The development of sympathetic neurons is controlled by a network of transcriptional regulators, including the paired homeodomain proteins Phox2a and Phox2b. To understand the role of Phox2 proteins in more detail, the effect of Phox2 overexpression was analysed in the avian peripheral nervous system. Phox2a expression in neural crest cultures elicited a strong increase in the number of sympathoadrenergic cells. Expression of Phox2a in the chick embryo promoted the generation of additional neurons expressing the noradrenergic marker genes DBH and TH, pan-neuronal genes SCG10 and NF160 and cholinergic genes ChAT and VAChT. Phox2a-induced neurons were found in ectopic locations such as dorsal root ganglia and peripheral nerve. Sympathoadrenergic development could be elicited in cultures of E5 dorsal root ganglia, demonstrating the presence of Phox2a-responsive cells in non-autonomic peripheral ganglia. Phox2b induced ectopic neurons in the chick embryo in the same way as Phox2a. These results show that Phox2 proteins are sufficient to promote sympathetic neuron generation and control, directly or indirectly, the expression of a large number of genes characteristic for sympathetic neurons.

Reference

Baroffio
A.
,
Dupin
E.
,
Le Douarin
N. M.
(
1988
)
Clone-forming ability and differentiation potential of migratory neural crest cells.
Proc. Natl. Acad. Sci. USA
85
,
5325
5329
Christie
D. C.
,
Forbes
M. E.
,
Maxwell
G. D.
(
1987
)
Phenotypic properties of catecholamine-positive cells that differentiate in avian neural crest cultures.
J. Neurosci
7
,
3749
3763
Duff
R. S.
,
Langtimm
C. J.
,
Richardson
M. K.
,
Sieber-Blum
M.
(
1991
)
In vitro clonal analysis of progenitor cell patterns in dorsal root and sympathetic ganglia of the quail.
Dev. Biol
147
,
451
459
Durbec
P. L.
,
Larsson-Blomberg
L. B.
,
Schuchardt
A.
,
Costantini
F.
,
Pachnis
V.
(
1996
)
Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts.
Development
122
,
349
358
Ensini
M.
,
Tsuchida
T. N.
,
Belting
H. G.
,
Jessell
T. M.
(
1998
)
The control of rostrocaudal pattern in the developing spinal cord: specification of motor neurons subtype identity is initiated by signals from paraxial mesoderm.
Development
125
,
969
982
Ernsberger
U.
,
Sendtner
M.
,
Rohrer
H.
(
1989
)
Proliferation and differentiation of embryonic chick sympathetic neurons:effects of ciliary neurotrophic factor.
Neuron
2
,
1275
1284
Ernsberger
U.
,
Patzke
H.
,
Tissier-Seta
J. P.
,
Reh
T.
,
Goridis
C.
,
Rohrer
H.
(
1995
)
The expression of tyrosine hydroxylase and the transcription factors cPhox-2 and Cash-1: Evidence for distinct inductive steps in the differentiation of chick sympathetic precursor cells.
Mech. Dev
52
,
125
136
Ernsberger
U.
,
Patzke
H.
,
Rohrer
H.
(
1997
)
The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation.
Mech. Dev
68
,
115
126
Fauquet
M.
,
Smith
J.
,
Ziller
C.
,
LeDouarin
N. M.
(
1981
)
Differentiation of autonomic neuron precursors in vitro: cholinergic and adrenergic traits in cultured crest cells.
J. Neurosci
1
,
478
492
Frank
E.
,
Sanes
J. R.
(
1991
)
Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus.
Development
111
,
895
908
Fraser
S. E.
,
Bronner-Fraser
M.
(
1991
)
Migrating neural crest cells in the trunk of the avian embryo are multipotent.
Development
112
,
913
920
Goridis
C.
,
Brunet
J.-F.
(
1999
)
Transcriptional control of neurotransmitter phenotype.
Curr. Opin. Neurobiol
9
,
47
53
Groves
A. K.
,
George
K. M.
,
Tissier-Seta
J.-P.
,
Engel
J. D.
,
Brunet
J.-F.
,
Anderson
D. J.
(
1995
)
Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.
Development
121
,
887
901
Guillemot
F.
,
Lo
L.-C.
,
Johnson
J. E.
,
Auerbach
A.
,
Anderson
D. J.
,
Joyner
A. L.
(
1993
)
Mammalian achaete-scute homolog 1 is required forthe early development of olfactory and autonomic neurons.
Cell
75
,
463
476
Heller
S.
,
Finn
T. P.
,
Huber
J.
,
Nishi
R.
,
Geissen
M.
,
Puschel
A. W.
,
Rohrer
H.
(
1995
)
Analysis of function and expression of the chick GPA receptor (GPAR) suggests multiple roles in neuronal development.
Development
121
,
2681
2693
Henion
P. D.
,
Weston
J. A.
(
1997
)
Timing and pattern of cell fate restrictions in the neural crest lineage.
Development
124
,
4351
4359
Hirsch
M. R.
,
Tiveron
M. C.
,
Guillemot
F.
,
Brunet
J. F.
,
Goridis
C.
(
1998
)
Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system.
Development
125
,
599
608
Hughes
S. H.
,
Kosik
E.
(
1984
)
Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors.
Virology
136
,
89
99
Iacovitti
L.
,
Joh
T. H.
,
Albert
V. R.
,
Park
D. H.
,
Reis
D. J.
,
Teitelman
G.
(
1985
)
Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia:studies in vivo and in vitro.
Dev. Biol
110
,
402
412
Kim
H. S.
,
Seo
H.
,
Yang
C. Y.
,
Brunet
J. F.
,
Kim
K. S.
(
1998
)
Noradrenergic-specific transcription of the dopamine-hydroxylase gene requires synergy of multiple cis -acting elements including at least two Phox2a-binding sites.
J. Neurosci
18
,
8247
8260
Le Douarin
N. M.
,
Teillet
M. A.
,
Ziller
C.
,
Smith
J.
(
1978
)
Adrenergic differentiation of cells of the cholinergic ciliary and Remak ganglia in avian embryo after in vivo transplantation.
Proc. Natl. Acad. Sci. USA
75
,
162
184
Le Lievre
C. S.
,
Schweizer
G. G.
,
Ziller
C. M.
,
LeDouarin
N. M.
(
1980
)
Restrictions of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments.
Dev. Biol
77
,
362
378
Lo
L. C.
,
Tiveron
M. C.
,
Anderson
D. J.
(
1998
)
MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity.
Development
125
,
609
620
Lo
L.
,
Morin
X.
,
Brunet
J.-F.
,
Anderson
D. J.
(
1999
)
Specification of neurotransmitter identity by Phox2 proteins in neural crest stem cells.
Neuron
22
,
693
705
Lyon
L. M.
,
Hogan
B. L. M.
,
Robertson
E. J.
(
1995
)
Colocalization of BMP7 and BMP2 RNAs suggest that these factors cooperatively mediate tissue interactions during murine development.
Mech. Dev
50
,
71
83
Mackey
H. M.
,
Payette
R. F.
,
Gershon
M. D.
(
1988
)
Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches.
Development
104
,
205
217
Marusich
M. F.
,
Furneaux
H. M.
,
Henion
P. D.
,
Weston
J. A.
(
1994
)
Hu neuronal proteins are expressed in proliferating neurogenic cells.
J. Neurobiol
25
,
143
155
Morgan
B. A.
,
Fekete
D. M.
(
1996
)
Manipulating gene expression with replication-competent retroviruses.
Meth. in Cell Biology
51
,
185
218
Morin
X.
,
Cremer
H.
,
Hirsch
M.-R.
,
Kapur
R. P.
,
Goridis
C.
,
Brunet
J.-F.
(
1997
)
Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2.
Neuron
18
,
411
423
Morrison
S. J.
,
White
P. M.
,
Zock
C.
,
Anderson
D. J.
(
1999
)
Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells.
Cell,
96
,
737
749
Pattyn
A.
,
Morin
X.
,
Cremer
H.
,
Goridis
C.
,
Brunet
J. F.
(
1997
)
Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis.
Development
124
,
4065
4075
Pattyn
A.
,
Morin
X.
,
Cremer
H.
,
Goridis
C.
,
Brunet
J.-F.
(
1999
)
The homeobox gene Phox2b is essential for the development of all autonomic derivatives of the neural crest.
Nature,
399
,
366
370
Perez
S. E.
,
Rebelo
S.
,
Anderson
D. J.
(
1999
)
Early specification of sensory neuron fate revealed by expression and function of neurogenins in the chick embryo.
Development
126
,
1715
1728
Pomeranz
H. D.
,
Rothman
T. P.
,
Chalazonitis
A.
,
Tennyson
V. M.
,
Gershon
M. D.
(
1993
)
Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin.
Dev. Biol
156
,
341
361
Raible
D. W.
,
Eisen
J. S.
(
1994
)
Restriction of neural crest fate in the trunk of the embryonic zebrafish.
Development
120
,
495
503
Reissmann
E.
,
Ernsberger
U.
,
Francis-West
P. H.
,
Rueger
D.
,
Brickell
P. M.
,
Rohrer
H.
(
1996
)
Involvement of bone morphogenetic proteins-4 and-7 in the specification of the adrenergic phenotype in developing sympathetic neurons.
Development
122
,
2079
2088
Richardson
M. K.
,
Sieber-Blum
M.
(
1993
)
Pluripotent neural crest cells in the developing skin of the quail embryo.
Dev. Biol
157
,
348
358
Robertson
K.
,
Mason
I.
(
1995
)
Expression of ret in the chicken embryo suggests roles in regionalization of the vagal neural tube and somites and in development of multiple neural crest and placodal lineages.
Mech. of Development
53
,
329
344
Rohrer
H.
,
Acheson
A. L.
,
Thibault
J.
,
Thoenen
H.
(
1986
)
Developmental potential of quail dorsal root ganglion cells analyzed in vitro and in vivo.
J. Neurosci
6
,
2616
2624
Sieber-Blum
M.
,
Cohen
A. M.
(
1980
)
Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells.
Dev. Biol
80
,
96
106
Schweizer
G.
,
Ayer-Le Lievre
C.
,
Le Dourain
N. M.
(
1983
)
Restrictions of developmental capacities in the dorsal root ganglia during the course of development.
Cell Different
13
,
191
200
Sextier-Sainte-Claire Deville
F.
,
Ziller
F.
,
Le Douarin
N. M.
(
1994
)
Developmental potential of enteric neural crest-derived cells in clonal and mass cultures.
Dev. Biol
163
,
141
151
Smith
J.
,
Cochard
P.
,
Le Douarin
N. M.
(
1977
)
Development of choline acetyltransferase and cholinesterase activities in enteric ganglia derived from presumptive adrenergic and cholinergic levels of the neural crest.
Cell Different
6
,
199
216
Sommer
L.
,
Shah
N.
,
Rao
M.
,
Anderson
D. J.
(
1995
)
The cellular function of MASH1 in autonomic neurogenesis.
Neuron
15
,
1245
1258
Stocker
K. M.
,
Sherman
L.
,
Rees
S.
,
Ciment
G.
(
1991
)
Basic FGF and TGF-1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos.
Development
111
,
635
645
Swanson
D. J.
,
Zellmer
E.
,
Lewis
E. J.
(
1997
)
The homeodomain protein Arix interacts synergistically with cAMP to regulate expression of neurotransmitter biosynthetic genes.
J. Biol. Chem
272
,
27382
27392
Teitelman
G.
,
Joh
T. H.
,
Grayson
L.
,
Park
D. H.
,
Reis
D. J.
,
Iacovitti
L.
(
1985
)
Cholinergic neurons of the chick ciliary ganglia express adrenergic traits in vivo and in vitro.
J. Neurosci
5
,
29
39
Tiveron
M.-C.
,
Hirsch
M.-R.
,
Brunet
J.-F.
(
1996
)
The expression pattern of the transcription factor Phox2 delineates synaptic pathways of the autonomic nervous system.
J. Neurosci
16
,
7649
7660
Valarche
I.
,
Tissier-Seta
J.-P.
,
Hirsch
M.-R.
,
Martinez
S.
,
Goridis
C.
,
Brunet
J.-F.
(
1993
)
The mouse homeodomain protein Phox2 regulates NCAM promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype.
Development
119
,
881
896
Vogel
K. S.
,
Weston
J. A.
(
1990
)
The sympathoadrenal lineage in avian embryos. I. Adrenal chromaffin cells lose neuronal traits during embryogenesis.
Dev. Biol
139
,
1
12
Wakamatsu
Y.
,
Weston
J. A.
(
1997
)
Sequential expression and role of Hu RNA-binding proteins during neurogenesis.
Development
124
,
3449
3460
Xue
Z. G.
,
Smith
J.
,
LeDouarin
N. M.
(
1985
)
Differentiation of catecholaminergic cells in cultures of embryonic avian sensory ganglia.
Proc. Natl. Acad. Sci. USA
82
,
8800
8804
Yang
C.
,
Kim
H.-S.
,
Seo
H.
,
Kim
C.-H.
,
Brunet
J.-F.
,
Kim
K.-S.
(
1998
)
Paired -like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine-hydroxylase gene.
J. Neurochem
71
,
1813
1826
Zellmer
E.
,
Zhang
Z.
,
Greco
D.
,
Rhodes
J.
,
Cassel
S.
,
Lewis
E. J.
(
1995
)
A homeodomain protein selectively expressed in noradrenergic tissue regulates transcription of neurotransmitter biosynthetic genes.
J. Neurosci
15
,
8109
8120
Zopf
D.
,
Hermans-Borgmeyer
I.
,
Gundelfinger
E. D.
,
Betz
H.
(
1987
)
Identification of gene products expressed in the developing chick visual system:characterization of a middle-molecular-weight neurofilament cDNA.
Genes Dev
1
,
699
708
This content is only available via PDF.