First published online March 24, 2005
doi: 10.1242/10.1242/dev.01727
Development 132, 1971-1981 (2005)
Published by The Company of Biologists 2005
New regulatory interactions and cellular responses in the isthmic organizer region revealed by altering Gbx2 expression
James Y. H. Li1,2,*,
Zhimin Lao1 and
Alexandra L. Joyner1,2,3
1 Howard Hughes Medical Institute and 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
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Fig. 2. Expression of Gbx2 in rhombomere 4 using a Hoxb1
regulatory element partially rescues the anterior hindbrain of
Gbx2–/– embryos. (A) Schematic representation
of r4-Gbx2 and r4-Gbx2-Z transgenes. (B) Expression of
Gbx2 analyzed by RNA in situ hybridization in r4-Gbx2
transgenic embryos and a wild-type control (inset) at E9.5. (C) Expression of
r4-Gbx2-Z analyzed by X-gal staining. Inset shows a mid-sagittal
section of an embryo. Gbx2 is ectopically expressed in r4 (black
arrowhead) in r4-Gbx2 and r4-Gbx2-Z transgenic embryos.
Gbx2 /lacZ is also expressed in the head mesenchyme at the mid/hindbrain
junction (black arrow) in r4-Gbx2-Z line #26. (D-G) Morphology of
embryos of the indicated genotypes. The anterior hindbrain between the
posterior limit of the mesencephalon and the otic vesicles (red arrowheads)
are demarcated by a bracket. Asterisks indicate dorsal r1.
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Fig. 1. Partial overlapping of the expression domains of Otx2 and
Hoxb1 in embryos lacking Gbx2 at the five-somite stage.
(A-I) RNA in situ hybridization analysis of Otx2 (A-C),
Hoxb1 (D-F) and Krox20 (G-I) on adjacent sagittal sections
of embryos of indicated genotypes. Red arrowheads indicate posterior limit of
Otx2 expression, and green arrowheads indicate the anterior limit of
Hoxb1 expression. Arrows indicate r5. Inset in F shows that there are
only a few cells in r4 (in bracket) expressing r4-Gbx2-Z after
staining with X-gal.
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Fig. 3. The cerebellum and the posterior midbrain are missing in
Gbx2–/–; HG embryos. (A-C) Dorsal view of
whole-mount brains in embryos of the indicated genotypes at E18.5. The
cerebellum, which is demarcated by a broken line in the wild-type embryo, is
absent in both Gbx2–/– and
Gbx2–/–; HG embryos (indicated by arrowheads).
(D-I) Sagittal sections of embryos of the indicated genotypes at E12.5 (D-F)
and E18.5 (G-I). The posterior midbrain (arrows) is missing in
Gbx2–/–; HG embryos. cb, cerebellum; mb,
midbrain; sc, superior colliculus, ic, inferior colliculus.
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Fig. 4. R3 is rescued in Gbx2-null mutants by the HG transgenes.
(A-C) Krox20 expression in embryos of the indicated genotypes at the
eight-somite stages. Inset shows dorsal views of the embryo. The transverse
bands of Krox20 expression in Gbx2–/–
(B) or Gbx2–/–; HG (C) embryos is narrower
than those in the wild-type embryo (A). In addition, the lateral-most
expression of Krox20 (arrowhead) is missing or greatly reduced in
Gbx2–/– and
Gbx2–/–; HG embryos. (D-F) Hoxa2
expression at E9.5. Expression of Krox20 and Hoxa2 in r3 is
restored in Gbx2–/–; HG embryos by the
eight-somite stage and E9.5, respectively. (G-I) Kreisler expression
in embryos at the six-somite stage of the indicated genotypes. Kreisler
expression in r5-6 appears more restricted in
Gbx2–/– (H) and
Gbx2–/–; HG (I) embryos, and the expression in
the dorsal-most region (arrow) is missing.
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Fig. 5. A normal spatial relationship of Wnt1 and Fgf8 expression
is restored in Gbx2-null mutants by the HG transgenes. (A-L)
RNA in situ hybridization on sagittal sections of embryos of the indicated
genotypes at E10.5. cDNA probes used for hybridization are listed to the left.
Expression of Otx2 and Hoxb1 is juxtaposed in Gbx2
mutant embryos (B,C,E,F), and ectopic Otx2-expressing cells (black
arrowhead) are seen in Hoxb1 expression domain of
Gbx2–/– embryos (B). There is an additional
transverse band of cells expressing Hoxb1 in
Gbx2–/–; HG embryos (F). The region (indicated
by a black bracket) between these two Hoxb1 expression domains
probably represents a rescued r3 tissue. Inset in F shows RNA in situ
hybridization with a Gbx2 probe, indicating that expression of the
HG transgene (Gbx2) recapitulates the endogenous
Hoxb1 expression. Wnt1 and Fgf8 are expressed in
complimentary domains at the Otx2/Gbx2 border region in wild-type and
Gbx2–/–; HG embryos (G,I,J,L), whereas the
expression domains (indicated by red bracket) of Wnt1 and
Fgf8 largely overlap in Gbx2 null embryos (H,K). Overlapping
expression of Wnt1 and Fgf8 occurs specifically at a gap of
Hoxb1 (Gbx2) expression (marked by a arrow) in
Gbx2–/–; HG embryos. Broken lines demarcate
the posterior limit of Otx2 expression, read arrowhead indicates
r4.
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Fig. 6. Reduction or increase of Fgf8 signaling in the mid/hindbrain
junction leads to increased cell death in the posterior midbrain. (A-D)
Analysis of apoptosis by a TUNEL assay on sagittal sections of E10.5 embryos
of the indicated genotypes. There is a significant increase in the number of
TUNEL-positive cells (red) in the posterior midbrain of
Gbx2–/–; HG (C) and r1 of Gbx2-CKO
embryos (D). Insets in A, C and D show a higher magnification of the
demarcated region. (E-L) Analysis of Spry1 (E-H) and Fgf8
(I-L) expression by RNA in situ hybridization in sections or whole-mount
embryos of the indicated genotypes at E10.5. Insets in I-L show dorsal views
of embryos. The expression domains of Fgf8 and Spry1 are
diffuse in the mid/hindbrain region of Gbx2–/–
embryos, and expanded in Gbx2-CKO embryos. By contrast, the
expression domains of these genes are sharp but more restricted in
Gbx2–/–; HG embryos.
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Fig. 7. Schematic summary of the gene expression. In
Gbx2–/– embryos, there is an ill-defined area
designated as region X, in which Otx2, Hoxb1 are ectopically
expressed. In contrast to wild type, expression domains of Wnt1 and
Fgf8 overlap in region X. In Gbx2–/–;
HG embryos, the expression of Gbx2 mimics Hoxb1
expression at the five-somite stage and leads to repression of Otx2
and a new Otx2/Hoxb1 (Gbx2) border in region X at the
eight-somite stage. The normal spatial relationship of Wnt1 and
Fgf8 is established at the new Otx2/Hoxb1 (Gbx2)
border. Krox20 is induced in r3 between two stripes of Hoxb1
expression.
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© The Company of Biologists Ltd 2005
