The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

Reference

Adams
B.
,
Dorfler
P.
,
Aguzzi
A.
,
Kozmik
Z.
,
Urbánek
P.
,
Maurer-Fogy
I.
,
Busslinger
M.
(
1992
)
Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis.
Genes Dev
6
,
1589
1607
Alvarado-Mallart
R.-M.
(
1993
)
Fate and potentialities of the avian mesencephalic/metencephalic neuroepithelium.
J. Neurobiol
24
,
1341
1355
Asano
M.
,
Gruss
P.
(
1992
)
Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development.
Mech. Dev
39
,
29
39
Gruss
P.
(
1991
)
The molecular basis of the undulated/Pax-1 mutation.
Cell
66
,
873
884
Chalepakis
G.
,
Stoykova
A.
,
Wijnholds
J.
,
Tremblay
P.
,
Gruss
P.
(
1993
)
Pax: gene regulations in the developing nervous system.
J. Neurobiol
24
,
1367
1384
Condie
B. G.
,
Capecchi
M. R.
(
1994
)
Mice with targeted disruptions in the paralogues genes hoxa-3 and hoxd-3 reveal synergistic interactions.
Nature
370
,
304
307
Conlon
R. A.
,
Rossant
J.
(
1992
)
Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 gene in vivo.
Development
116
,
357
358
Czerny
T.
,
Busslinger
M.
(
1995
)
DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5).
Molec. Cellular Biol
15
,
2858
2871
Czerny
T.
,
Schaffner
G.
,
Busslinger
M.
(
1993
)
DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site.
Genes Dev
7
,
2048
2061
Davis
C. A.
,
Holmyard
D. P.
,
Miller
K. J.
,
Joyner
A. L.
(
1991
)
Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.
Development
111
,
287
298
Davis
C. A.
,
Joyner
A. L.
(
1988
)
Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development.
Genes Dev
2
,
1736
1744
Davis
C. A.
,
Noble-Topham
S. E.
,
Rossant
J.
,
Joyner
A. L.
(
1988
)
Expression of the homeo box containing gene En-2 delineates a specific region of the developing mouse brain.
Genes Dev
2
,
361
371
Devereux
J.
,
Haeberli.
P.
,
Smithies
O.
(
1984
)
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res
12
,
387
395
DiNardo
S.
,
O'Farrell
P. H.
(
1987
)
Establishment and refinement of segmental pattern in the Drosophila embryo: spatial control of engrailed expression by pair-rule genes.
Genes Dev
1
,
1212
1225
DiNardo
S.
,
Sher
E.
,
Heemskerk-Jongens
J.
,
Kassis
J. A.
,
O'Farrell
P. H.
(
1988
)
Two-tiered regulation of spatially patterned engrailed gene expression during Drosophila embryogenesis.
Science
332
,
604
609
Emerson
B. M.
,
Lewis
C. D.
,
Felsenfeld
G.
(
1985
)
Interaction of specific nuclear factor with the nuclease-hypersensitive region of the chick adult beta-globin gene: nature of the binding.
Cell
41
,
21
30
Epstein
J.
,
Cai
J.
,
Glaser
T.
,
Jepeal
L.
,
Maas
R.
(
1994
)
Identification of a Pax paired domain recognition sequence and evidence for DNA-dependent conformational changes.
J. Biol. Chem
269
,
8355
8361
Gruss
P.
,
Walther
C.
(
1992
)
Pax in development.
Cell
69
,
719
722
Hanks
M.
,
Wurst
W.
,
Anson-Cartwright
L.
,
Auerbach
A. B.
,
Joyner
A. L.
(
1995
)
Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2.
Science
269
,
679
682
Heemskerk
J.
,
DiNardo
S.
,
Kostriken
R.
,
O'Farrell
P. H.
(
1991
)
Multiple modes of engrailed regulation in the progression towards cell fate determination.
Nature
352
,
404
410
Ingham
P. W.
,
Martinez-Arias
A.
(
1992
)
Boundaries and fields in early embryos.
Cell
68
,
221
235
Joyner
A. L.
(
1996
)
Engrailed, Wnt and Pax regulation of midbrain-hindbrain development.
Trends Genet
12
,
15
20
Joyner
A. L.
,
Herrup
K.
,
Auerbach
A.
,
Davis
C. A.
,
Rossant
J.
(
1991
)
Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.
Science
251
,
1239
1243
Joyner
A. L.
,
Guillemot
F.
(
1994
)
Gene targeting and development of the nervous system.
Current Opinion in Neurobiology
4
,
37
42
Joyner
A. L.
,
Kornberg
T.
,
Coleman
K. G.
,
Cox
D. R.
,
Martin
G. R.
(
1985
)
Expression during embryogenesis of a mouse gene with sequence homology to the Drosophila engrailed gene.
Cell
43
,
29
37
Keller
S. A.
,
Jones
J. M.
,
Boyle
A.
,
Barrow
L.
,
Killen
P. D.
,
Green
D. G.
,
Kapousta
N. V.
,
Hithcock
P. F.
,
Swank
R. T.
,
Meisler
M. H.
(
1994
)
Kidney and retinal defects (Krd), a transgene-induced mutation with a deletion of mouse chromosome 19 that inclues the Pax-2 locus.
Genomics
23
,
309
320
Kothary
R.
,
Clapoff
S.
,
Darling
S.
,
Perry
M. D.
,
Moran
L. A.
,
Rossant
J.
(
1989
)
Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice.
Development
105
,
707
714
Krauss
S.
,
Johansen
T.
,
Korzh
V.
,
Fjose
A.
(
1991
)
Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis.
Development
113
,
1193
1206
Krauss
S.
,
Maden
M.
,
Holder
N.
,
Wilson
S. W.
(
1992
)
Zebrafish pax[b] is involved in the formation of the midbrain-hindbrain boundary.
Nature
360
,
87
89
Logan
C.
,
Hanks
M. C.
,
Noble-Tophan
S.
,
Nallainathan
D.
,
Provart
N. J.
,
Joyner
A. L.
(
1992
)
Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.
Dev. Genetics
13
,
345
358
Logan
C.
,
Khoo
W. K.
,
Cado
D.
,
Joyner
A. L.
(
1993
)
Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts.
Development
117
,
905
916
Maulbecker
C. C.
,
Gruss
P.
(
1993
)
The oncogenic potential of Pax genes.
EMBOJ
12
,
2361
2367
McGinnis
W.
,
Krumlauf
R.
(
1992
)
Homeobox genes and axial patterning.
Cell
68
,
283
302
McMahon
A. P.
,
Bradley
A.
(
1990
)
The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain.
Cell
62
,
1073
1085
McMahon
A. P.
(
1992
)
The Wnt family of developmental regulators.
Trends Genet
8
,
236
242
McMahon
A. P.
,
Joyner
A. L.
,
Bradley
A.
,
McMahon
J. A.
(
1992
).
The midbrain-hindbrain phenotype of Wnt-1/Wnt-1 mice results from stepwise deletion of engrailed -expressing cells by 9.5 day post-coitum.
Cell
69
,
581
595
Millen
K. J.
,
Wurst
W.
,
Herrup
K.
,
Joyner
A. L.
(
1994
)
Abnormal embryonic cerebellar development and patterning of postnatal foliation in two mouse Engrailed-2 mutants.
Development
120
,
695
706
Morrissey
D.
,
Askew
D.
,
Raj
L.
,
Weir
M.
(
1991
)
Functional dissection of the paired segmentation gene in Drosophila embryos.
Genes Dev
5
,
1684
1696
Noll
M.
(
1993
)
Evolution and role of Pax genes.
Current Opinion in Genetics Dev
3
,
595
605
Nusse
R.
,
Varmus
H. E.
(
1992
)
Wnt -genes.
Cell
69
,
1073
1087
Perimon
N.
(
1994
)
The genetic basis of patterned baldness in Drosophila.
Cell
76
,
781
784
Puelles
L.
,
Rubenstein
L. R.
(
1993
)
Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization.
Trends Neurosci
16
,
472
479
Puschel
A. W.
,
Westerfield
M.
,
Dressler
G. R.
(
1992
)
Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish.
Mech. Dev
38
,
197
208
Rowitch
D. H.
,
McMahon
A. P.
(
1995
)
Analysis of Wnt-1, En-1, En-2, Pax-2 and Pax-5 expression in the embryonic mouse brain suggests an evolutionarily conserved pathway of gene activation.
Mech. Dev
52
,
3
8
Sanyanusin
P.
,
Schimmenti
L. A.
,
McNoe
L. A.
,
Ward
T. A.
,
Pierpont
M. M.
,
Sullivan
M. J.
,
Dobyns
W. B.
,
Eccles
M. R.
(
1995
)
Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux.
Nature Genetics
9
,
358
364
Thomas
K. R.
,
Capecchi
M. R.
(
1990
)
Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellum development.
Nature
346
,
847
850
Treisman
J.
,
Harris
E.
,
Desplan
C.
(
1991
)
The paired box encodes a second DNA-binding domain in the paired homeo domain protein.
Genes Dev
5
,
594
604
Urbánek
P.
,
Wang
Z.-Q.
,
Fetka
I.
,
Wagner
E. F.
,
Busslinger
M.
(
1994
)
Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP.
Cell
79
,
901
912
Walther
C.
,
Guenet
J.-L.
,
Simon
D.
,
Deutsch
U.
,
Jostes
B.
,
Goulding
M. D.
,
Plachov
D.
,
Balling
R.
,
Gruss
P.
(
1991
)
Pax: A murine multigene family of paired box-containing genes.
Genomics
11
,
424
434
Wurst
W.
,
Auerbach
A. B.
,
Joyner
A. L.
(
1994
)
Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
Development
120
,
2065
2075
Zannini
M.
,
Francis-Lang
H.
,
Plachov
D.
,
Lauro
R. D.
(
1992
)
Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.
Molec. Cell. Biol
12
,
4230
4241
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