doi: 10.1242/10.1242/dev.00225
Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation
Mahua Mukhopadhyay1,*,
Andreas Teufel1,*,
Tsuyoshi Yamashita1,*,
,
Alan D. Agulnick1,
,
Lan Chen1,
Karen M. Downs2,
Alice Schindler1,
Alexander Grinberg1,
Sing-Ping Huang1,
David Dorward3 and
Heiner Westphal1,
1 Laboratory of Mammalian Genes and Development, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda,
Maryland 20892, USA
2 Department of Anatomy, University of Wisconsin-Madison Medical School,
Madison, WI 53706, USA
3 National Institutes of Allergy and Infectious Diseases, Rocky Mountain
Laboratories, Hamilton, Montana 59840, USA
Present address: Department of Obstetrics and Gynecology, Asahikawa Medical
College, Nishikagura 4-5-3-11, Asahikawa, Japan
Present address: CyThera Inc., 3550 General Atomics Court, San Diego, CA
92121, USA
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Fig. 1. Gene targeting at the Ldb1 locus. (A), Partial restriction map of
the wild-type Ldb1 locus, the targeting vector, and disrupted
Ldb1 allele. A gene conferring neomycin-resistance replaces the
deleted Ldb1 coding sequences (exons 3-9) following homologous
recombination. (B) Southern blot analysis of targeted ES cells. Two
independent clones TS17 (lanes 2, 5) and TS153 (lanes 3, 6) were used for
blastocyst injection. TS12 is a wild-type control (lanes 1, 4).
EcoRV- and HindIII-digested ES cell DNA was used for
Southern analyses with 5' and 3' probes respectively. The 5'
probe hybridized to 4.7 kb and 6.2 kb restriction fragments generated from the
wild-type and the mutated alleles respectively, while the 3' probe
detected a 6.1 kb wild-type and a 6.6 kb mutant fragment.
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Fig. 2. Anterior-posterior axis phenotypes of Ldb1 null mutant embryos.
(A) Wild-type and (B) mutant E7.5 embryos. A constriction at the
embryonic-extraembryonic junction of the mutant embryo is clearly visible
(arrows in B). (C-E) E8.5 embryos showing in situ expression patterns of the
forebrain marker Six3. Frontal (C) and lateral (D) views of a
wild-type embryo, showing heart (arrow) and head (arrowhead) structures.
Frontal view (ventral side up, E) of an E8.5 mutant embryo with defects in
head and heart development, truncation of forebrain structures and absence of
Six3 expression. (F-H) En2 expression that marks
midbrain-hindbrain boundary at E8.5 is present in a wild-type embryo (F) but
absent in mutant embryos (G,H). (I,J) Krox2 expression marking rhombomeres 3
and 5 in wild-type (I) and mutant (J) embryos. Expression of this gene shows
partial duplication of rhombomeres 3 and 5 in the mutant (J). (K,L)
Hematoxylin and Eosin-stained horizontal sections of E8.5 wild-type and mutant
embryos, respectively. The mutant embryo (L) lacks all head structures
anterior of the otic vesicles (arrows). The kinks in the neural tube indicate
defective longitudinal extension of the neural epithelium. (M) Hematoxylin and
Eosin-stained cross section taken from the trunk level of an E8.5 mutant
embryo showing abnormal development of two neural grooves (arrows) within a
single continuous neuroepithelial layer. (N,O) Scanning electron micrographs
presenting ventral (N) and dorsal (O) views of an E8.5 mutant embryo. Abnormal
development of four rows of somites is indicated with arrows. (P,Q) TUNEL
staining of histological sections taken from comparable regions of E8.5
wild-type and mutant embryos, respectively. Arrows point to extensive cell
death in the mesenchyme, and to a lesser degree in the neuroepithelium, of the
mutant embryo (Q). The scale bars represents 100 µm in A-J,N,O; 25 µm in
K,L; 10 µm in M; and 2 µm in P,Q.
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Fig. 3. Defects in extraembryonic tissue development of the Ldb1 mutant.
(A,B) Benzidine staining reveals development of a network of blood islands and
primitive vessels (arrow) in the wild-type conceptus at E8.5 (A), which do not
exist in the Ldb1-/- mutant (B). (C,D) Alkaline
phosphatase staining of a wild-type and a mutant E7.5 conceptus, respectively.
Primordial germ cells at the base of the allantois are stained in the control
(arrow in C, magnified in the inset), while this signal is absent in the
mutant (D). (E,F) Extraembryonic tissue morphology. The mesodermal and
ectodermal layers (arrow) of the amnion are properly extended at the
embryonic/extraembryonic junction of a wild-type embryo (E). During its
outgrowth the allantois maintains contact with the embryonic tissue (large
arrow in E). In the Ldb1-/- mutant embryo (F) the
ectodermal layer (arrowhead) of the amnion fails to expand, thus creating a
contriction at the embryonic-extraembryonic junction. Although the mesodermal
layer of the amnion is expanded, it fails to extend at the constricted
embryonic/extraembryonic junction and forms pockets at the anterior and
posterior ends (small arrows in F). The mutant allantois (large arrow in F)
appears to have lost contact with the embryonic tissue.
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Fig. 4. Expression of heart markers. (A,B) Nkx2.5 expression at E7.75. At this
stage Nkx2.5 is normally expressed in the heart precursor cells that form a
crescent surrounding the heart field (A). The crescent shaped region of
Nkx2.5 expression is absent in a representative mutant embryo shown
in B. (C,D), Mesp1 expression in gastrulating embryos. In the
wild-type E6.75 embryo, Mesp1 expression is seen in early ingressing
mesodermal cells along the length of the primitive streak (C). An abnormal
V-shaped expression pattern of the Mesp1 gene in early ingressing
mesodermal cells is observed in a stage-matched Ldb1 null mutant
embryo (D; posterior is facing up). The arrows in B and D indicate the
embryonic-extraembryonic junction.
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Fig. 5. Marker analyses of anterior-posterior axis formation. (A-C),
Brachyury (T) expression in E7.5 embryos. The embryo shown
in B is one of about 60% (32/56) of the Ldb1 mutant embryos in which
proximal-distal extension of the primitive streak appears normal. The embryo
shown in C represents mutant embryos in which the expression pattern of
T is abnomal. In this lateral view we observe a twisted expression
domain of T, with seemingly normal distal extension. (D-F) T
expression in E8.5 embryos. (D) Wild-type T expression pattern
marking the region of the primitive streak (arrowhead) and the notochord
(arrow). (E,F), Ldb1 null mutant embryos with two primitive streaks
(arrowheads) connected distally. Two notochords are visible as well (arrows).
(G-J), Otx2 expression at E7.75 (G,H) and E7.5 (I,J) is restricted in
the mutant (H,J) compared to the wild type (G,I). (K,L), Hnf3ß
expression in E7.75 embryos. Two posterior expression domains were found in
the mutant (L). (K) The wild-type expression of Hnf3ß in
anteriorly migrated endomesodermal cells is indicated by an arrow. This
expression domain is absent in the mutant embryo (L). (M,N), Lim1
expression at E7.5 showing presence of one node in the wild-type (M, arrow)
and two nodes in the mutant (N, arrows). (O,P) Dkk1 expression at
mid-streak stage is absent in the mutant embryo (P). (Q,R), Hesx1
expression at E7.75 is not detectable in the ANE of the mutant. The scale bars
represent 100 µm.
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Fig. 6. Expression of Wnt inhibitors in E7.5 embryos. Wild-type embryos
are on the left, Ldb1-/- mutants on the right. (A,B)
Frzb expression. Expression is undetectable in the primitive streak
and the prospective cardiac mesoderm regions of the mutant. (C,D), Expression
of Sfrp1. The signal is greatly reduced in the anterior ectoderm of
the mutant. (E,F) Expression of Sfrp2. This signal is absent in the
mutant. (G,H) Cer1 expression. The signal, marking the AVE, the axial
mesendoderm and the definitive endoderm of late streak embryos, is absent in
the mutant. (I) Variation in the expression level of the Cer1 gene in
Ldb1 mutant and wild-type embryos. The scale bar represents 100
µm.
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Fig. 7. Analysis of maternal Ldb1 expression. Lanes 1-3, RT-PCR analysis
for Ldb1 mRNA. Lane 1, oocytes; lanes 2 and 3, E7.5 embryos.
Ldb1 mRNA is detected in wild-type (lane 2), but not in Ldb1
null mutant embryos (lane 3). Lanes 4-6, expression of control mRNA. The
control mRNA is absent in the egg (lane 4), but present in E7.5 wild-type
(lane 5) and Ldb1 mutant (lane 6) embryos.
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© The Company of Biologists Ltd 2003
