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The effect of the ret. mutation on the normal
of the central
Division at Developmental
The c.,et proto-oncogene,
Neurobiology,
and parasympathetic
Came\:a MARCOS and Vassilis PACHNIS
Nat!~..nal Institute for Medical Research,
a member of the receptor tyrosine
kinase (RTK)superfamilyI plays a critical role in tumour formation
in t/16 d~weloping
spinal
cor<=:
development
systems
The Ridgeway,
London
~
I
and embryonic organogenesis. Germ-/ine mutations have been
identified in patients with multiple endocrine neoplasia types 2A and
28 (characterized
by meduHary thyroid
carciror,1a
and
pheochromocytoma) and familial r,lBduilary carcinoma (FMTC), 8S
weU as in patients with congenital megacolon (Hjr~hprung's
disease, characterized by abser.ce of a subset of enh,ric ganglia).
Consistent with these studies In humans, targeted ipactivation of
the murine c.,et locus (ref.) results in kidney hypodj.'spJasia and
agenesis of the enteric and superior cervical ganglia (Sr.huchardt et
aI., 1994: D\;-bec et ai, 19948). A number of laboratories have
recently established that glial cell line-derived neurotrophic factor
(GDNF), a distant ~
of the TGF-~ family of growth 'actors, is a
functional ligand of the Rat receptor. Given the po:ent neurotrophic
activity of GOOFon midbrain dcpaminergic and motor n~ulons of the
central nervous system (CNS), it was of interest to examine the
potential in ~ivorole of c-retin the develo~1)9nt of the C~S.
c-ret ex Dression in the CNS
To investigate the potential role of c-re~ function in the CNS, we
first characterized the d;strtbution of c-ret rrRNA in this system.
Our results revealed that c-ret ~N.~, in the CNS is expressed
predominantly
nervous
NW7 1M,
UK
B
,
~r
r
~-
c
otdITlfl.ll;
~D
.f
--
,.~.XII
.,to ,.
d ,
.V1..
I
.A!
and brain s:~ln (Fig 1).
In the spina! cord, c-ret expression was. mai, Ity dp+ected in the
fig 1. c-rerexpresslon In transverse (A, B, E) and coronal (C, 0, F) CNS sections.
lateral and medial somatic motor columns, and in the visceral motor (A) E13.5 mouse embryo. Spinal motor neurons (mns) and dorsal root ganglia j()"g)
(B) Mesencephalic region of a neonate. Expression is
column. Within the bra!fl stem, high levels of co£etexpression were expressc-l8l1ransatpts.
detected In the vemral tegmental area (ViA) and the OOJIofooIt>'nucleus (III). ~q
observed in cranial motor and sensory nuclei throughout
embryonic,
Caudal brain stem region 01a neona1e. Expression Is observed in the dorsal lTIOtor
perinatal 2nd adult deve:opment. I:~crania! motor neurons, cofet is nucleus 01the vagus (dmnx),hypoglossal nucleus (XU), nucleus arnblguos (pm),
spII1B.ltrigeminal nudeus (Soli); .retbJlar format'.on (rf) and A1JC1 8dreoer~ ceil
primarily expressed in branchiomr:or and SOfT1.-;.tiC
motor nuclei and, groop. (0) Pontine region 01 a neonate. The locus C8fuleus (Ie), ladal (IIII),
abducens (Vi)an~ mesencephalic tngttmlnal (MMV) nuclei express c-lBt. (E a.;d F)
to a lesser extent, in Itjsceral motor nuclei. In the cranial sensory
E12.5 and neonatal midbrain region respectlvety. c-rot staining IS observed in the
system, c.,etoxpression is restricted to the general somatic
VTA and substantia nigra (SN).
alerent column, including the neural C~f..lt~erived mesencephaF::
trigeminaJ nucleus and the spinal trigeminal nucleus.
c.,et transcripts were also observed in the reticular formajon arid the
I
..)(tJ
XII
.
main monoaminergic systems of the brain stem: serotonergic Md
VI
catecholaminergic (dopaminergic, noradrenergic and adrenergic). Among
these systems, the highest levels of c-ret expression were detected ir.
the developing midbrain dopaminergic neurons, namely the sl:bstamia
ilv
nigra (SN) and ventral tegmAntal area (VTA).
.
~::... The Q\jS of ret- homozvaousmice
D
To gain insight into the role of c-ret function in ihe CNS, ':19 examined
the development of c.,et
positive cell ;)roups in the O-lS of n;thomozygou& neonatal mice. For this purpose, we emr;;'Jyed in ~itu
''':.VII
hybridization and immunochemical methods usirl9 r variety of
established neuronal markers (Fig 2).
A-
B.
4#:
#'
.
c
I
.
To examine
heterozygous
w-
,.f
i
L.
VT'
'
a"',
,";:' .'.
'~SN
~"~
:,
~\
.
+I~'
L
as a specific
$0
VTA
*'
.*
of midbrain d.Jpam;ilergic
r~311rons in retmice, we used tyrosine hydroxylase
(TH)
rnari<er for this group of cells.
immunoreactivity,
these cell groups
F
...
the development
and homozygous
As revealed by TI-i
a normal complement
of dopaminergic
neurons
was observed.
Moreover, thEtir axonal projections
in
to
the striatum also appeared normal.
To study of the elect of the ret- mutation M motor neurons, we
examined the expression
pattern of i5/-1 rrRNA in the CNS by in situ
hybridization.OUrresults indicate similarnumbersof dilerentiated motor
-I neurons are present in the brain stem and spinal cord off.':JrFig2. 151-' and THexpression Inthe braInstem of E16.5 (8l. heterozygous (+/-) heterozygous and homozygous mice.
(VI), facta! (VII) Overall, our findings revealed that none of the CNS populations
and homozygous (-1-) mice. (A-D) The trigeminal (V). abdu~s
and hypoglossal (XII) motor nude! are not affected in -1-mice. (E, F) A normal
complemenl 01the VTA and SN is seen in _/-mic.e.
analysed
..
are either absent or display
.
any gross morphologIcal
I3RS
De\'e/op1l1enr({/
neurohi%gy
abnormalities in ret-homozygous mice.
ParasvmDathetic nervous svstem
Given the dramatic elect of the ret- mutation in the enteric
nervous system (ENS), 'M:Iwished to examine the function of c,retin other parasympatkftic ganglia. Higllevels of expression
were detected normally in the ciliary, pterygopalatine I
submandibular, otic, car.1iac and pelvic ganglia. Furthermore,
our findings revealed c~et function is required for the
development of at least a subset of parasympathetic ganglia.
Using SCG10 and Phox-2 as specific cellular markers, we have
observed the ciliary and ptelygopS:atine ganglia do not develop
in ret- mut8J1tmice (fig3).
I
Pt~
Conclusions
['i
The spatiotemporaJ dl3tribution of c~et transcripts in the
I
developing mouse a.JS :~ complex and comprises a variety of
..:
i
+/-t-I
cell populations of distinct emblyonic origins. Overall, the
highest levels of c-rt;-t rrI1.NAue detected in two cell
populations highly respor."iive to GDNF: midbrain dopaminergic
neurons and motor neurons. c-ret expression in GOOFresponsive tissues
suggests
the GDNF-ret signalling
mechanism is involved in the specification and/or maintenance
of these neuronal popul3tions.
Ar:dlysis of ret-homozygous mice revealed that c-ret function
is not requ:.ect for the d~v()lopment of catecholamlnergic, motor
".-J~j
or serotonergk neurops during embryonic development.
Similarly, GONF-defici.~:1tembryonic mice also show no FIg 3. SCG10 expression In aanlal parasympathetic ~nglla of rer-heU¥."ozygou~
:.tainlng,
apparent morphologies! abnormalities in motor Of midbrain and homozygous neonatal mice. (A-O) As Indlcatcod by SCG10
(pt) ar.1 dUary
homozygous
mice lack a normal complement
01 ptel'yOOpal8tine
dopamir.ergic neurons (~lIOOfeet ai, 1996; Pichel at ai, 1996; ganglia. Abbrevlallons: e, eye: 011, olfactory eptthelium.
Sanchez e: aJ. 1996; ).
These findings s(rongl.': contradict the elects of exogenously supplied GDNFon rrndbrain dopaminergic neurons mrJtor neurons i1 vivo
and in vitro. To reconcile these apparently contradictory findings, two possibilities are being suggested. First, the GDNF-Ret ligandreceptor siQnallingsystem is required for the development of specific CNS populations after birth and not during embryogenesis. Given
that GDNF and c-ret null mice die in their first day of Hfe, the functional analysis of these genes thus far is limited to embryonic
devdlopment.To study the reMof GDNFand/or c-retinthe postnatal and adult CNS,the creooxP system could be used to cisttJptthese
lo(~ wrthpreciClespatiotemporaJcontro\' A second alternative possibility for the lack of a CNS phenotype in ret- mL."!80tsis that
compensation occurs by means of other neurotrophin-neurotrophin receptor signalling system(s). tt is possith~ that thl' GONF-Ret
signalling system acts synergistically with another ligand-receptor complex on neuronal survival in the CNS during normal development.
A
-
Suct! complex could compensate
!'IT-3 or NT-4I5,
and th":lir receptors,
for the loss of GONF and c-retfunction.
may playa
role in such
For in!';~ance, known neurotrophic
factors,
such as CNTF, BOtJF,
functional
redundancy.
Also, it is possible
that other GDNF-like or Ret-like
molecule might compensate for the loss of the GDNF-Ret signalling pathway. However, no such molecu~s have loeen reportej $v far.
',Ve have shown that c-r,'tfunction is r(lquired!or 1he neural development of a subset of parasympathetic
neurons, name~1 the ciliary and
pteryr;opalatin&
ganglia. These gangfla are derived from cranial neural crest. Interestingly, the other two main gloutJs Jf autonomic neurons:
by the ret-mutation, the ENS and superior cervical ganglia (SCG), Sie also cranial neural crest derivatives (SCluchardt et
aI., 1994: Durbec et a!., 19968). These findings suggest c~etfunction
is specifically
required fer the survival and/or differentiation
of
autonomic. nourons of cranial Mural crest origin and that the development
of autonomic
neurons derived from trunk neural crest is
ir:d~pendent of r:-retfu:1ction.
alsc affected
Fele.-ences
Durt,ec, P.L, Larsson Blomtel';), LB., Schuchardt, A..,Cost lIfItinJ, F., and Pachnis, V. (19968) Common orh;1inand deWlopmental dependmce
ano sympathetIc neuroblast~ DevelOpment 122:J.49, 358 .
on c-nlt of MJ':>setsof eoter;c
Durbec. P., Marcos, Gut\efre.l, C.V., ~llkenn)', C., Gi1Qoriu, M., WartlOwaara, K., Suvanto, P., Smith, D., Pond&.', B., Costantini, F., Saarma, M., Sariola, H., !L".d Pachnis,
v.,
(199.:1:.: GONf 8Igl'.:1l11og 1I'1rOYoj\the Ret reo'tptOf l)1'oslne kinase. Nature 381 :789, 793
Jing, S., Wen ~., Vu, V.,I-:oIst 0.l., Luo, 'f.,I=ang, M., Tamir, R., Antonio S., Hu, z., CuPOIes. A., louis, J.e., Hu, 5., Ahrock, e:h. and fox, d_M. (199(\ GDNF_indul>"CI
activation 01the Rei protein tyrosine kina!Wt Is mediated by GDNFR,a novel receptor tor l;DNF. Cell 85:~113, 1124
Moore, M,W., Klein, R.D., Farinas, I., Sauer, H., Armanin!, M., Phillips, H., Reichardt, LF., Ryan, A.M., CBNef, Moore, K. and Rosenthal, A. (1996) Renel and neuronal
abnormalitIes In mice lacking GONF. Nature 382: 76, 9
Pichel, J.G., Shan, L, Sheng, H.l., Granholm, A.C., Drago, J.. Gmberg, A., Lee, E.J., Huang, SP., Saarma, M., Hofer, B.J., Sat101a, H. and Westphal, H. (1996) D9Iects in
enterIC Innervation ~
kkJney developmMIln
mi08 lacking GOOF. Nature 382: 73, 6
Pachn~!I, V., Mankoo, B.S., 8'":<1Costantini, F. (1993) ExpreSSIon of the c-ret prOCo,oncogene during mouse 6fTlbry0genesis. DeYI!Iopment 119:1005, 1017.
Sanchez,
MP, Silos, Santi~,
I., Fr.~, J., He, B., Ura, S.A. and Barbadd, M. (1996) Renal age')eSls a.<'Idthe absence of enteric neurons In mice lack!n... GOOF. N.rJre
382: 70, 3
SChuchardt, A., D'I'Ji'ltI, ..., ~ar1son Blomberg, L, Costantini, F., and Pachnls, V. (1994) Defects In the kidney ana enteric nervou6 system 04 mice laddng the tyrosine
klna56 receptor Rat. Nator"'! 367:380, 383.
Treana, J.J , Goodman, L, de, S8t'Vage, F., Stone, D.M., Poulsen, K.T., Bec::k,C.D., Gray, C., Armanlni, MP., Pollock, A.A., HeftI, F., Phil:lps, H.S., GOddatd, A., Moofe,
MW., Buj, delIo, A., Davies, A.M., Asai, N., TakahashI. M_,Vancllen, R., Henderson, C_E. and Rosenthal, A. (1996) Characterizallor; of a mu:ticompooent -eceptOf lor
GDNF. NlllUre 382: 80. 3
Trupp, M., A.""as., E., Falnzl:Def, M., Nilsson, A.S., Sieber, B.A., GI1gortou, M., Kilkenny, C., Salazar, Gru9!O, E., Pad1nls, V., Arumae, U., Sari~a, H., Saarma, M., and
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