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First published online May 30, 2007
doi: 10.1242/10.1242/dev.000620
,
1 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine,
New York University School of Medicine, 540 First Avenue, New York, NY 10016,
USA.
2 Department of Cell Biology, New York University School of Medicine, 540 First
Avenue, New York, NY 10016, USA.
3 Department of Physiology and Neuroscience, New York University School of
Medicine, 540 First Avenue, New York, NY 10016, USA.
Author for correspondence (e-mail:
joynera{at}mskcc.org)
Accepted 11 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cerebellum, Foliation, Tectum, Patterning, En1, En2, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zervas et al., 2005).
Expression of the engrailed transcription factors (En1 and En2) before E13 is
regulated by Fgf8, and En1/2 are crucial for mes/r1 development. It has been
challenging to determine the full spectrum of functions of each En gene in
mouse because there is an early loss of the mes/r1 in En1 mutants,
and the two genes have overlapping functions. It is also unclear whether the
two En proteins have equivalent functions in brain development.
Following specification of the mes/r1 region during neural tube closure,
the mouse tectum and cerebellum (Cb) develop from the dorsal mes and r1,
respectively (Zervas et al.,
2005). The tectum of the Mb forms as a layered structure that is
divided morphologically and functionally into the anterior superior colliculus
and posterior inferior colliculus that process visual and auditory
information, respectively. Although expansion of the tectum along the A-P axis
is tightly linked to the level of isthmic organizer signaling, the molecular
basis of differential allocation of the inferior and superior colliculi is not
understood. The Cb is the center for motor control. Differentiated cells of
the mouse Cb begin to be generated at E10.5 and form a multi-laminar structure
consisting of the deep nuclei surrounded by a dense layer of granule cells, a
monolayer of Purkinje cells and an outer molecular layer. The granule cell
precursors form a proliferative external granule layer at E13.5 and then
migrate past the Purkinje cell layer to form the inner granule layer (IGL)
from birth until postnatal day (P) 14. Beginning at E17.5, fissures form in a
stereotyped manner and generate a highly foliated Cb. In terms of how early
A-P patterning could influence the final structure of the Cb, it is important
to note that a morphogenetic rotation of dorsal r1 transforms the A-P axis of
r1 into the medial-lateral (M-L) axis of the Cb primordium by E12.5
(Sgaier et al., 2005) (see
Fig. 8). Globally, the adult Cb
is subdivided into a medial vermis and two lateral hemispheres, with the
vermis divided along the A-P axis by 8-10 folia in different inbred mouse
strains (referred to as I-X) and the hemispheres divided by 6 folia
(Larsell, 1952). Preservation
of the general pattern of folia across mammals suggests that there is an
evolutionarily conserved genetic program that patterns folia of the Cb
(Altman and Bayer, 1997;
Herrup and Kuemerle,
1997).
The mouse En1 and En2 genes provide a unique tool for
gaining access to the genetic regulation of Cb and tectum patterning. The
dynamic expression patterns of the En genes (see
Fig. 1) and their mutant
phenotypes reflect each successive stage of Cb and tectum development
(Joyner, 1996). En1
is first expressed in the mes/r1 at E8.5, 
12 hours before En2,
and is later expressed in the absence of En2 in a number of other
tissues. En1-null mutant mice die at birth and have an almost
complete deletion of the Mb and Cb owing to tissue loss by E9.5
(Wurst et al., 1994), which is
caused, at least in part, by cell death
(Chi et al., 2003). Thus,
En1 is required for the initial establishment of the mes/r1 region.
By contrast, En2-null mutants have a mild phenotype - they are viable
and have defects limited to growth of the Cb and patterning of particular
folia (Joyner et al., 1991;
Millen et al., 1994). An
overlap in En gene function has been demonstrated by the complete
absence of the tectum and Cb in En1;En2 double mutants
(Liu and Joyner, 2001;
Simon et al., 2004), and a
rescue of the En1 mutant brain phenotype when En1 is
replaced with En2 using gene targeting
(Hanks et al., 1995).
Surprisingly, we found that Drosophila en also can rescue the
En1 mutant brain defects in knock-in mouse mutants, although
en cannot rescue other defects
(Hanks et al., 1998). An
important question is whether En1 has any later roles in tectum and
Cb patterning, as has been suggested by the Cb phenotype of En1-null
mutants that survive on a C57BL/6 genetic background
(Bilovocky et al., 2003), and
the degree to which such functions overlap with En2.
In order to study the temporal requirement for En1 in mes/r1
development, we generated a conditional mutant allele of En1. We find
that if En1 is removed at E9, only the posterior tectum is
depleted, and two copies of En2 are required to sustain Cb
development in these conditional En1 mutants. We next compared the
function of Drosophila and mouse En proteins in the mouse brain using
a sensitive genetic assay. We provide evidence that En2 is more potent at
supporting Cb development than En1, and demonstrate that Drosophila
En cannot rescue the En1 mutant brain defects in the absence of
endogenous En2. Curiously, our analysis of knock-in mutants and En1/2
double-null mutants uncovered that both genes are preferentially required in
particular functional domains of the tectum and cerebellum. We propose an `En
code' that divides the tectum and Cb into functional systems based on the dose
of En required for the development of each domain.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The En1flox targeting construct was linearized with
SalI and electroporated into W4 embryonic stem (ES) cells
(Auerbach et al., 2000) as
described previously (Matise et al.,
2000). Clones were screened by Southern blot analysis using
5' external and 3' internal probes to identify targeted clones
(see Fig. S1 in the supplementary material). One positive clone was obtained
and injected into C57BL/6 blastocysts to generate ES cell chimeric mice
(Papaioannou and Johnson,
2000). Chimeric mice were mated with Black Swiss mice to generate
En1flox-neo/+ mice. The neo cassette was removed
by mating En1flox-neo/+ mice with hACTB-Flpe mice
(Rodriguez et al., 2000),
which expresses Flpe broadly under the control of the human ß-actin
promoter. The wild-type (337 bp) and En1flox (380 bp)
alleles were detected by PCR with the following primers: En1flox1A,
5'-GCCAAACTGCTTACGACCG-3'; En1flox1B,
5'-TGGGTGGGTAGAGAAGAGGC-3'.
Mes/r1-specific En1 conditional mutant mice (En1flox/Cre) were generated by crossing En1flox/+ mice with En1Cre/+ mice. En1Cre/Cre and En1flox/Cre mice were generated within the same litter by crossing En1flox/Cre mice with En1Cre/+ mice. En1flox/+;En2-/+ were bred to En2-/+ mice to generate En1flox/+;En2-/- and En1Cre/+;En2-/+ were bred to En1flox/+;En2-/- mice to generate En1flox/Cre;En2-/+ and En1flox/Cre;En2-/- mice. To eliminate the possibility that the observed mild phenotype in En1flox/Cre mice was due to a genetic background effect, we generated En1Cre/Cre-null mutants in the same litters as En1flox/Cre mice by mating En1flox/Cre mice to En1Cre/+ mice. We screened a total of 39 mice from seven separate litters in which the expected frequency of all genotypes is 25%. Whereas nine En1flox/Cre mice were found, only one En1Cre/Cre mouse that survived to adulthood was observed (2.6%).
Breeding and genotyping of En1/En2 double mutants
En2ntd (Millen et
al., 1994), En2tau-lacZ
(Sgaier et al., 2005),
En1lki (Hanks et al.,
1995), En1Cre
(Kimmel et al., 2000) or
En1CreERT1 (Sgaier et
al., 2005) mutant alleles were used as En2- or
En1-null alleles on an outbred Swiss Webster (SW) genetic background.
Various combinations of En1- and En2-null alleles were
interbred to generate the required genotypes. En12ki
(Hanks et al., 1995) and
En1Denki (Hanks et al.,
1998) mice were bred with En2ntd and
En2tau-lacZ, respectively, on an outbred SW genetic
background to generate the required genotypes. For embryonic analysis, noon of
the day on which a vaginal plug was detected was designated as E0.5.
Genotyping was carried out by PCR. The R26R, En1Cre and
En1CreERT1 alleles were detected as previously described
(Li et al., 2002;
Sgaier et al., 2005;
Soriano, 1999). The primers
used for genotyping the En2 wild-type and En2ntd
alleles were: 1, 5'-TGCTCTTTGACGCTTCGGTG-3'; 2,
5'-CCTTGGATGGAGTGCTCAAAGC-3'; and 3,
5'-TCATGCTGGAGTTCTTCGCC-3'. PCR with primers 1 and 2 detected a
300 bp wild-type allele, whereas primers 1 and 3 detected a 500 bp
En2ntd mutant allele. The primers used to genotype
En1 wild-type, En12ki and
En1Denki alleles were: A,
5'-AGCTGCACCGCACCACCAAC-3'; B,
5'-GCACACAAGAGCGAGGCAGC-3'; C,
5'-CCCTGTGCCTTCGCTGAGG-3'; D,
5'-TGCCTGGCGCCTGTAGGACC-3'; and E,
5'-TTGTAGGGTAATGGGGCTGGG-3'. PCR with primers A and B detected the
232 bp En1 wild-type allele, whereas primers C and D detected the 230
bp En12ki allele, and primers E and C detected the 280 bp
En1Denki allele.
Histological analysis, ß-gal histochemistry and RNA in situ hybridization
Tissue processing, RNA in situ hybridization and ß-galactosidase
(ß-gal) analysis were performed as described on the Joyner website
(http://www.mskcc.org/mskcc/html/75282.cfm)
using Fgf8 (Crossley and Martin,
1995), Fgf17 (Xu et
al., 1999), Spry1 (courtesy of Gail Martin),
Otx2 (Simeone, 1993),
Gbx2 (Bouillet, 1995), Wnt1 (Parr, 1993), En1
(Joyner and Martin, 1987)
antisense RNA probes.
Fate mapping
En1CreERT1/+;En2-/+;R26R/R26R adult males
were bred with 5- to 6-week-old En2-/+ females and
tamoxifen (Sigma T-5648) administered at 5 mg per 40 g of body weight via
gavage at 18.00 h of E10.5 (Sgaier et al.,
2005).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E9.0 for development of the Cb and superior colliculus) indicates
that the two En proteins are functionally interchangeable and that the brain
defects in En1 and En2 single mutants arise from cells that
express only one En gene at critical time points in development. To address
the question of whether En2 can compensate for En1 after the mes/r1 is
specified, we first delineated the dynamic expression patterns of the En genes
using En1lacZ (Hanks et
al., 1995; Matise and Joyner,
1997) and En2tau-lacZ
(Sgaier et al., 2005) knock-in
alleles. En1-lacZ expression was first detected at the two-somite
stage broadly spanning the mes/r1 region
(Fig. 1A) and then
progressively narrowed around the mes/r1 junction (isthmic organizer)
(Fig. 1B-D).
En2-tau-lacZ expression commenced at the five-somite stage in a
subdomain of the En1-positive region
(Fig. 1G) and quickly broadened
(Fig. 1G-J). Within the
bilateral wing-like structure of the E12.5 cerebellar primordium (CbP),
En1-lacZ was restricted to the medial-most region (vermis anlage),
whereas En2-tau-lacZ was expressed in all but the most-lateral CbP
(Fig. 1E,K). Similarly, in the
Mb, En2-tau-lacZ expression extended more rostrally than
En1-lacZ (into the anlage of the superior colliculus).
Furthermore, expression of both En genes formed a mirror image double gradient
across the mes/r1 region, with highest levels in the isthmus. Starting at
E15.5, En1 and En2 expression changed to a sagittal striped
pattern along the M-L axis of the CbP, owing to regional downregulation
(Fig. 1F,L)
(Millen et al., 1995). Thus,
the En genes have dynamic gene expression patterns, but after E9
(including postnatal stages; data not shown) the En1 expression
domain appears to be encompassed within the En2 expression
domain.
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E9. To determine
whether this is the case, we generated a conditional mutant allele of
En1 (En1flox) in which the coding sequences in
the second exon are flanked with loxP sites (see Fig. S1 in the
supplementary material). Germline chimeras were bred to Black Swiss mice to
avoid rescue of the En1 mutant phenotype by the C57BL/6 background
(Bilovocky et al., 2003). In
order to delete En1 at E9.0, we combined the
En1flox allele with the null En1Cre
knock-in allele (Kimmel et al.,
2000). Within the mes/r1 region, Cre function is first detected at
the five-somite stage and is active in all mes/r1 cells by 15 somites (E9.0)
(Li et al., 2002). In
En1flox/Cre mice, the second exon of the
En1flox allele thus begins to be deleted shortly after its
expression is induced, and En1 is expressed for only approximately 24
hours (Fig. 2A).
Indeed, En1flox/Cre mice were found to be viable and
survive to adulthood. En1flox/Cre mutants had a limb
phenotype similar to the rare En1 mutants that survive
(Loomis et al., 1996) (data
not shown). The brains of all but one adult En1flox/Cre
mouse analyzed (n=8) appeared grossly normal in whole-mount
(Fig. 2B,C). The one mouse that
was different had a partial deletion of the Mb and Cb (data not shown).
Analysis of sagittal sections of the remaining En1flox/Cre
mice revealed that the inferior colliculus (posterior tectum) was partially
truncated (in all eight) (Fig.
2E,F and Table 1;
compare also with Fig. 3B,I).
In addition, five of the seven En1flox/Cre mice had a mild
foliation defect in the anterior vermis (medial Cb), and the overall size of
the vermis was slightly smaller than normal. The fissure between the
anterior-most folia (I/II and III) either failed to form (in two of five)
(Fig. 2F), or was shallower
than normal (in three of five) in these mutants. Of significance, in two of
the seven mutants analyzed, the fissure between folia I/II and III appeared as
deep as in wild-type brains (Fig.
2D,E). One likely possibility for the variable rescue in the Cb is
that in the two En1flox/Cre mutants that had a normal Cb,
En1 was ablated at a slightly later stage. Interestingly, in all
eight of the En1flox/Cre mice analyzed, the superior
colliculus and the hemispheres (lateral Cb) appeared normal
(Fig. 2D-I). Thus, our analysis
of the requirement for En1 after E9 demonstrates that two copies
of En2 are sufficient to support Cb development, but despite being
expressed in a broader domain of the tectum than En1, En2 alone is
not able to fully regulate inferior colliculus development.
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E9, we removed one copy of En2 on the
En1flox/Cre background. Strikingly,
En1flox/Cre;En2-/+ mutant mice were not found
at weaning (42 mice were analyzed from five litters of a cross between
EnCre/+;En2-/+ and
Enflox/+;En2-/+ mice). We therefore analyzed
the phenotypes of En1flox/Cre embryos lacking one
En2 allele at E18.5, and compared them with
En1-/- and En1-/-;En2-/-
double mutants (Fig. 3). As
expected, at E18.5, the vermis of
En1flox/Cre;En2+/+ mice was either normal or
slightly delayed in forming folia (Fig.
3B,I and Table 1).
By contrast, En1flox/Cre;En2-/+ mice
(Fig. 3J) displayed a complete
deletion of the Cb that was very similar to
En1-/-;En2-/+
(Fig. 3G) and
En1-/-;En2-/-
(Fig. 3H) mutant embryos and
more severe than En1-/-;En2+/+ mutants
(Fig. 3F) that had some lateral
Cb tissue remaining (see also Table
1). However, unlike En1-/-;En2-/+
(Fig. 3G) and
En1-/-;En2-/- mutants
(Fig. 3H), which have no
tectum, in En1flox/Cre;En2-/+ mutant mice
(Fig. 3J) some superior
colliculus tissue remained, similar to the phenotype of
En1-/-;En2+/+ mice
(Fig. 3F and
Table 1). By contrast,
En1flox/Cre;En2-/- mutants had a complete
deletion of the Cb and tectum (Fig.
3K and Table 1). In
summary, when En1 is expressed until E9.0
(En1flox/Cre), two copies of En2 can support
superior colliculus and Cb development and partial development of the inferior
colliculus, whereas one copy of En2 is not sufficient to support any
development of the inferior colliculus or Cb, and in the absence of all
En2 the superior colliculus also does not form.
|
).
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), were found to survive to adulthood.
En1-/+;En2-/- mutants
(Fig. 3B,E) had a more severe
delay in Cb foliation than En2-/- mutants
(Fig. 3D). Consistent with
this, the Cb of adult En1-/+;En2-/- mutants
(Fig. 4D,H,L) had a variable
but greater size reduction and more foliation defects than
En2-/- (Fig.
4B,F,J) or En1-/+;En2+/- mice
(Fig. 4C,G,K and see
Table 1). In the majority of
the mutants (six out of eight) the five anterior-most folia (I-V) of the
vermis were replaced by a single fold. In addition, folium VIII was more
defective than in En2-/- mutants: it was absent or
decreased in size and misaligned with folium IX. The inferior colliculus also
was partially truncated. In one of the mutants, folia I-V were almost
completely lost and fused to a more profoundly truncated tectum (data not
shown). One other En1-/+;En2-/- mutant had an
even more severe phenotype that included a complete deletion of the vermis and
inferior colliculus (data not shown). In contrast to the vermis, although the
overall size of the En1-/+;En2-/- hemispheres
(Fig. 4I,L) was reduced
compared with En2-/- mutants
(Fig. 4J), the foliation defect
was the same (fusion of the crusII and paramedian folia) or, paradoxically,
partially rescued in one mutant. Interestingly, in all the viable
En1/2 mutants the relative thickness of the remaining molecular and
granule cell layers and Purkinje cell density were normal. Our analysis of
viable En1/2 mutants provides additional evidence that the anterior
vermis (folia I-V) and inferior colliculus are most sensitive to a reduction
in En1, and also reveals redundant functions for En1 and
En2 in folia I-V and VIII.
En2 can sustain more extensive Cb vermis development than En1
In order to directly compare the in vivo functions of En1 and En2 proteins,
we took advantage of our En1En2ki knock-in mice
(Hanks et al., 1995). A
sensitive assay for comparing protein function is to combine knock-in alleles
with loss-of-function alleles (Wang and
Jaenisch, 1997). If the two En proteins have equivalent
properties, then removal of En2 in En1En2ki/En2ki
mice (En1En2ki/En2ki;En2-/-) should only cause
the foliation defects seen in En2-/- mice. Indeed, when
expressed in two copies from the En1 locus, En2 is
sufficient to support development of the inferior colliculus. Unlike
En1flox/Cre;En2+/+ mice, which have a partial
truncation of the tectum, the inferior colliculus appeared normal in
En1En2ki/En2ki;En2-/- adult or early postnatal
mice (Fig. 5A,C and data not
shown). Furthermore, consistent with En1 expression being restricted
to the primordium of the vermis, as compared with the broader En2
expression in the hemispheres after E9.5, the hemispheres of
En1En2ki/En2ki;En2-/- mice
(Fig. 5G,I) had the
characteristic En2-/- phenotype
(Fig. 4J). However, unlike
En2-/- mice (Fig.
4F), in which folium VIII is associated with folium IX, in three
out of four adult En1En2ki/En2ki;En2-/- mice
(Fig. 5A,C), folium VIII was
normal. This shows that the vermis foliation defects in
En2-/- mice can be rescued by expressing the En2
gene, but not the En1 gene, from the En1 locus. This
suggests that En1 and En2 proteins are not equivalent, but rather that En2
activity is either specifically required in the posterior Cb (folium VIII) or
more active in the Cb.
We further tested whether En2 is generally more potent than En1 by comparing the phenotype of En1-/+;En2-/- mice with En1En2ki/-;En2-/- mice. Strikingly, we found that En1En2ki/-;En2-/- mice (n=3) had a much milder anterior Cb phenotype than En1-/+;En2-/- mutants. The anterior vermis of En1En2ki/-;En2-/- mutants (Fig. 5A,D) had three distinct folia (I/II, III and IV/V) compared with one in En1-/+;En2-/- mice (Fig. 4H). In addition, in the posterior vermis of En1En2ki/-;En2-/- mice (Fig. 5D), folium VIII was only partially displaced toward folium IX, in contrast to En1+/-;En2-/- mutants (Fig. 4H), which have a substantial deletion of folium VIII. This sensitive dosage assay of one copy of En2 or En1 expressed from the En1 locus in the absence of all other En function thus indicates that En2 is generally more active than En1 in regulating Cb development. Finally, the posterior tectum of En1En2ki/-;En2-/- mice (Fig. 5D) was partially truncated (Fig. 4H and data not shown), seemingly less than in En1-/+;En2-/- mutants (Figure 4H and data not shown), indicating that the dose of both En proteins contributes to the truncation of the inferior colliculus in the various En mutants.
|
). Whereas
En1En2ki/En2ki;En2-/- and
En1Denki/Denki;En2+/+ mice are viable, we did
not detect any En1Denki/Denki;En2-/- mice after
birth. Section analysis of E18.5
En1Denki/Denki;En2-/- embryos revealed either
the same phenotype as En1-/-;En2-/- embryos
(n=1 of 3) or a similar phenotype to
En1flox/Cre;En2-/+ mutants (n=2 of 3;
data not shown). Interestingly, half the expected number of
En1Denki/Denki;En2-/+ mice survived to
adulthood, indicating that Drosophila En has some En1-like activity
as En1-/-;En2-/+ die at birth. Histological
analysis of the brains of three
En1Denki/Denki;En2-/+ mice
(Fig. 5A,E and
Table 1) that survived
surprisingly revealed that two of the mutants had a normal tectum and Cb,
whereas one had a partial deletion of the posterior tectum and a vermis
foliation defect similar to En1+/-;En2-/-
mutants (Fig. 4H). The five
En1Denki/+;En2-/- mice analyzed had a phenotype
that was milder than En1-/+;En2-/- mice, with a
distinct IV/V folium (Fig. 5A,F
and Fig. 4H). All but one of
the five En1Denki/+;En2-/- mutants analyzed had
an En2-/- foliation pattern in the hemispheres
(Fig. 4J,
Fig. 5G,L). These results
indicate that Drosophila En function can partially replace normal En1
function in the initiation of normal En2 expression (before
E9.0) in En1Denki/Denki;En2+/+ mice, after
which mouse En2 contributes a necessary function for continued normal
development of mes/r1-derived structures.
Medial-lateral patterning of the Cb appears to be altered in En1-/+;En2-/- mutants
One possible reason for the loss of the posterior tectum and anterior
vermis in En1-/+;En2-/- adult mutants is
specific loss of the cells that give rise to the these two regions (caudal mes
and rostral r1) (Sgaier et al.,
2005; Zervas et al.,
2004). To determine whether this is the case, we fate mapped the
posterior mes and anterior r1 in En1-/+;En2-/-
mutants by genetic inducible fate mapping (GIFM)
(Joyner and Zervas, 2006)
using our null En1CreERT1/+ allele
(Sgaier et al., 2005) and the
R26R lacZ reporter allele
(Soriano, 1999). When
tamoxifen (TM) is administered at 18.00 h to E10.5 wild-type
(En1CreERT1/+;En2+/+;R26R/+) embryos
(marking cells at E11-12), the posterior mes and medial-most domain of the
E12.5 CbP are marked (Fig. 6A)
and give rise to the vermis and inferior colliculus of the adult, respectively
(Fig. 6G,I)
(Sgaier et al., 2005). GIFM in
En1CreERT1/+;En2-/-;R26R/+ mutants also marked
the posterior mes and medial-most domain of the E12.5 CbP
(Fig. 6B). A difference that
was apparent between the mutants and wild types was that despite an overall
reduction in the size of the mes and r1 in the mutants, the size of the
initial marked domain in the mes/r1 at E12.5 was similar to that in the wild
type, and the regions devoid of marked cells were smaller in the mutants as
compared with the wild type (Fig.
6A,B). At E16.5, the size of the marked population of cells in the
Cb appeared wider in En1CreERT1/+;En2-/-;R26R/+
embryos (Fig. 6D) than in wild
types (Fig. 6C). By contrast,
the size of the marked domain in the tectum was smaller in mutants than in
wild types (Fig. 6C-F).
Similarly, in adult En1CreERT1/+;En2-/-;R26R/+
mutants, the marked domain in the Cb was broader than normal, whereas the size
of the marked domain in the tectum was greatly reduced compared with wild
types as it was restricted to the remaining inferior colliculus
(Fig. 6G-J). The fate mapping
results in the mes suggest that in En1/2 mutants, the posterior mes
cells marked at E12.5 do not expand normally and this results in a smaller
inferior colliculus in the adult. By contrast, because the domain of marked
cells in the adult Cb is larger than normal even though the vermis is reduced
in size, this suggests that the anterior r1 cells marked at E12.5 are not only
retained but contribute to more lateral regions of the vermis than normal.
Thus, the loss of tectum and vermis tissue in
En1-/+;En2-/- adult mutants is not simply owing
to loss of the cells that give rise to the these two regions.
|
), one
possible reason for the defects in En2-/- and
En1-/+;En2-/- mutants is a disruption of the
isthmic organizer (decrease in Fgf signaling). However, our previous
whole-mount RNA in situ analysis of
En1-/+;En2-/- embryos from the six-somite stage
to E9.5 did not reveal any obvious changes in the expression of Fgf8
or mes/r1 morphology (Liu and Joyner,
2001). Based on section analysis, En2-/-
embryos had a slight reduction in the size of the Cb by E11.5, and
En1-/+;En2-/- mutants had a greater reduction
in the CbP and truncation of the tectum (see
Fig. 7). We therefore performed
RNA in situ analysis at E11.5, a day before Fgf8 expression is
normally terminated in the isthmus. Consistent with the fate mapping study, we
found that the expression domain of En1 was not obviously altered
along the A-P axis in the mutants (Fig.
7G-I). The only obvious difference in the expression domains of
Fgf8 and Fgf17 (a related organizer gene) and of the direct
target gene Spry1 (Liu et al.,
2003) between En2-/- and
En1-/+;En2-/- mutants as compared with
wild-type mice (En1-/+) was a slight reduction in the size
of the domains, which correlated with the reduction in mes/r1 tissue in each
mutant (Fig. 7A-F and data not
shown). Consistent with retention of organizer activity, the expression
domains of two mes genes (Otx2 and Wnt1) and one r1 gene
(Gbx2) regulated by Fgf8, were not altered (data not shown). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), raising the
possibility that a key role of En1/2 is to maintain Fgf8
expression (Liu et al., 2003).
We found that Fgf8 expression is maintained as long as one allele of
En1 is present, although there are subtle decreases in
Fgf8/17 expression. Since En1/2 expression persists after
E12.5, when Fgf8 expression is terminated, and
En1-/+;En2-/- mutants have a much more severe
loss of the tectum and Cb folia than
Fgf8-/+;Fgf17-/- mutants, it is possible that
En1/2 do not control mes/r1 development solely through regulating
Fgf8/17 expression.
En1/2 differentially regulate retention of cells in the mes and r1
By determining the fate of the En1-expressing cells at E11,
which normally give rise to the vermis and inferior colliculus, in
En1-/+;En2-/- mutants using GIFM, we uncovered
an unexpected differential role for En1/2 in regulating growth and
survival of cells in the tectum versus the Cb. In En1/2 mutants, the
posterior mes cells marked at E12.5 do not expand normally and this results in
a smaller inferior colliculus in the adult. By contrast, the anterior r1 cells
marked at E12.5 are not only retained but contribute to more lateral regions
of the vermis than normal. If the lineage restriction at the mes/r1 border
that restricts mes and r1 cells from mixing
(Zervas et al., 2004) is
disrupted in En1/2 mutants, then it is possible that the population
of marked mes cells in
En1CreERT1/+;En2-/-;R26R/+ embryos move into r1
and expand the marked population in the Cb. Another possibility is that the
precursors of the lateral Cb are selectively lost in the mutants. However,
this is not in accordance with our observation that the hemispheres of
En1-/+;En2-/- adults are less compromised than
the vermis. Although the ultimate overall loss of cells in the mes and r1 of
En1-/+;En2-/- mutants could be accounted for by
cell death, similar to the situation in En1 mutants
(Chi et al., 2003), our fate
mapping study shows that it is not as simple as the cells being lost equally
on either side of the isthmus.
|
En1 and En2 are differentially required in subregions of the Cb
Our analysis of En1/2 double-mutant combinations (null, knock-in
and conditional) uncovered additional differential requirements for
En1 and En2 in specific regions of the Cb (see
Table 1 and
Fig. 8B). En1/2
functions are normally uncoupled in the hemispheres as only En2 is
required to divide the posterior region into two folds (crusII and
paramedian). However, the partial rescue of the hemisphere phenotype in rare
En1-/+;En2-/- and
En1Denki/+;En2-/- mutants indicates that En1
can support hemisphere development when expressed more laterally than normal.
A comparison of the phenotypes of these mice with
En1flox/Cre;En2+/+ mutants (which have normal
posterior foliation) indicated that En2 plays a greater role than
En1 in formation of folium VIII. We demonstrated that this difference
is not owing to a difference in gene expression, but instead to a difference
in protein activity because the vermis foliation defect seen in
En1+/+;En2-/- mutants is rescued in
En1En2ki/En2ki;En2-/- mice. Furthermore,
En1En2ki/-;En2-/- mice have a milder phenotype
than En1-/+;En2-/- mutants. Thus, En2 appears
to be more effective in promoting development of the vermis (folia I-V and
VIII) than En1. We further discovered that the two En genes act concomitantly
to divide the anterior Cb into five folia.
En1-/+;En2-/+ double heterozygotes and the
majority of En1flox/Cre;En2+/+ mutants have a
fusion of the anterior three folia (I-III) and the anterior defect is greatly
exaggerated in En1-/+;En2-/- mutants (fusion of
folia I-V), despite En2-/- mutants having normal anterior
foliation. Since some En1flox/Cre mice have normal
anterior folia, this indicates a crucial requirement for expression of
En1 only at E9, when En1 expression is fading out in
the mutants and En2 is initiating. To our knowledge, this is the
first evidence that the pattern of Cb folia can be influenced by genetic
events that occur at such an early embryonic stage.
|
;
Zervas et al., 2004), the
anterior and posterior mes give rise to the superior and inferior colliculi,
respectively (Fig. 8A).
Consistent with strong and sustained expression of the En genes in the
primordium of the inferior colliculus, this region is most sensitive to the
dose of En genes, and in particular to that of En1. However, by using
the sensitive assay of En12ki knock-in alleles combined
with removal of the endogenous En2 gene, we found that En2 is at
least as potent as En1 at promoting inferior colliculus development. Given the
transient expression of the En genes in the superior colliculus, it is perhaps
surprising that this region is dependent at all on the combination of the two
genes. This indicates a requirement for a short burst of En function (En1 or
En2) before E9.5. The remaining inferior colliculus tissue in En mutants is
likely to correlate with tectum cells that are normally in the low end of the
En gradient, suggesting that they are least sensitive to loss of En alleles.
There also is a general correlation between the domains of En gene expression
and the requirement for each gene in the Cb (vermis versus hemispheres). After
E9.5, En1 is only maintained in anterior r1 and the medial Cb
primordium (the anlage of the vermis), consistent with no function in the
hemispheres. The limit of the En2 expression domain extends more
posterior early in r1 and laterally later in the CbP, correlating with a role
in the hemispheres. It is not clear, however, why the En genes do not play a
major role in the anterior hemispheres or in folia VI/VII and IX/X in the
vermis.
An `En code' divides the tectum and Cb into subregions
Taken together, our analysis of a series of En mutants provides evidence
that functional domains of the Cb are genetically encoded by the engrailed
genes, as specific regions of the tectum and Cb have differential
sensitivities to reducing En gene dosage. The phenotypes of multiple mutants
point to a genetic division of the tectum into two regions and of the Cb into
six. We propose that this represents an `En code' that is used to partition
the mes/r1 region into domains that in the adult regulate related neural
functions (Fig. 8B). The two
functional divisions of the tectum, the inferior and superior colliculi, are
delineated based on a temporal requirement for En1 and sensitivity to
the overall dose of En protein. Within the vermis of the Cb, the anterior five
folia (I-V) and folium VIII are particularly sensitive to a reduction of En
genes, and preferentially to En2, thus dividing the vermis into four
broad regions (folia I-V, VI/VII, VIII, IX/X). Strikingly, this division of
the Cb is very similar to the transverse zones recently proposed based on four
different domains of parasagittal gene expression
(Armstrong et al., 2005;
Ozol et al., 1999). The fact
that two independent genetic measures of regionalization of the vermis (mutant
phenotypes and gene expression) point to the same subdivisions of the vermis
strongly argues that patterning of the folia is fundamental to organization of
Cb function. Consistent with this, each transverse zone receives afferent
inputs from distinct regions of the spinal cord and/or particular hindbrain
nuclei. We predict that, likewise, the division of the hemispheres into
regions based on a need for En2 only in two folia (crusII and
paramedian) (Fig. 8B)
represents genetic partitioning into related functional systems.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2325/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Developmental Biology Program, Sloan-Kettering Institute,
Rockefeller Research Labs, 1275 York Avenue, New York, NY 10021, USA
|
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