close

Вход

Забыли?

вход по аккаунту

?

Slug expression during organogenesis in mice.

код для вставкиСкачать
EXPRESSions
THE ANATOMICAL RECORD PART A 271A:189 –191 (2003)
Slug Expression During
Organogenesis in Mice
KATHLEEN F. ORAM, ETHAN A. CARVER, AND THOMAS GRIDLEY*
Jackson Laboratory, Bar Harbor, Maine
ABSTRACT
The vertebrate Slug gene encodes a zinc finger-containing transcriptional repressor. Here we report expression of the mouse Slug gene during
organogenesis and late fetal development using histochemical detection of
␤-galactosidase expressed from a targeted SluglacZ knock-in allele. The Slug
gene is highly expressed in the mesenchymal or stromal component of
numerous organs. It is also highly expressed in craniofacial mesenchyme, in
bone of both mesodermal and neural crest origin, and in the outflow tract
and the endocardial cushions of the heart. Anat Rec Part A 271A:189 –191,
2003. © 2003 Wiley-Liss, Inc.
Key words: bone; Slug; heart; lacZ reporter; zinc finger protein
The Slug gene encodes a zinc finger-containing transcriptional repressor homologous to the product of the
Drosophila Snail gene (Hemavathy et al., 2000; Nieto,
2002). Evidence in tissue culture cells suggests that Slug
expression may regulate epithelial–mesenchymal transitions (Savagner et al., 1997). In several vertebrate species,
expression of the Slug gene is implicated in regulating the
formation and delamination of the mesoderm and neural
crest, both of which are formed by an epithelial–mesenchymal transition (Nieto, 2001). The Slug gene has also
been identified as a downstream target of the E2A-HLF
fusion oncoprotein, and evidence in this system indicated
that Slug acts as an anti-apoptotic agent in mammalian
pro-B cells (Inukai et al., 1999). We previously described
the phenotype of a targeted null mutation of the mouse
Slug gene (Jiang et al., 1998). Slug mutant mice exhibited
postnatal growth deficiency, but, surprisingly, Slug was
not required for either mesoderm or neural crest generation, apparently because of differences in the early expression pattern of Slug in mice as compared to other vertebrates (Jiang et al., 1998; Sefton et al., 1998). In this
report we describe Slug expression during organogenesis
in mice, utilizing sensitive histochemical detection of ␤-galactosidase protein expressed from a targeted SluglacZ
knock-in allele (Jiang et al., 1998).
MATERIALS AND METHODS
The official allele designation for the SluglacZ targeted
mutant allele (Jiang et al., 1998) is Slughtm2Grid. Embryos
were collected from timed matings of a Slughtm2Grid/⫹
heterozygous male with wild-type females from a mixed
C57BL/6J X 129S1/SvImJ background. Embryos were dissected in ice-cold phosphate-buffered saline (PBS), fixed in
4% paraformaldehyde and suspended in 20% sucrose over©
2003 WILEY-LISS, INC.
night at 4°C. Embryos were mounted in OTC medium,
frozen in liquid nitrogen, and cryosectioned at 18 ␮m.
Sections were stained for ␤-galactosidase activity, as described by Hogan et al. (1994). Sections were counterstained with eosin.
RESULTS AND DISCUSSION
In order to analyze expression of the Slug gene during
later stages of embryogenesis and organogenesis in mice,
we used histochemical detection of ␤-galactosidase protein
expressed from a targeted SluglacZ knock-in allele. We
demonstrated previously that expression of the SluglacZ
knock-in allele faithfully reflects the expression pattern of
Slug RNA during early embryogenesis (Jiang et al., 1998).
However, histochemical detection of the ␤-galactosidase
fusion protein was more sensitive and had less background compared to detection of Slug RNA by in situ
hybridization. Therefore, we used histochemistry to assess
expression of the Slug gene during organogenesis and late
fetal development in mice.
Overall, SluglacZ expression levels were highest during
the early stages of organogenesis and declined markedly
by embryonic days 17 (E17) and E18 (Fig. 1A–C). We
Grant sponsor: NIH; Grant numbers: RO1 HD34883; P60
DE13078; T32 CA09217; P30 CA34196.
*Correspondence to: Thomas Gridley, Jackson Laboratory, 600
Main St., Bar Harbor, ME 04609. Fax: (207) 288-6077.
E-mail: gridley@jax.org
Received 10 October 2002; Accepted 25 October 2002
DOI 10.1002/ar.a.10027
Published online 27 January 2003 in Wiley InterScience
(www.interscience.wiley.com).
190
ORAM ET AL.
Figures 1–3.
191
SLUG EXPRESSION DURING ORGANOGENESIS IN MICE
lacZ
previously showed that at E10, the Slug
gene was
highly expressed in craniofacial mesenchyme, particularly
in the frontonasal region (Jiang et al., 1998). The frontonasal region continued to be a major site of SluglacZ expression through E16 (Fig. 1A–D). The SluglacZ gene was
highly expressed in developing bone, including bones undergoing endochondral or intramembranous ossification
(Fig. 1D and E). In developing long bones, SluglacZ expression was highest in proliferating chondrocytes, and was
downregulated in hypertrophic chondrocytes (Fig. 1E).
Expression was also observed in both vibrissae (Fig. 1D)
and hair follicles.
SluglacZ expression was observed in the outflow tract of
the heart, in the endocardial cushions, and later in the
heart valves (Fig. 2). Slug expression was previously observed in the endocardial cushions of the avian heart,
where it is proposed to regulate the epithelial–mesenchymal transformation involved in development of the endocardial cushions (Romano and Runyan, 1999, 2000; Carmona et al., 2000).
lacZ
Very high levels of Slug
expression were observed in
the mesenchymal or stromal component of many developing organs. These included the kidney (Fig. 3A), lung (Fig.
3B), and submandibular gland. High levels of SluglacZ
expression were observed in the wall of the stomach and
intestines, although expression was excluded from the
epithelial lining of the gut (Fig. 3C). SluglacZ expression
was notably excluded from the central and peripheral
nervous systems. In the brain, the only SluglacZ expression observed was in mesenchymal cells in the choroid
plexus (Fig. 3D).
In summary, SluglacZ expression levels were highest
during the early stages of organogenesis and declined
markedly by E17 and E18. The highest levels of SluglacZ
expression during organogenesis were observed in craniofacial mesenchyme, proliferating chondrocytes in bone of
both neural crest and mesodermal origin, intestinal and
stomach walls, and mesenchyme of the lung and kidney.
LITERATURE CITED
Fig. 1. SluglacZ expression during organogenesis stages and in
developing bones. A–C: SluglacZ expression is highest at E13–E15,
and then begins to decline. The regions of most intense ␤-galactosidase activity include the frontonasal region, developing bone, lung,
kidney, and gut. SluglacZ expression is notably excluded from the
central nervous system. D: Expression in the frontonasal region.
SluglacZ is expressed in the nasal cartilages (nc), craniofacial mesenchyme/connective tissue, and vibrissae follicles (arrowheads).
SluglacZ expression is excluded from the olfactory sensory epithelium
(arrows) and the vomeronasal organ (vo). E: In the developing long
bones, SluglacZ is expressed in proliferating chondrocytes (pc) and
the perichondrium (arrows). SluglacZ expression is downregulated in
hypertrophic chondrocytes (hc). SluglacZ is also expressed in isolated
cells (arrowheads) in the bone matrix. All sections in all figures were
stained for ␤-galactosidase activity. A–C, and E: Parasagittal sections. D: Transverse section.
Fig. 2. SluglacZ expression during heart development. A: SluglacZ is
expressed in the atrioventricular cushions (arrowhead) at E13.5. B:
SluglacZ expression is observed in the outflow tract (ot). C: SluglacZ
expression is observed in the mitral (mt) and tricuspid (tv) valves at
E15.5. All sections are parasagittal.
Fig. 3. SluglacZ expression in mesenchyme of numerous organs. A:
SluglacZ is expressed in kidney mesenchyme, but is not expressed in
epithelial cells of developing tubules or glomeruli (arrowheads). Expression is also observed in the perichondrial fibroblasts of the ribs (arrows).
B: SluglacZ is highly expressed in lung mesenchyme, but not in alveolar
epithelium. C: SluglacZ expression was observed in the intestinal wall
(arrow), but is not expressed in the epithelial lining of the gut. D: Expression is observed in mesenchyme of the choroid plexus. E: SluglacZ
expression is observed in the tongue in a subepithelial layer (arow) and
in isolated cells in the tongue epithelium (arrowheads). A–D: Parasagittal
sections. E: Transverse section.
Carmona R, González-Iriarte M, Macı́as D, Pérez-Pomares JM, Garcı́a-Garrido L, Munoz-Chápuli R. 2000. Immunolocalization of the
transcription factor Slug in the developing avian heart. Anat Embryol 201:103–109.
Hemavathy K, Ashraf SI, Ip YT. 2000. Snail/Slug family of repressors:
slowly going into the fast lane of development and cancer. Gene
257:1–12.
Hogan B, Beddington R, Costantini F, Lacy E. 1994. Manipulating the
mouse embryo. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. p 373–375.
Inukai T, Inoue A, Kurosawa H, Goi K, Shinjyo T, Ozawa K, Mao M,
Inaba T, Look AT. 1999. SLUG, a ces-1-related zinc finger transcription factor gene with antiapoptotic activity, is a downstream
target of the E2A-HLF oncoprotein. Mol Cell 4:343–352.
Jiang R, Lan Y, Norton CR, Sundberg JP, Gridley T. 1998. The Slug
gene is not essential for mesoderm or neural crest development in
mice. Dev Biol 198:277–285.
Nieto MA. 2001. The early steps of neural crest development. Mech
Dev 105:27–35.
Nieto MA. 2002. The Snail superfamily of zinc finger transcription
factors. Nat Rev Mol Cell Biol 3:155–166.
Romano LA, Runyan RB. 1999. Slug is a mediator of epithelial–
mesenchymal cell transformation in the developing chicken heart.
Dev Biol 212:243–254.
Romano LA, Runyan RB. 2000. Slug is an essential target of TGF␤2
signaling in the developing chicken heart. Dev Biol 223:91–102.
Savagner P, Yamada KM, Thiery JP. 1997. The zinc-finger protein
Slug causes desmosome dissociation, an initial and necessary step
for growth factor-induced epithelial–mesenchymal transition.
J Cell Biol 137:1403–1419.
Sefton M, Sánchez S, Nieto MA. 1998. Conserved and divergent roles
for members of the Snail family of transcription factors in the chick
and mouse embryo. Development 125:3111–3121.
Документ
Категория
Без категории
Просмотров
0
Размер файла
558 Кб
Теги
expressions, sluga, mice, organogenesis
1/--страниц
Пожаловаться на содержимое документа