all expressed at

the anterior end of the developing telencephalon

[113,118,177].

7.1.2. FGFR

Although over 20 genes encode FGF ligands, there

are only five known genes encoding FGF receptors

[53,183]. In mouse development, Fgfr1, Fgfr2, and

Fgfr3, but not Fgfr4, are expressed in the progenitor

cells lining the telencephalic ventricles [144,145,217].

‘‘Knock-out’’ models show that embryos deficient for

either Fgfr1 or Fgfr2 die during the early stages oftelencephalic vesicle development [8,216,217], whereas

Fgfr3-deficient mice survive and show no obvious

Table 3

FGFR1 loss of function mutations and their associated symptoms

Mutation Exon/intron Kallmann�s syndrome associated symptoms Ref.

G97D Exon 3 [41]

Y99C Exon 3 [41]

S107X Exon 3 [162]

303–304 ins CC Exon 3 [41]

A167S Exon 5 Cleft palate, corpus callosum agenesis, unilateral hearing loss [41]

C277Y Exon 7 [41]

936 G to A Exon 7 (donor splice site) Multiple dental agenesis [41]

V607M Exon 13 Bimanual synkinesis [41]

R622X Exon 14 Cleft lip or palate [41]

1970–1971 delCA Exon 14 [41]

W666R Exon 15 Cleft palate [41]

IVS15+ 1G to A intron 15 (donor splice site) [41]

M719R Exon 16 [41]

P745S Exon 17 [162]

P772S Exon 18 Cleft palate, unilateral absence of nasal cartilage, iris coloboma [41]


telencephalic defects [40]. Fgfr5 is expressed in adult

kidney, brain and lung but to date the phenotype of

the Fgfr5 has not been established [183].

Analysis of Fgfr1-deficient mice has shown a largely

normal cerebral cortex antero-posterior pattern. How-

ever, the olfactory bulbs do not form normally, implicat-ing an essential role for Fgfr1 in patterning and

morphogenesis of the rostral telencephalon, which could

directly affect the GnRH neuronal migratory activity

[74]. The role of FGFR1 in human olfactory bulb devel-

opment and GnRH neuronal ontogeny is described else-

where in this review.

7.2. FGFR1: another KS gene

Loss of function mutations of FGFR1, on chromo-

some 8p11–12 have recently been incriminated in the

AKS (see Table 3) [41,162]. The FGFR-1 also is involved

in autosomal dominant craniosynostosis syndromes

such as the Pfeiffer syndrome characterized by craniofa-

cial anomalies and characteristic broad thumbs and big

toes [14].The involvement of FGFR-1 (KAL-2) and KAL-1 in

the same developmental disease raises the possibility

that these gene products might functionally interact

[41], although whether this represents a direct or indirect

interaction is currently unclear. It is already established

that anosmin-1 binds to HSPGs, and HSPGs are

essential for dimerization of the binary FGF–FGFR

complex, thereby creating a heteropentameric or hetero-hexameric molecular complex [41]. This extracellular

interaction between FGF, the FGF receptor and hepa-

ran sulphate proteoglycans is not only necessary for

receptor dimerization but triggers autophosphorylation

of several intracellular domain tyrosine residues. These

phosphotyrosines either stimulate receptor protein

tyrosine kinase activity or serve as docking sites for

downstream signalling molecules [141,163]. Although a

direct functional anosmin-1 interaction with the FGFR

signaling pathway can be envisaged, facilitating signal

transduction, there is no direct proof of this at present.

Although very little is known about the downstream

FGFR pathway, Six3 and Irx3 enable switches in the

signal transduction pathways that control rostral fore-brain development [88]. Moreover, PLCc-dependentand Raf-dependent signaling pathways downstream of

FGFR are both involved in the distinct aspects of the

CNS patterning [201]. Furthermore, the Rho family of

small guanosine triphosphatases family, including,

Rho, Rac, and Cdc42 permit the generation of membra-

nous extensions, thereby controlling filopodial and lam-

ellipodial activity in neuronal growth cones andcontrolling cell polarity towards or away from different

sources of chemoattractive/chemorepellent molecules

[68,130]. The clinical relevance of this interaction is rein-

forced by the observation that synkinesis, a defect

thought to be due to aberrant corticospinal pathway

generation, is also demonstrable in some pedigrees af-

fected by AKS.

8. Conclusion

GnRH neuronal and olfactory axonal migrations are

interlinked in early embryogenesis. Disturbance of anos-

min-1 function does not appear to perturb these early

migratory events, but rather leads to failed OB develop-

ment, with the secondary consequence, at least in man,of GnRH neuronal arrest at the level of the cribriform

plate. The earlier migratory pathway appears regulated

by a number of key molecules, which represent potential

candidate genes disrupted in other euosmic forms of

HH, and include the ephrins, DCC and netrins, semaph-

orins, and NELF, as well as ARK. Not only are these

molecules likely to play a role in migration, but they

may also play a role in GnRH terminal differentiation,


and exert an anti-apoptotic effect. The later migratory

route appears to be influenced by the activity of the

anosmin-1-FGFR1 pathways, which if disrupted, leads

to failed OB development and arrest of GnRH neuronal

migration. Direct evidence for an anosmin-1-FGFR1

interaction is lacking at present. The distribution ofanosmin-1 immunoreactivity in the fetal brain however

suggests that this system may also play a role in GnRH

neurones ontogeny affecting the outgrowth of their neu-

rites and their phenotype.

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