First published online 5 November 2003
doi: 10.1242/dev.00846
Development 130, 6209-6220 (2003)
Published by The Company of Biologists 2003
Regulation of outgrowth and apoptosis for the terminal appendage: external genitalia: development by concerted actions of BMP signaling
Kentaro Suzuki1,
Daniel Bachiller2,
YiPing P. Chen3,
Mami Kamikawa1,
Hidenao Ogi1,
Ryama Haraguchi1,
Yukiko Ogino1,
Yasuhiro Minami4,
Yuji Mishina5,
Kyung Ahn6,
E. Bryan Crenshaw, III6 and
Gen Yamada1,*
1 Center for Animal Resources and Development (CARD), Graduate School of Medical
and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto
860-0811, Japan
2 Molecular Genetics Laboratory, Division of Head and Neck Surgery, UCLA School
of Medicine, Los Angeles, CA 90095-7795, USA
3 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA
70118, USA
4 Department of Genome Sciences, Graduate School of Medicine, Kobe University,
Kobe, Japan
5 Molecular Developmental Biology Group, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental Health Sciences,
NC 27709, USA
6 Mammalian Neurogenetics Group, Center for Childhood Communication, The
Children's Hospital of Philadelphia, PA 19104, USA
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Fig. 1. Expression of Bmp and BMP antagonist genes during mouse genital tubercle
(GT) development. (Top) SEM picture of an embryonic GT at 12.5 dpc (green line
indicates urethral plate). The arrowhead indicates the distal urethral
epithelium (DUE; which is covered with an epithelial layer). (A) Bmp2
expression was localized to the urethral epithelium (black arrowhead) and
proximolateral mesenchyme (white arrows) of the GT at 12.5 dpc. (B) By 12.5
dpc, the expression of Bmp4 was detected in the mesenchyme along the
urethral plate and also around the DUE (white arrows). (C) Bmp7 was
expressed in the DUE (white arrow) and in the urethral epithelium with its
expression level moderately higher in the distal and lower in the proximal
regions. (D) Nog was expressed in the mesenchyme around the DUE
(white arrows). Its expression was also localized in the proximal mesenchyme
around the urethral plate (white arrowhead). (E) Bambi was expressed
in the mesenchyme with its expression level moderately higher in the distal
and lower in proximal regions. All figures are ventral views. Scale bar: 250
µm.
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Fig. 2. BMP4 and BMP7 promote apoptosis, whereas NOG inhibits apoptosis in vitro.
Apoptosis was confirmed by performing both Nile Blue staining and TUNEL assays
(one of the two assays was shown). (A-C) GT explants were micro-dissected from
12.0 dpc embryos, implanted with BMP4 (B4) or control (c) BSA beads, and
cultured for 24 hours. BMP4 beads inhibited GT outgrowth when compared with
the opposite side of the same specimen implanted with control BSA beads (A).
Implanted BMP4 beads (C) in the murine GT mesenchyme increased apoptosis
compared with control beads (B). (D-F) BMP-treated GT displayed increased
apoptosis in the distal GT region compared with the control (D, control; E,F,
BMP4- and BMP7-treated GT, respectively). (G,H) TUNEL analysis was performed
on NOG treated specimens. A clear reduction in the number of apoptotic cells
within the distal GT mesenchyme was observed by the NOG application (G,
control section; H, NOG treated section). (A-C) Ventral views. (D-F) Frontal
views. (G,H) Coronal sections. The upper side is dorsal and bottom side is
ventral. Scale bars: in A, 125 µm in A-C and 250 µm in D-F; in G, 50
µm in G,H.
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Fig. 4. Exogenous BMP4 downregulates the expression of Fgf8, Wnt5a and
cyclin D1 and suppresses cell proliferation. GTs from 12.0 dpc embryos were
cultured for 3 days (A,B) or for 24 hours (C-H) with BMP4 protein or BSA in
the medium. (A,B) BMP4-treated GT displayed retarded GT outgrowth. (C-H)
Fgf8, Wnt5a and cyclin D1 expression were suppressed by BMP4
treatment. Shh expression was unaltered by BMP4 treatment indicating
specific effects by BMP4 (data not shown). (I) Reduced cell proliferation of
BMP4-treated GT. The number of phospho-histone H3 immunopositive cells
decreased to 60% compared with those of control GT explants. (J) Alteration of
Fgf8 expression was observed 6.5 hours after the BMP4 treatment by
RT-PCR analysis. (A-H) Ventral views. Scale bar: 250 µm in A,B; 325 µm
in C-H.
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Fig. 3. Retarded GT outgrowth associated with an increase of apoptosis. (A-F) GTs
at 12.0 dpc were cultured for 3 days (A,B) or for 24 hours (C-F). Apoptotic
cells were increased in the GT epithelium and its adjacent mesenchyme after
the staurosporine treatment, which was associated with retarded GT outgrowth
proximodistally. (G,H) GT explants between 11.5 dpc and 12.0 dpc treated with
anti-SHH antibody (5E1) showed a reduction in Fgf8 expression after
24 hours of culture. (I,J) An increase of apoptosis was observed by the 5E1
treatment. The number of Nile Blue-positive cells was significantly increased
by 5E1 treatment. (A-D,G,H) Ventral views; (E,F,I,J) frontal views. Scale bar:
250 µm.
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Fig. 5. GT hypoplasia of Nog mutant mice. (A,B) Nog mutant mice
showed GT hypoplasia at 16.5 dpc. (C-F) A reduction of Wnt5a and
Fgf8 expression in the developing external genitalia region of
Nog mutants compared with wild type at 12.5 dpc (for Wnt5a)
and at 12.0 dpc (for Fgf8). (G) Reduced cell proliferation of
Nog mutant mice GT. The number of phospho-histone H3 immunopositive
cells decreased to 60% compared with wild type. (H,I) TUNEL analysis was
performed on Nog mutant at 12.5 dpc. Apoptotic-cells were not
detected in distal mesenchyme of Nog mutants. (A,B) Lateral views;
(C-F) ventral views; (H,I) coronal sections. Scale bars: 650 µm in A,B; 250
µm in C-F; 200 µm in H,I.
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Fig. 6. GT agenesis of Wnt5a mutant mice. (A,B) Wnt5a mutant mice
displayed external genitalia agenesis at 18.5 dpc. (C) The number of
phospho-histone H3 immunopositive cells decreased to 40% compared with wild
type at 10.5 dpc. Scale bar: 550 µm in A,B.
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Fig. 7. GT hyperplasia of Bmpr1a conditional mutant mice. (A)
Brn4-Cre-mediated expression of lacZ was detected in the
outermost epithelial layer adjacent to the DUE and the GT surface ectoderm but
not in the DUE at 12.5 dpc. (B) lacZ expression was already detected
at 10.5 dpc before GT outgrowth in the cloacal regions (within the white
arrows). (C) Reduction ( 80%) of pSMAD1 signals in the mutant was detected
at 10.5 dpc before GT formation and more prominently GT at E12.5 dpc (cell
number of wild type is shown as 100%). (D,E) Bmpr1a mutant mice
displayed augmented GT outgrowth proximodistally. Note that the DUE region of
Bmpr1a mutant mice was enlarged compared with that of wild type, as
clearly observed by SEM analysis. The arrow indicates the DUE covered with
epithelia (D,E, inset). (F,G) Augmented Fgf8 expression in the
Bmpr1a mutant mice GT compared with wild type. F also shows
non-specific background signals in addition to the distal DUE expression.
(H,I) A marked decrease of apoptosis was found in the distal GT region of
Bmpr1a mutants. (J,K) GT hyperplasia was prominently observed in
Bmpr1a mutant newborns. (A,H,I) Coronal sections; (B,D-G) ventral
views; (J,K) lateral views. N.D., not detected. Scale bar: 200 µm in A; 300
µm in B; 600 µm in D,E; 750 µm in F,G; 100 µm in H,I; 950 µm in
J,K.
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Fig. 8. Possible signaling crosstalks in the whole distal GT region including the
DUE. (A) SEM picture of an embryonic genital tubercle (GT) at 12.5 dpc. The
yellow region shows the location of the DUE. (B) Coronal sections of the boxed
region in A. DUE, which expresses Fgf8, locates adjacent to the
outer-most epithelial layer (yellow region) aligned with normal GT ectoderm
(region between black broken lines). (C) Possible crosstalks in the distal GT
mesenchymes including BMP4, WNT5A may underlie the alteration of cell survival
or of cell proliferation. Previous studies suggested Fgf8 as a growth
stimulatory factor (Haraguchi et al.,
2000 ). This study indicated a possibility that it could also work
as a survival factor. (D) Ablation of BMP signaling in the Bmpr1a
mutant distal GT region is shown, which may be modulated by distal GT
epithelia (double-headed arrows; possibly also by distal-dorsal epithelia) and
GT mesenchyme. Epithelial derived (either from the outer-most epithelial layer
or from the adjacent GT ectoderm) signals may affect Fgf8 expression
in the DUE and consequently affect apoptosis. Putative positive signaling
factors are indicated by blue characters and negative signaling factors are
indicated by red characters. The antagonist, noggin, is shown in green.
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© The Company of Biologists Ltd 2003
