小鼠牙齿形态发生过程中Msx1/2、BMP-4的表达及细胞凋亡的研究
摘要
牙齿形态发生包括一系列决定器官发育启始和结构调控的过程。在
细胞水平，它主要表现为细胞的增殖和分化；在分子水平调控牙齿形态发
生的主要包括信号分子、信号分子受体和转录因子，它们形成了调节牙齿
发育的信号网络系统。细胞在这一网络系统的调节下发生增殖、分化、聚
集以及凋亡。近来homeobox基因家族成员在发育中的作用受到了越来越多
的重视。Homeobox基因是一类在进化过程中高度保守的基因家族，它编
码一组特殊的转录因子。Msx基因（Muscle segment homeobox gene）是
Homeobox基因家族的重要成员，是器官发育过程中的主调控基因。BMP-4
在牙齿发育中既是生长因子又是重要的信号分子。细胞凋亡可促进衰老细
胞和组织的自身清除以及组织塑形。但在牙齿形态发生过程中转录因子
Msx1/2、信号分子BMP-4及细胞生理性凋亡的系统性研究国内外尚未见报
道。本研究采用原位杂交技术和原位末端标记（TUNEL）法，观察Msx1/2、
BMP-4在小鼠牙齿形态发生各阶段的时空表达变化及生理性凋亡细胞的
分布区域，并探讨其调控作用及生理意义。牙齿的形成过程与修复过程具
有一定的相似性，同样的信号途径可能在其中发挥作用。本实验初步揭示
了牙齿发育调控网络的部分细节，为进一步研究牙齿修复机制奠定了基
础。实验内容及结果如下：
一．小鼠牙齿形态发生过程中Msx基因的表达及意义
本实验采用原位杂交的方法，观察小鼠牙齿形态发生各阶段（蕾状
期，帽状期，钟状期）Msx基因的动态表达情况。结果发现Msx1与Msx2
的表达具有如下模式：
第四军医大学硕士学位论文
一
——
从以上结果推测：蕾状期，MSXI参与了外胚间充质细胞的聚集和信
号分子BMP4从上皮向间充质转移的过程；牙蕾上皮表达的MSXZ则通
过激活钙依赖性粘附分子来调控间充质细胞的分化和增殖。帽状期，
MSXI诱导BMP－4生成，从而参与了成牙本质细胞的分化；MSXZ与牙
乳头、牙囊间充质细胞以及内外釉上皮的进一步分化有关，而其在釉结
区域的过表达与釉结的生长停滞和凋亡有着密切联系。钟状期，MSXI
与牙本质基质蛋白的分泌相关；MSXZ则继续参与成牙本质细胞和成釉
细胞的分化。
二．小鼠牙齿形态发生过程中BMP－4表达及凋亡细胞分布
1．采用原位杂交的方法，观察小鼠牙齿形态发生各阶段（蕾状期，
帽状期，钟状期）中BMP－4的表达情况。结果显示：蕾状期时，牙胚BMP－4
在上皮蕾和其周围聚集的牙间充质中均有表达；帽状期中，BMP4阳性细
胞主要位于内外釉上皮、釉结及靠近内外釉上皮的牙囊和牙乳头细胞；而
在钟状期，BMP－4阳性细胞则位于内外釉上皮、前成牙本质细胞中。这一
结果提示：BMP－4具有多重的效应，它在作为生长因于发挥作用的同时，
其更为重要的生物学作用则是充当信号分子，调节上皮间充质的相互作
用，以及牙胚细胞的凋亡。
2．采用原位末端标记法（TUNEL），观察小鼠牙齿形态发生各阶段凋
亡细胞的分布情况。结果显示：蕾状期，凋亡细胞主要位于蕾状突的头部：
帽状期，凋亡细胞集中在内釉上皮凹面中心区域（即釉结）和内外釉上皮
交界的颈环处；钟状期，凋亡细胞在未来牙尖部位（即继发性釉结处）和
－3－
第四军医大学硕士学位论文
一
颈环处较密集。表明在牙齿形态发生过程中，凋亡被上皮间充质相互作用
调节，它通过控制细胞的增殖数量，清除衰老的细胞和暂时性结构，发挥
重要的塑形作用。对照前面实验的结果，推测上皮性信号中心釉结的清除
过程中，BMP－4是通过MsXZ来调控其细胞的凋亡。
Tooth morphogenesis is a series of process including the initiation of organogenesis and the regulation of structures. Morphogenesis in cellular level mainly means the proliferation and differentiation of cells. The migration of ectomesenchymal cells derived from neurocrest plays a vital role in the initiation of dental development. Apoptosis has a great value on the maintenance of histo-differatiation, organogenesis and homestasis of multicellular organ. During dental development, the regulatory molecules include signaling molecules and recepotors, transcription factors, which involve in the signaling networks. The signaling networks mediate cellular proliferation, differentiation, adhesion, apoptosis.
Recently the homeobox genes family is found during the course of embryogenesis. Homeobox genes are the highly conservative DNA sequences throughout evolution, which encode the homeodomain proteins binding with DNA. The homeobox genes are regulators of place-dependent morphogenesis during embryogenesis. Muscle segment homeobox genes (Msx) are sort of master regulator gene during embryogenesis, including
Msxl, Msx2, and Msx3, which participate in embryogenesis. The functional deficiency of human Msx genes can result in prenatal dysgenesis.
So in our research, we observed the dynamic expression of Msx 1/2, BMP-4, and the distribution of apoptosis in the growth phase of mouse teeth by molecular biological experimental technology, then exploring the regulatory roles of Msxl/2, BMP-4, the significance of apoptosis and their reciprocal relationship. The study consisted of two parts described as below:
l.The study on the expression of Msx gene during mouse odontogenesis
In this research, we mapped out the dynamic localization of Msxl/2 during every phase of mouse tooth germ (bud stage, cap stage, bell stage) by in situ hybridization. The results were :
Localization of Msx 1 Localization of Msx2
Bud stage dental mesenchyme epithelium of dental bud
Cap stage dental sac, dental papilla near enamel epithelium inner and outer enamel epithelium, enamel knot
Bell stage preodontoblast inner and outer enamel epithelium, preodontoblast
It suggested that there were apparent difference in regulatory roles of tooth development between Msxl and Msx2. At bud stage, Msxl participateed in the adhesion of ectomesenchymal cells and migration of signaling BMP-4 from epithelium to mesenchyme; Msx2 could induce Ca +-dependent adhesion molecules to mediate the proliferation and
differentiation of mesenchyme cells. At cap stage, Msxl could induce BMP-4 to play a role in the differentiation of odontoblast, and Msx2 could induce differentiation of dental papilla cells, dental sac cells and enamel epithelium cells, and it also initiated the apoptosis of enamel knots. At bell stage, Msxl could induce the dentinogenesis; Msx2 could participate in the differientiation of odontoblast and ameloblasts.
2.The study on localization of BMP-4 and apoptosis
2.1 In this research, we mapped out the localization of BMP-4 during every phase of rat tooth germ (bud stage, cap stage, bell stage) by in situ hybridization. The results indicated that BMP-4 was confined to dental epithelium and adhered dental mesenchyme at bud stage, and BMP-4 positive signals distribute in inner and outer enamel epithelium, enamel knot, dental sac and preodontoblast. It suggested that BMP-4 was not only growth factor but also the important signaling molecule to regulate the interaction between epithelium and mesenchyme.
2.2 In this research, we observed the localization of apoptosis during the growth phase of rat tooth germ by TUNEL. The results indicated that apoptosis distribute in head of bud protrusion at bud stage; primary enamel knot and cervical round at cap stage; secondary enamel knot and cervical round at bell stage. This suggested that apoptosis could be regulated by interaction of epithelium-mesenchyme, and could control the quantities of proliferating cells, remove the aging cells and temporary structures, and play a role in pat
细胞水平，它主要表现为细胞的增殖和分化；在分子水平调控牙齿形态发
生的主要包括信号分子、信号分子受体和转录因子，它们形成了调节牙齿
发育的信号网络系统。细胞在这一网络系统的调节下发生增殖、分化、聚
集以及凋亡。近来homeobox基因家族成员在发育中的作用受到了越来越多
的重视。Homeobox基因是一类在进化过程中高度保守的基因家族，它编
码一组特殊的转录因子。Msx基因（Muscle segment homeobox gene）是
Homeobox基因家族的重要成员，是器官发育过程中的主调控基因。BMP-4
在牙齿发育中既是生长因子又是重要的信号分子。细胞凋亡可促进衰老细
胞和组织的自身清除以及组织塑形。但在牙齿形态发生过程中转录因子
Msx1/2、信号分子BMP-4及细胞生理性凋亡的系统性研究国内外尚未见报
道。本研究采用原位杂交技术和原位末端标记（TUNEL）法，观察Msx1/2、
BMP-4在小鼠牙齿形态发生各阶段的时空表达变化及生理性凋亡细胞的
分布区域，并探讨其调控作用及生理意义。牙齿的形成过程与修复过程具
有一定的相似性，同样的信号途径可能在其中发挥作用。本实验初步揭示
了牙齿发育调控网络的部分细节，为进一步研究牙齿修复机制奠定了基
础。实验内容及结果如下：
一．小鼠牙齿形态发生过程中Msx基因的表达及意义
本实验采用原位杂交的方法，观察小鼠牙齿形态发生各阶段（蕾状
期，帽状期，钟状期）Msx基因的动态表达情况。结果发现Msx1与Msx2
的表达具有如下模式：
第四军医大学硕士学位论文
一
——
从以上结果推测：蕾状期，MSXI参与了外胚间充质细胞的聚集和信
号分子BMP4从上皮向间充质转移的过程；牙蕾上皮表达的MSXZ则通
过激活钙依赖性粘附分子来调控间充质细胞的分化和增殖。帽状期，
MSXI诱导BMP－4生成，从而参与了成牙本质细胞的分化；MSXZ与牙
乳头、牙囊间充质细胞以及内外釉上皮的进一步分化有关，而其在釉结
区域的过表达与釉结的生长停滞和凋亡有着密切联系。钟状期，MSXI
与牙本质基质蛋白的分泌相关；MSXZ则继续参与成牙本质细胞和成釉
细胞的分化。
二．小鼠牙齿形态发生过程中BMP－4表达及凋亡细胞分布
1．采用原位杂交的方法，观察小鼠牙齿形态发生各阶段（蕾状期，
帽状期，钟状期）中BMP－4的表达情况。结果显示：蕾状期时，牙胚BMP－4
在上皮蕾和其周围聚集的牙间充质中均有表达；帽状期中，BMP4阳性细
胞主要位于内外釉上皮、釉结及靠近内外釉上皮的牙囊和牙乳头细胞；而
在钟状期，BMP－4阳性细胞则位于内外釉上皮、前成牙本质细胞中。这一
结果提示：BMP－4具有多重的效应，它在作为生长因于发挥作用的同时，
其更为重要的生物学作用则是充当信号分子，调节上皮间充质的相互作
用，以及牙胚细胞的凋亡。
2．采用原位末端标记法（TUNEL），观察小鼠牙齿形态发生各阶段凋
亡细胞的分布情况。结果显示：蕾状期，凋亡细胞主要位于蕾状突的头部：
帽状期，凋亡细胞集中在内釉上皮凹面中心区域（即釉结）和内外釉上皮
交界的颈环处；钟状期，凋亡细胞在未来牙尖部位（即继发性釉结处）和
－3－
第四军医大学硕士学位论文
一
颈环处较密集。表明在牙齿形态发生过程中，凋亡被上皮间充质相互作用
调节，它通过控制细胞的增殖数量，清除衰老的细胞和暂时性结构，发挥
重要的塑形作用。对照前面实验的结果，推测上皮性信号中心釉结的清除
过程中，BMP－4是通过MsXZ来调控其细胞的凋亡。
Tooth morphogenesis is a series of process including the initiation of organogenesis and the regulation of structures. Morphogenesis in cellular level mainly means the proliferation and differentiation of cells. The migration of ectomesenchymal cells derived from neurocrest plays a vital role in the initiation of dental development. Apoptosis has a great value on the maintenance of histo-differatiation, organogenesis and homestasis of multicellular organ. During dental development, the regulatory molecules include signaling molecules and recepotors, transcription factors, which involve in the signaling networks. The signaling networks mediate cellular proliferation, differentiation, adhesion, apoptosis.
Recently the homeobox genes family is found during the course of embryogenesis. Homeobox genes are the highly conservative DNA sequences throughout evolution, which encode the homeodomain proteins binding with DNA. The homeobox genes are regulators of place-dependent morphogenesis during embryogenesis. Muscle segment homeobox genes (Msx) are sort of master regulator gene during embryogenesis, including
Msxl, Msx2, and Msx3, which participate in embryogenesis. The functional deficiency of human Msx genes can result in prenatal dysgenesis.
So in our research, we observed the dynamic expression of Msx 1/2, BMP-4, and the distribution of apoptosis in the growth phase of mouse teeth by molecular biological experimental technology, then exploring the regulatory roles of Msxl/2, BMP-4, the significance of apoptosis and their reciprocal relationship. The study consisted of two parts described as below:
l.The study on the expression of Msx gene during mouse odontogenesis
In this research, we mapped out the dynamic localization of Msxl/2 during every phase of mouse tooth germ (bud stage, cap stage, bell stage) by in situ hybridization. The results were :
Localization of Msx 1 Localization of Msx2
Bud stage dental mesenchyme epithelium of dental bud
Cap stage dental sac, dental papilla near enamel epithelium inner and outer enamel epithelium, enamel knot
Bell stage preodontoblast inner and outer enamel epithelium, preodontoblast
It suggested that there were apparent difference in regulatory roles of tooth development between Msxl and Msx2. At bud stage, Msxl participateed in the adhesion of ectomesenchymal cells and migration of signaling BMP-4 from epithelium to mesenchyme; Msx2 could induce Ca +-dependent adhesion molecules to mediate the proliferation and
differentiation of mesenchyme cells. At cap stage, Msxl could induce BMP-4 to play a role in the differentiation of odontoblast, and Msx2 could induce differentiation of dental papilla cells, dental sac cells and enamel epithelium cells, and it also initiated the apoptosis of enamel knots. At bell stage, Msxl could induce the dentinogenesis; Msx2 could participate in the differientiation of odontoblast and ameloblasts.
2.The study on localization of BMP-4 and apoptosis
2.1 In this research, we mapped out the localization of BMP-4 during every phase of rat tooth germ (bud stage, cap stage, bell stage) by in situ hybridization. The results indicated that BMP-4 was confined to dental epithelium and adhered dental mesenchyme at bud stage, and BMP-4 positive signals distribute in inner and outer enamel epithelium, enamel knot, dental sac and preodontoblast. It suggested that BMP-4 was not only growth factor but also the important signaling molecule to regulate the interaction between epithelium and mesenchyme.
2.2 In this research, we observed the localization of apoptosis during the growth phase of rat tooth germ by TUNEL. The results indicated that apoptosis distribute in head of bud protrusion at bud stage; primary enamel knot and cervical round at cap stage; secondary enamel knot and cervical round at bell stage. This suggested that apoptosis could be regulated by interaction of epithelium-mesenchyme, and could control the quantities of proliferating cells, remove the aging cells and temporary structures, and play a role in pat
引文
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37． Graham, A., Heyman, I., Lumsden, A., 1993． Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain. Development 119,233-245．
38． Graham, A., Francis-West, P., Brickell, P., Lumsden, A., 1994． The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372, 684-686．
39． Hodgkinson, J.E., Davidson, C.L., Beresford, J., Sharpe, P.T., 1993． Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells. Biochim. Biophys. Acta 1174, 11-16．
40． Holland, P.W., Hogan, B.L., 1988． Expression of homeobox genes during mouse development: a review. Genes Dev. 2, 773-282．
41． Hu, G., Vastardis, H., Bendall, A.J., Wang, Z., Logan, M., Zhang, H., Nelson, C., Stein, S., Greenfield, N., Seidman, C.E., Seidman, J.G., Abate-Shen, C., 1998． Haploinsufficiency of MSX1: A mechanism for selective tooth agenesis. Mol. Cell. Biol. 18, 6044-6051．
42． Jernvall, J., Aberg, T., Kettunun, P., Keraen, S., Thesleff, I., 1998． The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161-169．
43． Johnson, R.L., Tabin, C.J., 1997． Molecular models for vertebrate limb development. Cell 90, 979-990．
44． Jowett, A.K., Vainio, S., Ferguson, M.W., Sharpe, P.T., Thesleff, I., 1993． Epithelial-mesenchymal interactions are required for Msxl and Msx2 gene expression in the developing murine molar tooth. Development 117, 461-470．
45． Kessel, M., Gruss, P., 1990． Murine developmental control genes. Science 249, 374-379．
46． Liu, J.K., Ghattas, I., Liu, S., Chen, S., Rubenstein, J.L., 1997． Dlx genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev. Dyn. 210, 498-512．
47． Lumsden, A., Sprawson, N., Graham, A., 1991． Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113, 1281-1291．
48． Maeda, R., Kobayashi, A., Sekine, R., Lin, J.J., Kung, H.-F., Maeno, M., 1997． Xmsxl modifies mesodermal tissue pattern along
dorsoventral axis in Xenopus laevis embryo. Development 124, 2553-2560．
49． Marazzi, G., Wang, Y., Sassoon, D., 1997． Msx2 is a transcriptional regulator in the BMP4-mediated programmed cell death pathway. Dev. Biol. 186, 127-138．
50． Miyama, K., Yamada, G., Yamamoto, T.S., Takagi, C., Miyado, K., Sakai, M., Ueno, N., Shibuya, H., 1999． A BMP-inducible gene, Dlx5, regulates osteoblast differentiation and mesoderm induction. Dev. Biol. 208, 123-133．
51． Morasso, M.I., Jamrich, M., Sargent, T.D., 1993． The homeodomam gene Xenopus Distal-less-like-2 (Xdll-2) is regulated by a conserved mechanism in amphibian and mammalian epidermis. Dev. Biol. 162, 267-276．
52． Morasso, M.I., Markova, N.G., Sargent, T.D., 1996． Regulation of epidermal differentiation by a Distal-less homeodomam gene. J. Cell Biol. 135, 1879-1887．
53． Morasso, M.I., Yonescu, R., Griffin, C.A., Sargent, T.D., 1997． Localization of human DLX8 to chromosome 17q21． 3-q22 by fluorescence in-situ hybridization. Mamm. Genome 8, 302-303．
54． Papalopulu, N., Kintner, C., 1993． Xenopus Distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals. Development 117, 961-975．
55． Pavlova, A., Boutin, E., Cunha, G., Sassoon, D., 1994． Msxl (Hox7． 1) in the adult mouse uterus: cellular interactions underlying regulation of expression. Development 120, 335-346．
56． Pera, E., Stein, S., Kessel, M., 1999． Ectodermal patterning in the avian embryo: epidermis versus neural plate. Development 126, 63-73．
57． Peters, H., Balling, R., 1999． Teeth. Where and how to make them. Trends Genet. 15,59-65．
58． Phippard, D.J., Weber-Hall, S.J., Sharpe, P.T., Naylor, M.S., Jayatalake, H., Maas, R., Woo, I., Roberts-Clark, D., Francis-West, P.H., Liu, Y.H., Maxson, R., Hill, R.E., Dale, T.C., 1996． Regulation of Msxl, Msx2, BMP-2 and BMP-4 during foetal and postnatal mammary gland development. Development 122, 2729-2737．
59． Porteus, M.H., Bulfone, A., Ciaranello, R.D., Rubenstein, J.L.R., 1991． Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain. Neuron 7, 221-229．
60． Porteus, M.H., Bulfone, A., Liu, J.K., Puelles, L., Lo, L.C., Rubenstein, J.L., 1994． Dlx2, Mashl, and Map2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain. J. Neurosci. 14, 6370-6383．
61． Price, M., Lemaistre, M., Pischetola, M., Di Lauro, R., Duboule, D., 1991． A mouse gene related to Distal-less shows a restricted expression in the developing forebrain. Nature 351, 748-751．
62． Price, M., 1993． Members of the Dlx-and Nkx2-gene families are regionally expressed in the developing forebrain. J. Neurobiol. 24, 1385-1399．
63． Robinson, G.W., Wray, S., Mahon, K.A., 1991． Spatially restricted expression of a member of a new family of murine Distal-less
homeobox genes in the developing forebrain. New Biol. 3, 1183-1194．
64． Salinas, P.C., Nusse, R., 1992． Regional expression of the Wnt-3 gene in the developing mouse forebrain in relationship to diencephalic neuromeres. Mech. Dev. 39, 151-160．
65． Sharpe, P.T., 1995． Homeobox genes and orofacial development. Connect. Tissue Res. 32, 17-25．
66． Sheng, H.Z., Bertuzzi, S., Chiang, C., Shawlot, W., Taira, M., Dawid, I., Westphal, H., 1997． Expression of murine Lhx5 suggests a role in specifying the forebrain. Dev. Dyn. 208, 266-277．
67． Smith, S.T., Jaynes, J.B., 1996． A conserved region of engrailed, shared among all en-, gsc-, Nkl-, Nk2-, and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141-3150．
68． Suzuki, A., Ueno, N., Hemmati-Brivanlou, A., 1997． Xenopus Msxl mediates epidermal induction and neural inhibition by BMP-4． Development 124, 3037-3044．
69． Takahashi, Y., Le Douarin, N., 1990． cDNA cloning of a quail homeobox gene and its expression in neural crest-derived mesenchyme and lateral plate mesoderm. Proc. Natl. Acad. Sci. USA 87, 7482-7486．
70． Thesleff, I., Nieminen, P., 1996． Tooth morphogenesis and cell differentiation. Curr. Opin. Cell Biol. 8, 844-850．
2． Kratochwil,K.,Dull,M.,Farinas,I.,Galceran,J.Grosschedl,R.Lefl
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3． Chen Y, Zhao X, Shaping limbs by apoptosis. J Exp Zool 1998 Dec 15;282(6) :691-702．
4． Ferrari D, Lichtler AC, Pan ZZ, Dealy CN, Upholt WB, Kosher RA , Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis. Dev Biol 1998 May 1;197(1) :12-24
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7． Thesleff,I.and Sharpe,P. Signalling networks regulating dental development.Mech Dev 67: 111-123; 1997．
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9． Vainio,S.,Karavanova,I.,Jowett,A.,and Thesleff,I.Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during tooth development. Cell 75:45-58;1993．
10． Kettunen,P.and Thesleff,I.Expression and function of FGFs-4,-8,-9
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11． YiPing Chen,Marianna Bei,Ian Woo,Ichiro Satokata and Richard Mass.Msxlcontroles inductive signaling in mammalian tooth morphogenesis.Development 122:3035-3044;1996．
12． Zhou p,Byrne C,Jacobs J,Fuchs e.Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate.Genes Dev 9:700-713;1995．
13． Lsot,H.,Vonesch,J.,Peterka,M.,Tureckova,J.,Peterkova,R. and Ruch, J.V. Mouse molar morphogenesis revisited dy three-dimensional reconstructionll. Spatial distribution of mitoses and apoptosis in cup to bell staged first and second upper molar teeth. Int.J.Dev.Biol. 40:1017-1031;1996．
14． Graham,A., Francis,W.P., Brickell,P. and Lumsden,A. The signalling molecule BMP-4 mediated apoptosis in the rhombencephalic neural crest.Nature 372:684-686; 1994．
15． Jukka Jernvall,Thomas Aberg,Paivi Kettunen,Soile Keranen and Irma Thesleff.Thelife history of an embryonic signaling center:Bmp4 induces P21 and is associated with apoptosis in the mouse tooth enamel knot Development 125:161-169;1998．
16． Acampora, D., Merlo, G.R., Paleari, L., Zerega, B., Postiglione, M.P., Mantero, S., Bober, E., Barbieri, O., Simeone, A., Levi, G., 1999． Craniofacial, vestibular and bone defects in mice lacking theDistal-less-related gene Dlx5． Development 126, 3795-3809．
17． Anderson, S.A., Eisenstat, D.D., Shi, L., Rubenstein, J.L., 1997a.
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18． Asano, M., Emori, Y., Saigo, K., Shiokawa, K., 1992． Isolation and characterization of a Xenopus cDNA which encodes a homeodomain highly homologous to Drosophila Distal-less. J. Biol. Chem. 267, 5044-5047．
19． Beauchemin, M., Savard, P., 1992． Two distal-less related homeobox-containing genes expressed in regeneration blastemas of the newt. Dev. Biol. 154, 55-65．
20． Begbie, J., Brunet, J.F., Rubenstein, J.L., Graham, A., 1999． Induction of the epibranchial placodes. Development 126, 895-902．
21． Bei, M., Maas, R., 1998． FGFs and BMP4 induce both Msxl-independent and Msxl-dependent signaling pathways in early tooth development. Development 125, 4325-4333．
22． Bulfone, A., Kim, H.J., Puelles, L., Porteus, M.H., Grippo, J.F., Rubenstein, J.L., 1993a. The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos. Mech. Dev. 40, 129-140．
23． Bulfone, A., Puelles, L., Porteus, M.H., Frohman, M.A., Martin, G.R., Rubenstein, J.L., 1993b. Spatially restricted expression of Dlxl, Dlx2 (Tesl), Gbx2, and Wnt3 in the embryonic day 12． 5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13,3155-3172．
24． Catron, K.M., Iler, N., Abate, C., 1993． Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three
murine homeodomain proteins. Mol. Cell. Biol. 13, 2354-2365．
25． Chen, Y., Zhao, X., 1998． Shaping limbs by apoptosis. J. Exp. Zool. 282, 691-702．
26． Cohen, S.M., Bronner, G., Kuttner, F, Jurgens, G., Jackie, H., 1989． Distal-less encodes a homoeodomain protein required for limb development in Drosophila. Nature 338, 432-434．
27． Davidson, D.R., Crawley, A., Hill, R.E., Tickle, C., 1991． Positiondependent expression of two related homeobox genes in developing vertebrate limbs. Nature 352, 429-431．
28． Mina, M., Gluhak, J., Upholt, W.B., Kollar, E.J., Rogers, B., 1995． Experimental analysis of Msx-1 and Msx-2 gene expression during chick mandibular morphogenesis. Dev. Dyn. 202, 195-214．
29． Depew, M.J., Liu, J.K., Long, J.E., Presley, R., Meneses, J.J., Pedersen, R.A., Rubenstein, J.L.R., 1999． Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126, 3831-3846．
30． Dirksen, M.L., Mathers, P., Jamrich, M., 1993． Expression of a Xenopus Distal-less homeobox gene involved in forebrain and craniofacial development. Mech. Dev. 41, 121-128．
31． Dirksen, M.L., Morasso, M.I., Sargent, T.D., Jamrich, M., 1994． Differential expression of a Distal-less homeobox gene Xdll-2 in ectodermal cell lineages. Mech. Dev. 46, 63-70．
32． Ekker, M., Akimenko, M.A., Bremiller, R., Westerfield, M., 1992． Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos. Neuron 9, 27-35．
33． Ekker, M., Akimenko, M.A., Allende, M.L., Smith, R., Drouin, G., Langille, R.M., Weinberg, E.S., Westerfield, M., 1997． Relationships among msx gene structure and function in zebrafish and other vertebrates. Mol. Biol. Evol. 14, 1008-1022．
34． Fang, H., Elinson, R.P., 1996． Patterns of distal-less gene expression and inductive interactions in the head of the direct developing frog Eleutherodactylus coqui. Dev. Biol. 179, 160-172．
35． Feledy, J.A., Morasso, M.I., Jang, S.I., Sargent, T.D., 1999b. Transcriptional activation by the homeodomain protein distal-less 3． Nucleic Acids Res. 27, 764-770．
36． Graham, A., Papalopulu, N., Krumlauf, R., 1989． The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57, 367-378．
37． Graham, A., Heyman, I., Lumsden, A., 1993． Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain. Development 119,233-245．
38． Graham, A., Francis-West, P., Brickell, P., Lumsden, A., 1994． The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372, 684-686．
39． Hodgkinson, J.E., Davidson, C.L., Beresford, J., Sharpe, P.T., 1993． Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells. Biochim. Biophys. Acta 1174, 11-16．
40． Holland, P.W., Hogan, B.L., 1988． Expression of homeobox genes during mouse development: a review. Genes Dev. 2, 773-282．
41． Hu, G., Vastardis, H., Bendall, A.J., Wang, Z., Logan, M., Zhang, H., Nelson, C., Stein, S., Greenfield, N., Seidman, C.E., Seidman, J.G., Abate-Shen, C., 1998． Haploinsufficiency of MSX1: A mechanism for selective tooth agenesis. Mol. Cell. Biol. 18, 6044-6051．
42． Jernvall, J., Aberg, T., Kettunun, P., Keraen, S., Thesleff, I., 1998． The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161-169．
43． Johnson, R.L., Tabin, C.J., 1997． Molecular models for vertebrate limb development. Cell 90, 979-990．
44． Jowett, A.K., Vainio, S., Ferguson, M.W., Sharpe, P.T., Thesleff, I., 1993． Epithelial-mesenchymal interactions are required for Msxl and Msx2 gene expression in the developing murine molar tooth. Development 117, 461-470．
45． Kessel, M., Gruss, P., 1990． Murine developmental control genes. Science 249, 374-379．
46． Liu, J.K., Ghattas, I., Liu, S., Chen, S., Rubenstein, J.L., 1997． Dlx genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev. Dyn. 210, 498-512．
47． Lumsden, A., Sprawson, N., Graham, A., 1991． Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo. Development 113, 1281-1291．
48． Maeda, R., Kobayashi, A., Sekine, R., Lin, J.J., Kung, H.-F., Maeno, M., 1997． Xmsxl modifies mesodermal tissue pattern along
dorsoventral axis in Xenopus laevis embryo. Development 124, 2553-2560．
49． Marazzi, G., Wang, Y., Sassoon, D., 1997． Msx2 is a transcriptional regulator in the BMP4-mediated programmed cell death pathway. Dev. Biol. 186, 127-138．
50． Miyama, K., Yamada, G., Yamamoto, T.S., Takagi, C., Miyado, K., Sakai, M., Ueno, N., Shibuya, H., 1999． A BMP-inducible gene, Dlx5, regulates osteoblast differentiation and mesoderm induction. Dev. Biol. 208, 123-133．
51． Morasso, M.I., Jamrich, M., Sargent, T.D., 1993． The homeodomam gene Xenopus Distal-less-like-2 (Xdll-2) is regulated by a conserved mechanism in amphibian and mammalian epidermis. Dev. Biol. 162, 267-276．
52． Morasso, M.I., Markova, N.G., Sargent, T.D., 1996． Regulation of epidermal differentiation by a Distal-less homeodomam gene. J. Cell Biol. 135, 1879-1887．
53． Morasso, M.I., Yonescu, R., Griffin, C.A., Sargent, T.D., 1997． Localization of human DLX8 to chromosome 17q21． 3-q22 by fluorescence in-situ hybridization. Mamm. Genome 8, 302-303．
54． Papalopulu, N., Kintner, C., 1993． Xenopus Distal-less related homeobox genes are expressed in the developing forebrain and are induced by planar signals. Development 117, 961-975．
55． Pavlova, A., Boutin, E., Cunha, G., Sassoon, D., 1994． Msxl (Hox7． 1) in the adult mouse uterus: cellular interactions underlying regulation of expression. Development 120, 335-346．
56． Pera, E., Stein, S., Kessel, M., 1999． Ectodermal patterning in the avian embryo: epidermis versus neural plate. Development 126, 63-73．
57． Peters, H., Balling, R., 1999． Teeth. Where and how to make them. Trends Genet. 15,59-65．
58． Phippard, D.J., Weber-Hall, S.J., Sharpe, P.T., Naylor, M.S., Jayatalake, H., Maas, R., Woo, I., Roberts-Clark, D., Francis-West, P.H., Liu, Y.H., Maxson, R., Hill, R.E., Dale, T.C., 1996． Regulation of Msxl, Msx2, BMP-2 and BMP-4 during foetal and postnatal mammary gland development. Development 122, 2729-2737．
59． Porteus, M.H., Bulfone, A., Ciaranello, R.D., Rubenstein, J.L.R., 1991． Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain. Neuron 7, 221-229．
60． Porteus, M.H., Bulfone, A., Liu, J.K., Puelles, L., Lo, L.C., Rubenstein, J.L., 1994． Dlx2, Mashl, and Map2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain. J. Neurosci. 14, 6370-6383．
61． Price, M., Lemaistre, M., Pischetola, M., Di Lauro, R., Duboule, D., 1991． A mouse gene related to Distal-less shows a restricted expression in the developing forebrain. Nature 351, 748-751．
62． Price, M., 1993． Members of the Dlx-and Nkx2-gene families are regionally expressed in the developing forebrain. J. Neurobiol. 24, 1385-1399．
63． Robinson, G.W., Wray, S., Mahon, K.A., 1991． Spatially restricted expression of a member of a new family of murine Distal-less
homeobox genes in the developing forebrain. New Biol. 3, 1183-1194．
64． Salinas, P.C., Nusse, R., 1992． Regional expression of the Wnt-3 gene in the developing mouse forebrain in relationship to diencephalic neuromeres. Mech. Dev. 39, 151-160．
65． Sharpe, P.T., 1995． Homeobox genes and orofacial development. Connect. Tissue Res. 32, 17-25．
66． Sheng, H.Z., Bertuzzi, S., Chiang, C., Shawlot, W., Taira, M., Dawid, I., Westphal, H., 1997． Expression of murine Lhx5 suggests a role in specifying the forebrain. Dev. Dyn. 208, 266-277．
67． Smith, S.T., Jaynes, J.B., 1996． A conserved region of engrailed, shared among all en-, gsc-, Nkl-, Nk2-, and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122, 3141-3150．
68． Suzuki, A., Ueno, N., Hemmati-Brivanlou, A., 1997． Xenopus Msxl mediates epidermal induction and neural inhibition by BMP-4． Development 124, 3037-3044．
69． Takahashi, Y., Le Douarin, N., 1990． cDNA cloning of a quail homeobox gene and its expression in neural crest-derived mesenchyme and lateral plate mesoderm. Proc. Natl. Acad. Sci. USA 87, 7482-7486．
70． Thesleff, I., Nieminen, P., 1996． Tooth morphogenesis and cell differentiation. Curr. Opin. Cell Biol. 8, 844-850．