The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3′RARE)/Hoxb1(3′RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3′RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3′RARE) and Hoxa1(null)/Hoxb1(null)double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.

REFERENCES

Barrow
J. R.
,
Capecchi
M. R.
(
1996
)
Targeted disruption of the Hob-2 locus in mice interferes with expression of Hoxb −1 and Hoxb −4.
Development
122
,
3817
3828
Carpenter
E. M.
,
Goddard
J. M.
,
Chisaka
O.
,
Manley
N. R.
,
Capecchi
M. R.
(
1993
).
Loss of Hox -A1 (Hox −1.6) function results in the reorganization of the murine hindbrain.
Development
118
,
1063
1075
Chavrier
P.
,
Zerial
M.
,
Lemaire
P.
,
Almendral
J.
,
Bravo
R.
,
Charnay
P.
(
1988
)
A gene encoding a protein with zinc fingers is activated during G0/G1transition in cultured cells.
EMBO J
7
,
29
35
Chisaka
O.
,
Capecchi
M. R.
(
1991
).
Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene Hox −1.5 [see comments].
Nature
350
,
473
479
Chisaka
O.
,
Musci
T. S.
,
Capecchi
M. R.
(
1992
).
Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox −1.6 [see comments].
Nature
355
,
516
520
Cooke
M.
,
Gould
A.
,
Brand
N.
,
Davies
J.
,
Strutt
P.
,
Shaknovich
R.
,
Licht
J.
,
Waxman
S.
,
Chen
Z.
,
Gluecksohn-Waelsch
S.
, et al. 
(
1995
)
Expression of the zinc-finger gene PLZF at rhombomere boundaries in the vertebrate hindbrain.
Proc. Nat. Acad. Sci. USA
92
,
2249
2253
Cordes
S. P.
,
Barsh
G. S.
(
1994
)
The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor.
Cell
79
,
1025
1034
Couly
G. F.
,
Coltey
P. M.
,
Le Douarin
N. M.
(
1993
)
The triple origin of skull in higher vertebrates: a study in quail-chick chimeras.
Development
117
,
409
429
Crossley
P. H.
,
Martin
G. R.
(
1995
)
The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo.
Development
121
,
439
451
D'Amico-Martel
A.
,
Noden
D. M.
(
1983
)
Contributions of placodal and neural crest cells to avian cranial peripheral ganglia.
Am. J. Anat
166
,
445
468
Dolle
P.
,
Lufkin
T.
,
Krumlauf
R.
,
Mark
M.
,
Duboule
D.
,
Chambon
P.
(
1993
)
Local alterations of Krox-20 and Hox gene expression in the hindbrain suggest lack of rhombomeres 4 and 5 in homozygote null Hox a-1 (Hox −1. 6) mutant embryos.
Proc. Nat. Acad. Sci. USA
90
,
7666
7670
Drucker
B. J.
,
Goldfarb
M.
(
1993
)
Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues.
Mech. Dev
40
,
155
163
Dupe
V.
,
Davenne
M.
,
Brocard
J.
,
Dolle
P.
,
Mark
M.
,
Dierich
A.
,
Chambon
P.
,
Rijli
F. M.
(
1997
)
In vivo functional analysis of the Hox a-1 3retinoic acid response element (3 RARE).
Development
124
,
399
410
Fraser
S.
,
Keynes
R.
,
Lumsden
A.
(
1990
)
Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions.
Nature
344
,
431
435
Frohman
M. A.
,
Boyle
M.
,
Martin
G. R.
(
1990
)
Isolation of the mouse Hox −2. 9 gene; analysis of embryonic expression suggests that positional information along the anterior-posterior axis is specified by mesoderm.
Development
110
,
589
607
Frohman
M. A.
,
Martin
G. R.
,
Cordes
S. P.
,
Halamek
L. P.
,
Barsh
G. S.
(
1993
)
Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr).
Development
117
,
925
936
Gale
E.
,
Prince
V.
,
Lumsden
A.
,
Clarke
J.
,
Holder
N.
,
Maden
M.
(
1996
)
Late effects of retinoic acid on neural crest and aspects of rhombomere.
Development
122
,
783
793
Gaunt
S. J.
,
Blum
M.
,
De Robertis
E. M.
(
1993
)
Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall.
Development
117
,
769
778
Gavalas
A.
,
Davenne
M.
,
Lumsden
A.
,
Chambon
P.
,
Rijli
F. M.
(
1997
)
Control of axon guidance and rostral hindbrain patterning by Hoxa-2.
Development
124
,
3693
3702
Gendron-Maguire
M.
,
Mallo
M.
,
Zhang
M.
,
Gridley
T.
(
1993
)
Hox a-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest.
Cell
75
,
1317
1331
Goddard
J. M.
,
Rossel
M.
,
Manley
N. R.
,
Capecchi
M. R.
(
1996
)
Mice with targeted disruption of Hox b-1 fail to form the motor nucleus of the VIIth nerve.
Development
122
,
3217
3228
Graham
A.
,
Francis-West
P.
,
Brickell
P.
,
Lumsden
A.
(
1994
)
The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest.
Nature
372
,
684
686
Graham
A.
,
Heyman
I.
,
Lumsden
A.
(
1993
)
Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain.
Development
119
,
233
245
Guthrie
S.
,
Lumsden
A.
(
1991
)
Formation and regeneration of rhombomere boundaries in the developing chick hindbrain.
Development
112
,
221
229
Guthrie
S.
,
Prince
V.
,
Lumsden
A.
(
1993
)
Selective dispersal of avian rhombomere cells in orthotopic and heterotopic grafts.
Development
118
,
527
538
Heikinheimo
M.
,
Lawshe
A.
,
Shackleford
G. M.
,
Wilson
D. B.
,
MacArthur
C. A.
(
1994
)
Fgf-8 expression in the post-gastrulation mouse suggests roles in the development of the face, limbs and central nervous system.
Mech. Dev
48
,
129
138
Hunt
P.
,
Guilisano
M.
,
Cook
M.
,
Sham
M.-H.
,
Faiella
A.
,
Wilkinson
D.
,
Boncinelli
E.
,
Krumlauf
R.
(
1991
)
A distinct Hox code for the branchial region of the vertebrate head.
Nature
353
,
861
864
Kessel
M.
(
1993
)
Reversal of axonal pathways from rhombomere 3 correlates with extra Hox expression domains.
Neuron
10
,
379
393
Keynes
R.
,
Krumlauf
R.
(
1994
)
Hox genes and regionalization of the nervous system.
Annu. Rev. Neurosci
17
,
109
132
Keynes
R.
,
Lumsden
A.
(
1990
)
Segmentation and the origin of regional diversity in the vertebrate central nervous system.
Neuron
4
,
1
9
Köntges
G.
,
Lumsden
A.
(
1996
)
Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny.
Development
122
,
3229
3242
Kurihara
Y.
,
Kurihara
H.
,
Suzuki
H.
,
Kodama
T.
,
Maemura
K.
,
Nagai
R.
,
Oda
H.
,
Kuwaki
T.
,
Cao
W. H.
,
Kamada
N.
, et al. 
(
1994
)
Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1 [see comments].
Nature
368
,
703
710
La Rosa
G. J.
,
Gudas
L. J.
(
1988
)
Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox.
Mol. Cell Biol
8
,
3906
3917
Lambert
P. R.
,
Dodson
E. E.
(
1996
)
Congenital malformations of the external auditory canal.
Otolaryngol. Clin. North Am
29
,
741
760
Le Lievre
C. S.
,
Le Douarin
N. M.
(
1975
)
Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos.
J. Embryol. exp. Morph
34
,
125
154
Lufkin
T.
,
Dierich
A.
,
Le Meur
M.
,
Mark
M.
,
Chambon
P.
(
1991
)
Disruption of the Hox −1. 6 homeobox gene results in defects in a region corresponding to its rostral domain of expression.
Cell
66
,
1105
110
Lumsden
A.
,
Keynes
R.
(
1989
)
Segmental patterns of neuronal development in the chick hindbrain.
Nature
337
,
424
428
Lumsden
A.
,
Krumlauf
R.
(
1996
)
Patterning the vertebrate neuraxis.
Science
274
,
1109
1115
Lumsden
A.
,
Sprawson
N.
,
Graham
A.
(
1991
)
Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo.
Development
113
,
1281
1291
Maconochie
M.
,
Nonchev
S.
,
Studer
M.
,
Chan
S.-K.
,
Pöpperl
H.
,
Sham
M.-H.
,
Mann
R.
,
Krumlauf
R.
(
1997
)
Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.
Genes Dev
11
,
1885
1896
Maden
M.
,
Horton
C.
,
Graham
A.
,
Leonard
L.
,
Pizzey
J.
,
Siegenthaler
G.
,
Lumsden
A.
,
Eriksson
U.
(
1992
)
Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.
Mech. Dev
37
,
13
23
Mahmood
R.
,
Kiefer
P.
,
Guthrie
S.
,
Dickson
C.
,
Mason
I.
(
1995
)
Multiple roles for FGF-3 during cranial neural development in the chicken.
Development
121
,
1399
1410
Manley
N. R.
,
Capecchi
M. R.
(
1995
)
The role of Hox a-3 in mouse thymus and thyroid Development.
Development
121
,
1989
2003
Manzanares
M.
,
Cordes
S.
,
Kwan
C.-T.
,
Sham
M. H.
,
Barsh
G. S.
,
Krumlauf
R.
(
1997
)
Segmental regulation of Hox b-3 by kreisler.
Nature
387
,
191
195
Mark
M.
,
Lohnes
D.
,
Mendelsohn
C.
,
Dupe
V.
,
Vonesch
J. L.
,
Kastner
P.
,
Rijli
F. M.
,
Bloch-Zupan
A.
,
Chambon
P.
(
1995
)
Roles of retinoic acid receptors and of Hox genes in the patterning of the teeth and of the jaw skeleton.
Int. J. Dev. Biol
39
,
111
121
Mark
M.
,
Lufkin
T.
,
Vonesch
J. L.
,
Ruberte
E.
,
Olivo
J. C.
,
Dolle
P.
,
Gorry
P.
,
Lumsden
A.
,
Chambon
P.
(
1993
)
Two rhombomeres are altered in Hox a-1 mutant mice.
Development
119
,
319
338
Marshall
H.
,
Nonchev
S.
,
Sham
M. H.
,
Muchamore
I.
,
Lumsden
A.
,
Krumlauf
R.
(
1992
)
Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity [see comments].
Nature
360
,
737
741
McKay
I. J.
,
Muchamore
I.
,
Krumlauf
R.
,
Maden
M.
,
Lumsden
A.
,
Lewis
J.
(
1994
)
The kreisler mouse: a hindbrain segmentation mutant that lacks two rhombomeres.
Development
120
,
2199
2211
Mitchell
P. J.
,
Timmons
P. M.
,
Hebert
J. M.
,
Rigby
P. W.
,
Tjian
R.
(
1991
)
Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.
Genes Dev
5
,
105
10
Moody
S. A.
,
Heaton
M. B.
(
1983
)
Developmental relationships between trigeminal ganglia and trigeminal motoneurons in chick embryos. I. Ganglion development is necessary for motoneuron migration.
J. Comp. Neurol
213
,
327
343
Murphy
P.
,
Hill
R. E.
(
1991
)
Expression of the mouse labial-like homeobox-containing genes, Hox 2. 9 and Hox 1. 6, during segmentation of the hindbrain.
Development
111
,
61
74
Niederlander
C.
,
Lumsden
A.
(
1996
)
Late emigrating neural crest cells migrate specifically to the exit points of cranial branchiomotor nerves.
Development
122
,
2367
2374
Niswander
L.
,
Martin
G. R.
(
1992
)
Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse.
Development
114
,
755
768
Nittenberg
R.
,
Patel
K.
,
Joshi
Y.
,
Krumlauf
R.
,
Wilkinson
D. G.
,
Brickell
P. M.
,
Tickle
C.
,
Clarke
J. D.
(
1997
)
Cell movements, neuronal organisation and gene expression in hindbrains lacking morphological boundaries.
Development
124
,
2297
2306
Noden
D. M.
(
1988
)
Interactions and fates of avian craniofacial mesenchyme.
Development
103
,
121
140
Noden
D. M.
(
1983
)
The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues.
Dev. Biol
96
,
144
165
Nonchev
S.
,
Vesque
C.
,
Maconochie
M.
,
Seitanidou
T.
,
Ariza-McNaughton
L.
,
Frain
M.
,
Marshall
H.
,
Sham
M. H.
,
Krumlauf
R.
,
Charnay
P.
(
1996
)
Segmental expression of Hox a-2 in the hindbrain is directly regulated by Krox-20.
Development
122
,
543
554
Pöpperl
H.
,
Bienz
M.
,
Studer
M.
,
Chan
S.-K.
,
Aparicio
S.
,
Brenner
S.
,
Mann
R. S.
,
Krumlauf
R.
(
1995
)
Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent on exd/pbx.
Cell
81
,
1031
1042
Rijli
F. M.
,
Mark
M.
,
Lakkaraju
S.
,
Dierich
A.
,
Dolle
P.
,
Chambon
P.
(
1993
)
A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hox a-2, which acts as a selector gene.
Cell
75
,
1333
1349
Rivera-Perez
J. A.
,
Mallo
M.
,
Gendron-Maguire
M.
,
Gridley
T.
,
Behringer
R. R.
(
1995
)
Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development.
Development
121
,
3005
3012
Ruberte
E.
,
Friederich
V.
,
Morriss-Kay
G.
,
Chambon
P.
(
1992
)
Differential distribution patterns of CRABP I and CRABP II transcripts during mouse embryogenesis.
Development
115
,
973
987
Schneider-Maunoury
S.
,
Seitanidou
T.
,
Charnay
P.
,
Lumsden
A.
(
1997
)
Segmental and neuronal architecture of the hindbrain of Krox-20 mouse mutants.
Development
124
,
1215
1226
Schneider-Maunoury
S.
,
Topilko
P.
,
Seitandou
T.
,
Levi
G.
,
Cohen-Tannoudji
M.
,
Pournin
S.
,
Babinet
C.
,
Charnay
P.
(
1993
)
Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain.
Cell
75
,
119
214
Schörle
H.
,
Meier
P.
,
Buchert
M.
,
Jaenisch
R.
,
Mitchell
P. J.
(
1996
)
Transcription factor AP-2 essential for cranial closure and craniofacial development.
Nature
381
,
235
238
Sechrist
J.
,
Serbedzija
G. N.
,
Scherson
T.
,
Fraser
S. E.
,
Bronner-Fraser
M.
(
1993
)
Segmental migration of the hindbrain neural crest does not arise from its segmental generation.
Development
118
,
691
703
Seitanidou
T.
,
Schneider-Maunoury
S.
,
Desmarquet
C.
,
Wilkinson
D. G.
,
Charnay
P.
(
1997
)
Krox-20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain.
Mech. Dev
65
,
31
42
Sham
M. H.
,
Vesque
C.
,
Nonchev
S.
,
Marshall
H.
,
Frain
M.
,
Gupta
R. D.
,
Whiting
J.
,
Wilkinson
D.
,
Charnay
P.
,
Krumlauf
R.
(
1993
)
The zinc finger gene Krox20 regulates Hox B2 (Hox 2. 8) during hindbrain segmentation.
Cell
72
,
183
196
Studer
M.
,
Lumsden
A.
,
Ariza-McNaughton
L.
,
Bradley
A.
,
Krumlauf
R.
(
1996
)
Altered segmental identity and abnormal migration of motor neurons in mice lacking Hox b-1.
Nature
384
,
630
634
Studer
M.
,
Gavalas
A.
,
Marshall
H.
,
Ariza-McNaughton
L.
,
Rijli
F. M.
,
Chambon
P.
,
Krumlauf
R.
(
1998
)
Genetic interactions between Hoxa1 and Hoxb1 reveal additional roles in regulation of early hindbrain patterning.
Development
125
,
1025
1036
Swiatek
P. J.
,
Gridley
T.
(
1993
)
Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20.
Genes Dev
7
,
2071
2084
Taneja
R.
,
Thisse
B.
,
Rijli
F. M.
,
Thisse
C.
,
Bouillet
P.
,
Dolle
P.
,
Chambon
P.
(
1996
)
The expression pattern of the mouse receptor tyrosine kinase gene MDK1 is conserved through evolution and requires Hox a-2 for rhombomere-specific expression in mouse embryos.
Dev. Biol
177
,
397
412
Trainor
P. A.
,
Tam
P. P.
(
1995
)
Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches.
Development
121
,
2569
2582
Vesque
C.
,
Maconochie
M.
,
Nonchev
S.
,
Ariza-McNaughton
L.
,
Kuroiwa
A.
,
Charnay
P.
,
Krumlauf
R.
(
1996
)
Hox b-2 transcriptional activation in rhombomeres 3 and 5 requires an evolutionarily conserved cis-acting element in addition to the Krox-20 binding site.
EMBO J
15
,
5383
5396
Wilkinson
D. G.
(
1995
)
Genetic control of segmentation in the vertebrate hindbrain.
Perspect. Dev. Neurobiol
3
,
29
38
Wilkinson
D. G.
,
Bhatt
S.
,
Chavrier
P.
,
Bravo
R.
,
Charnay
P.
(
1989
)
Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse.
Nature
337
,
461
464
Wilkinson
D. G.
,
Bhatt
S.
,
Cook
M.
,
Boncinelli
E.
,
Krumlauf
R.
(
1989
)
Segmental expression of Hox −2 homoeobox-containing genes in the developing mouse hindbrain.
Nature
341
,
405
409
Wilkinson
D. G.
,
Peters
G.
,
Dickson
C.
,
McMahon
A. P.
(
1988
)
Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.
EMBO J
7
,
691
695
Wingate
R. J.
,
Lumsden
A.
(
1996
)
Persistence of rhombomeric organisation in the postsegmental hindbrain.
Development
122
,
2143
2152
Yamada
G.
,
Mansouri
A.
,
Torres
M.
,
Stuart
E. T.
,
Blum
M.
,
Schultz
M.
,
De Robertis
E. M.
,
Gruss
P.
(
1995
)
Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death.
Development
121
,
2917
2922
Zhang
J.
,
Hagopian-Donaldson
S.
,
Serbedzija
G.
,
Elsemore
J.
,
Plehn-Dujowich
D.
,
McMahon
A. P.
,
Flavell
R. A.
,
Williams
T.
(
1996
)
Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2.
Nature
381
,
238
241
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