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The essential roles of the small GTPase Rac1 in limb development
Journal of Oral Biosciences 55 (2013) 116–121
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Review
The essential roles of the small GTPase Rac1 in limb development Dai Suzuki n, Atsushi Yamada, Ryutaro Kamijo Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 7 March 2013 Received in revised form 4 April 2013 Accepted 23 April 2013 Available online 21 June 2013
Vertebrate limbs develop through complicated interactions between various types of cells of the ectoderm and mesenchyme, which facilitate the proper steps of growth and formation by cell proliferation, differentiation, and apoptosis. These processes are regulated by several signaling proteins such as Fgf, Bmp, and Wnt. Recently, several types of Rac1 conditional knockout mice were created, and Rac1 was observed to play essential roles in each of the developing limb tissues. Mice with genetic deletion of Rac1 in the chondrocytes, the limb bud ectoderm, or the limb bud mesenchyme exhibit dwarfism with short limbs, severe truncations of limbs, or syndactyly with short limbs, respectively. Analyses of these mice demonstrated that Rac1 regulates limb development by controlling many kinds of gene expression and various cellular functions. & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Conditional knockout mice Limb development Rac1
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 1.1. Limb development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2. Rac1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 2.1. The roles of Rac1 in chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 2.2. The roles of Rac1 in the limb bud ectoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 2.3. The roles of Rac1 in the limb bud mesenchyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 3. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
1. Introduction 1.1. Limb development The vertebrate limb is composed of various cells and tissues, including bones, muscles, blood vessels, and skin. Limb development in mice during the embryonic stages originates from extensions of the limb primordium (limb bud), which is composed of mesenchymal cells derived from the lateral plate mesoderm and ectoderm covering the mesenchyme. The limb bud has specialized regions such as the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). The AER, which is located at the tip of the limb buds, maintains outgrowth of the limb bud by expressing
n
Corresponding author. Tel.: +81 3 3784 8163; fax: +81 3 3784 5555. E-mail address: [email protected] (D. Suzuki).
fibroblast growth factor 8 and 4 (Fgf8, Fgf4), which in turn keeps the underlying mesenchymal cells in the progress zone (PZ) in an undifferentiated state [1–4]. A group of cells located in the posterior mesenchyme of the limb bud, the ZPA, acts as the organizer of the anterior–posterior (AP) polarity of the limb bud; the polarizing activity of the ZPA is mediated by sonic hedgehog (Shh) [5]. The analysis of various gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH (hedgehog), TGF-β/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning [6]. After maturation of the limb buds, mesenchymal cells are recruited via migration and condensed at the position of each of the bones during the patterning stage, and then the chondrocyte progenitors are differentiated from condensing mesenchyme to proliferate and differentiate in the fixed regions during the next step [7]. Cells undergoing chondrogenesis acquire a distinct
1349-0079/$ - see front matter & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.job.2013.05.002
D. Suzuki et al. / Journal of Oral Biosciences 55 (2013) 116–121
spherical cell morphology and initiate expression of the transcription factors Sox9 [8], Sox5, and Sox6, which regulate the genes encoding the extracellular matrix (ECM) molecules collagen II and aggrecan [9–11]. Chondrocytes differentiate by hypertrophy, downregulate the expression of collagen II, and initiate the expression of collagen X [12], the matrix molecule bone sialoprotein (Bsp) [13], and secreted factors such as matrix metalloproteases 9 and 13 (Mmp9, Mmp13). The metalloproteases degrade the ECM during the remodeling process for hypertrophic enlargement, proper vascularization, and ossification [14,15]. Hypertrophic chondrocytes mineralize their surrounding matrix and undergo apoptosis [16]. The vascular tissue is stimulated to invade into this region, allowing the entrance of osteoclast and osteoblast precursors, which remodel the remaining hypertrophic matrix and lay down the bone tissue [17].
2. Rac1 The proteins in the mammalian family of Rho proteins were initially isolated as Ras-like small GTP-binding proteins and they were found to be composed of the Rho, Rac, and Cdc42 subfamilies [18]. These proteins act as molecular switches; they are inactive when bound to GDP, but upon exchange of GDP for GTP, they bind to and activate a number of downstream effectors that regulate multiple cellular processes such as actin dynamics, gene expression, and cell cycle progression [19,20]. The switching of Rho GTPases between these two states is regulated by three sets of proteins: guanine nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and guanine nucleotide-dissociation inhibitors (GDIs). Rac proteins form a subfamily within the Rho GTPases. They occur in one of three isoforms, stimulate lamellipodium and membrane ruffle formation, and induce membrane extension during phagocytosis [21]. The three Rac isoforms have different expression patterns. Rac1 is the best-studied member of this family and is ubiquitously expressed, whereas Rac2 expression is mostly restricted to cells of hematopoietic origin, and Rac3 mRNA is most abundant in the brain [22–24]. Recently, analysis of the functions of genes in vivo by using genetically modified mice has become a routine procedure. In the case of Rac1 global knockout mice, the mice displayed the characteristic of embryonic lethality between embryonic days 8.5 and 9.5 because of an abnormal formation of the three germ layers during gastrulation [25]. Therefore, we had to create conditional knockout mice by using the Cre-loxP system, which have a deletion of Rac1 in specific tissues, for more detailed analyses of the functions of Rac1 in specific cells and organs during development [26,27]. This review focuses on the current knowledge of the roles of Rac1 during limb development and the factors that are involved in this process, according to the results of analyses using Rac1 conditional knockout mice. 2.1. The roles of Rac1 in chondrocytes Some in vitro data have shown important roles of Rac1 in the control of mesenchymal condensation, proliferation, and differentiation of chondrocytes, as well as apoptosis. Pharmacological inhibition of Rac1 expression by NSC23766 in micromass culture resulted in reduction of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6, decrease in the chondrogenic markers Col2a1 (collagen II) and Acan (aggrecan), and a decrease in the accumulation of glycosaminoglycans [28]. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased Sox9, Sox5, Sox6, Col2a1, and Acan expression, reduced cell numbers, and markedly accelerated hypertrophic differentiation and apoptosis
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[28,29]. Rac1 overexpression results in activation of the p38 MAP kinase (MAPK) pathway in ATDC5 cells. Inhibition of p38 MAPK signaling blocks the upregulation of collagen X promoter activity and activation of caspase-1 and -3 by Rac1 overexpression [29]. These results suggest that Rac1 signaling accelerates the progression of chondrocyte hypertrophy and apoptosis through a p38 MAPK-dependent mechanism (Fig. 1A). In addition, activation of Rac1 increased with maturation compared with immature primary chondrocytes. Activated Rac1 overexpression induced chondrocyte enlargement and increased matrix expression of Mmp9 and Mmp13, which are characteristic of mature chondrocytes. Conversely, Rac1 inactivation by expression of dominant negative forms of Rac1 diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes [30]. These data provide evidence that Rac1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process (Fig. 1A). Genetic deletion of Rac1 in proliferating collagen II-expressing chondrocytes using Col2-Cre transgenic mice resulted in skeletal deformities, severe kyphosis, and dwarfism (Fig. 1B) [31,32]. These Rac1-deficient mice (Rac1fl/fl; Col2-Cre) have disorganized growth plates, reduced proliferation, hypertrophic zones with chondrocytes of abnormal shape and size, reduced proliferation by expression of the cell cycle genes cyclin D1 and p57, and increased apoptosis. Moreover, phosphorylation of p38 MAPK is greatly reduced and Col10a1 (collagen X), Ibsp (bsp), and Ihh (Indian hedgehog), key regulators of cartilage development, are increased in Rac1fl/fl; Col2-Cre mice. In addition, a recent study showed the molecular pathways of reduced chondrocyte proliferation in Rac1fl/fl; Col2-Cre mice [33]. The Rac1fl/fl; Col2-Cre mouse growth plates have reduced inducible nitric oxide synthase (iNOS) and nitric oxide (NO) expression and increased activation of the transcription factor 3 (Atf3), a known suppressor of cyclin D1 expression in chondrocytes [34]. The growth plate of iNOS knockout mice showed reduced chondrocyte proliferation and expression of cyclin D1, resembling the phenotype of Rac1fl/fl; Col2-Cre growth plates, including increased Atf3. These data suggest that Rac1 is required for iNOS expression and NO production in chondrocytes. NO suppresses the expression of Atf3, which acts as a transcriptional repressor of cyclin D1, which in turn slows chondrocyte proliferation and induces premature exit of the cell cycle. There are some different results between in vitro and in vivo studies in this field. The most likely potential explanation for this discrepancy is that a standard two-dimensional cell culture system as used in vitro cannot recapitulate an in vivo model; e.g., the three-dimensional growth plate organization and tightly controlled cell-ECM interactions [32]. According to in vitro data, Rac1 regulates Sox9 expression [28,29]; however, Rac1fl/fl; Prx1Cre mice, which have Rac1 deleted in the limb mesenchyme before the initiation of Sox9 expression (more detailed information is indicated in the third main paragraph), did not show changes in the Sox9 expression pattern during mesenchymal condensation [35]. Although Rac1 overexpression in ATDC5 cells suppressed cell proliferation, Rac1fl/fl; Col2-Cre mice demonstrated reduced expressions of cyclin D1 and p57, BrdU-positive cells in the growth plates, and a shorter proliferative zone. Rac1fl/fl; Col2-Cre mice also had reduced phosphorylation of p38 MAPK and increased expression of the hypertrophic chondrocyte marker genes Col10a1 and Ibsp. Col2a1-MKK6EE mice, transgenic mice with expression of a constitutively active mutant of MKK6 (a MAPK kinase that specifically activates p38 MAPK) in chondrocytes, showed a reduced zone of hypertrophic chondrocytes and reduced expression of Ihh and Col10a1 [36]. These in vivo data imply that Rac1 inhibits chondrocyte hypertrophy by activation of p38 signaling even though in vitro data showed that Rac1 induced hypertrophy and apoptosis in chondrocytes through p38 MAPK (Fig. 1A).
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Control
Rac1 ; Col2-Cre
Fig. 1. Model of Rac1 functions during chondrocyte differentiation. (A) Chondrogenesis starts with the condensation of mesenchymal precursor cells, followed by chondrogenic differentiation within the condensations. A subset of chondroblasts then initiates proliferation, subsequently undergoes hypertrophic differentiation, and finally undergoes apoptosis. Each stage is characterized by the expression of specific marker genes shown in each of the cells. In vitro data, upper flow, indicated that Rac1 induces differentiation from the mesenchymal cell to the chondroblast and from the prehypertrophic chondrocyte to apoptosis through p38 MAPK. Rac1 inhibits chondrocyte proliferation, although differentiation of chondroblasts to proliferating chondrocytes is not clear, while p38 MAPK represses Ihh expression [54]. Activated Rac1 is increased along with chondrocyte differentiation. Rac1 upregulates chondrocyte proliferation and downregulates hypertrophy, including both prehypertrophic and hypertrophic chondrocytes, through activation of p38 MAPK; this has been indicated by in vivo data using genetically modified mice. Apoptosis in terminal differentiation of chondrocytes may be inhibited by Rac1; however, there is a possibility that the function can be changed depending on the developmental stages. Col2a1, collagen II; Acan, aggrecan; Ihh, Indian hedgehog; Col10a1, collagen X; Bsp, bone sialoprotein; Mmp13, matrix metalloproteases 13. (B) Skeletal preparations revealed that Rac1 conditional knockout mice in chondrocytes (Rac1fl/fl; Col2-Cre) have dwarfism and kyphosis. The control genotype is Rac1fl/fl or Rac1fl/+; Col2-Cre. Modified from Wang G et al. [32] (http:// dx.doi.org/10.1016/j.ydbio.2007.03.520).
Rac1fl/+; Msx2-Cre
Rac1fl/fl; Msx2-Cre
Fig. 2. Model of Rac1 functions in the apical ectodermal ridge (AER). (A) Skeletal preparations indicated that Rac1 conditional knockout in the limb bud ectoderm (Rac1fl/fl; Msx2-Cre) showed a lack of all hindlimb structures (upper panels, arrows) and exhibited truncations at various levels in the forelimb (lower panels). S: scapula; H: humerus; R: radius; U: ulna; P: phalanges. Modified from Wu X et al. [38] (http://dx.doi.org/10.1016/j.cell.2008.01.052). (B) Wnts stimulate receptors to transmit signals and activate Rac1. Activated Rac1 binds β-catenin and leads to nuclear accumulation of β-catenin, which regulates gene expression. In AER, Wnt/β-catenin signaling with Rac1 induces expression of Fgf8, Fgf4, and Bmp4. The combination of Fgf8 and Fgf4 inhibits apoptosis in the ectoderm and mesenchyme, and Fgf8, Fgf4, and Bmp4 advance the limb bud outgrowth by collaboration of the factors. In addition, this Wnt signaling may be negatively regulated by extracellular Dkk1 binding to LPR5/6 before binding to Wnts.
Taken together, despite discrepancies between in vitro and in vivo models, it is clear that Rac1 functions in chondrocyte proliferation and differentiation during endochondral bone development. 2.2. The roles of Rac1 in the limb bud ectoderm Genetic removal of Rac1 from the apical ectodermal ridge (AER) of mouse embryonic limb buds, accomplished by crossing Rac1 flox and Msx2-Cre transgenic mice, resulted in mice that had truncations at various levels in the forelimbs and that lacked all hindlimb structures (Fig. 2A) [37,38]. An increase in apoptosis was observed in both the mesenchyme and ectoderm in the forelimb of Rac1fl/−; Msx2-Cre embryos at E10.5. Moreover, the expression of Fgf8, Fgf4, and Bmp4 in the AER was markedly reduced in Rac1fl/-; Msx2-Cre
embryos at E10.5. Single conditional knockout mice, which were inactivated for each of the genes Fgf8, Fgf4, or Bmp4 by crossing Msx2-Cre mice, indicated extremely mild phenotypes in limbs compared to Rac1fl/−; Msx2-Cre limbs [37,39,40]; however, double conditional knockout mice for Fgf8 and Fgf4 (Fgf8fl/−; Fgf4fl/−; Msx2Cre) displayed severe forelimb truncations and lack of all hindlimb structures with marked increase in apoptotic cells in the limb buds of both the AER and the mesenchyme [4]. These results suggest that the absence of limb structures in Rac1fl/−; Msx2-Cre mice is a result of the failed outgrowth of the limb bud and increased apoptosis in the AER and mesenchyme caused by a marked decrease in the expression of growth and apoptosis controlling factors Fgf8, Fgf4, and Bmp4. Conversely, limb bud extension generally occurs with proper development of the AER and mesenchyme, which is based on strictly controlled apoptosis and
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Hind limb
Fore limb
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Rac1fl/fl
Rac1fl/fl; Prx1-Cre
Fig. 3. Model of Rac1 function in the limb bud mesenchyme. (A) Rac1 conditional knockout in the limb bud mesenchyme (Rac1fl/fl; Prx1-Cre) showed syndactyly in both foreand hind-limbs. Modified from Suzuki D et al. [35] (http://dx.doi.org/10.1016/j.ydbio.2009.09.014). (B) Rac1 controls apoptosis-related genes Bmp2, Bmp7, Msx1, and Msx2, which induce apoptosis in the interdigits of the mesenchyme, leading to webbing separation.
proliferation caused by regulation of the expressions of various genes such as Fgf8, Fgf4, and Bmp4, which have cooperative functions through Rac1 (Fig. 2B). Interestingly, mouse embryos lacking one copy each of Rac1 and β-catenin in the limb bud ectoderm (Rac1fl/+; β-cateninfl/+; Msx2-Cre) developed no hindlimbs and had severe truncations in the forelimbs [38]. Mice with removal of Wnt3a or β-catenin in the limb ectoderm also presented with severe limb phenotypes, including altered expression of Fgf8 and Bmp4 [41,42]. On the other hand, stabilized β-catenin in the limb ectoderm markedly increased the expression of Fgf8 and Bmp4 in the AER [42]. Moreover, mouse embryos lacking one copy of Rac1 and overexpressing one copy of Dkk1 (Dickkopf 1), which is another ligand of the Wnt receptors LRP5/6 that antagonizes the Wnt signaling pathway (Fig. 2B), in the limb bud ectoderm (Rac1fl/+; R26-Dkk1; Msx2-Cre) also displayed limb hypoplasia similar to the Rac1fl/−; Msx2-Cre and Rac1fl/+; β-cateninfl/+; and Msx2-Cre mouse limb phenotypes [38]. R26-Dkk1 mice are engineered so that Dkk1 can only be transcribed when the loxP sites, containing a stop signal, are recombined by Cre [43,44]. Heterozygous mice with a single modification of Rac1, β-catenin, or R26-Dkk1 showed no phenotype. Thus, Wnt signaling controls the limb outgrowth by the proportions of multiple regulators such as Rac1, β-catenin, and Dkk1 (Fig. 2B). These results are consistent with in vitro data that indicated that Rac1 binds β-catenin and prompts Wnt-induced βcatenin nuclear accumulation, leading to functions of Wnt signaling [38]. Overall, Rac1 plays a critical role in canonical Wnt signaling and is essential for the development of the distal ectoderm of the limb bud. 2.3. The roles of Rac1 in the limb bud mesenchyme To analyze Rac1 function in the limb bud mesenchyme, we generated Rac1 conditional knockout mice (Rac1fl/fl; Prx1-Cre), which have limb bud mesenchyme-specific inactivation of the Rac1 gene by using Prx1-Cre transgenic mice [35]. Prx1-Cre transgenic mice express Cre recombinase under the control of a Prx1 limb enhancer as the limb bud mesenchyme, not ectoderm, of fore- and hind-limbs after E9.5 and E10.5 respectively [45]. Rac1fl/fl; Prx1-Cre mice were shorter in body length and had a lower body weight compared to the controls. Rac1fl/fl; Prx1-Cre mice had short limbs and demonstrated delayed endochondral ossification in limb bones by an experiment of skeletal preparations. Although this phenotype of long bone development in limbs is similar to cartilage-specific Rac1 conditional knockout mice (Rac1fl/fl; Col2-Cre), detailed analysis of the molecular mechanism is needed in the future. The most striking feature of the fore- and
hind-limbs of Rac1fl/fl; Prx1-Cre mice was profound soft tissue syndactyly (Fig. 3A). Experiments of skeletal preparations and micro-CT analyses indicated that Rac1fl/fl; Prx1-Cre mice do not have an abnormal number of phalanges. These results introduced one possibility: a defect in apoptosis in the Rac1fl/fl; Prx1-Cre mouse interdigital regions during the process of webbing separation in limb development at embryonic stages. To determine whether Rac1fl/fl; Prx1-Cre webbing was due to a defect in apoptosis, TUNEL assays were performed on the control and Rac1fl/fl; Prx1-Cre embryos at E12.5-E14.5, and demonstrated a significant reduction in the degree of interdigital apoptosis in Rac1fl/fl; Prx1-Cre limbs. We subsequently investigated the major genes causing reduced apoptosis in knockout mice, and whole mount in situ hybridization analysis showed that expression levels of Bmp2, Bmp7, Msx1, and Msx2 were down-regulated in the Rac1fl/fl; Prx1-Cre mouse interdigital region. The implantation of BMP2- or BMP7-soaked beads in the interdigital mesenchyme promotes apoptosis in the chick limb and accelerates interdigital regression [46]. Msx1 and Msx2 expression increases in the chick limb mesenchyme in association with the cell death-inducing activity of Bmps [47]. Additionally, Bmp2fl/fl; Prx1-Cre mice had soft tissue syndactyly [48], and the interdigital webbing in Msx1−/−; Msx2−/− mice did not regress [49]. These results suggest that insufficient expression of Bmp2, Bmp7, Msx1, and Msx2 may be the root cause of the lack of interdigital apoptosis and associated syndactyly in Rac1fl/fl; Prx1-Cre mice. Our study infers that interdigit formation occurs with webbing separation that is dependent on the induction of apoptosis caused by a regulation of the expression of the apoptosis-related genes Bmp2, Bmp7, Msx1, and Msx2 through Rac1 in the limb bud mesenchyme (Fig. 3B). Interestingly, limb bud mesenchymespecific β-catenin conditional knockout mice (β-catenin fl/−; Prx1Cre) indicated more severe phenotypes in limbs compared to Rac1fl/fl; Prx1-Cre mice [50], even though there is a close resemblance between Rac1fl/−; Msx2-Cre and β-cateninfl/−; Msx2-Cre mice in the limb phenotypes and altered expressions of genes [41]. This observation implies that there may be different molecular mechanisms of intercellular signaling in limb bud mesenchymal cells compared to the relationship of Rac1 and β-catenin in the limb mesoderm.
3. Conclusion and future directions The analysis of Rac1 conditional knockout mice indicates the essential roles of Rac1 during limb development. Additionally, our group demonstrated that Cdc42, a member of the Rho family, also
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has crucial functions during limb development, including apoptosis in the interdigits, even though limb bud mesenchyme-specific Cdc42 conditional knockout mice (Cdc42fl/fl; Prx1-Cre) displayed a slightly different type of syndactyly compared to Rac1fl/fl; Prx1-Cre mice by having an altered expression pattern of Sox9 in the Cdc42fl/fl; Prx1-Cre limb bud [51]. In recent years, the significance of RAC1 and CDC42 during development, including that of the limb, has been shown in several human diseases and syndromes. Heterozygous gain-of-function mutations of ARHGAP31, a RAC1/CDC42 GTPase regulatory protein, cause Adams–Oliver syndrome (AOS), which is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD) [52]. Additionally, recessive mutations in the dedicator of cytokinesis 6 gene, DOCK6, which encodes an atypical guanidine exchange factor (GEF) known to activate RAC1 and CDC42, lead to AOS [53]. These data suggest that recessive and dominant AOS can be caused by mutations in the two modulators of the RAC1 and CDC42 GTPase activity, which makes it possible for other modulators of their signaling to be potential candidate genes in this genetically heterogeneous condition. Further analyses of the functions and interactions of Rho family-related factors and applications to diseases, including diagnosis and treatment as AOS, are expected in the near future.
Conflict of interest
[3]
[4] [5] [6] [7] [8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
The authors declare no conflict of interest. [16]
Acknowledgments We would like to thank Prof. T. Shiroishi, Dr. M. Tamura, Dr. T. Amano (National Institute of Genetics, Shizuoka, Japan), Prof. T. Nohno (Kawasaki Medical School, Okayama, Japan), and Dr. N. Wada (Tokyo University of Science, Chiba, Japan) for their kind instructions on the experimental techniques of whole mount in situ hybridization analysis and for giving useful advice; Prof. N. Tsumaki (Kyoto University, Kyoto, Japan), Dr. S. Takeda (Keio University, Tokyo, Japan), and Dr. A. Kimura (Tokyo Medical and Dental University, Tokyo, Japan) for kindly providing the Prx1-Cre transgenic mice and for critical advice; Prof. A. Aiba (Tokyo University, Tokyo, Japan) and Dr. M. Sakahara (Japanese Foundation for Cancer Research, Tokyo, Japan) for kindly providing the Rac1 flox mice and for their valuable advice; Prof. M. Nakamura (Showa University, Tokyo, Japan) for their instructions on the experimental techniques of immunohistochemistry and cell death analysis and for giving useful advice; Dr. R. Yasuhara (Showa University, Tokyo, Japan) for giving us the proper advice on the generation of mice; and all our other colleagues for encouraging us. This work was supported in part by the Project to Establish Strategic Research Center for Innovative Dentistry from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. D. Suzuki is also grateful for the award from the Japanese Association for Oral Biology (JAOB) at the 54th Annual Meeting of JAOB. References [1]
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Review
The essential roles of the small GTPase Rac1 in limb development Dai Suzuki n, Atsushi Yamada, Ryutaro Kamijo Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 7 March 2013 Received in revised form 4 April 2013 Accepted 23 April 2013 Available online 21 June 2013
Vertebrate limbs develop through complicated interactions between various types of cells of the ectoderm and mesenchyme, which facilitate the proper steps of growth and formation by cell proliferation, differentiation, and apoptosis. These processes are regulated by several signaling proteins such as Fgf, Bmp, and Wnt. Recently, several types of Rac1 conditional knockout mice were created, and Rac1 was observed to play essential roles in each of the developing limb tissues. Mice with genetic deletion of Rac1 in the chondrocytes, the limb bud ectoderm, or the limb bud mesenchyme exhibit dwarfism with short limbs, severe truncations of limbs, or syndactyly with short limbs, respectively. Analyses of these mice demonstrated that Rac1 regulates limb development by controlling many kinds of gene expression and various cellular functions. & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Conditional knockout mice Limb development Rac1
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 1.1. Limb development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2. Rac1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 2.1. The roles of Rac1 in chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 2.2. The roles of Rac1 in the limb bud ectoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 2.3. The roles of Rac1 in the limb bud mesenchyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 3. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
1. Introduction 1.1. Limb development The vertebrate limb is composed of various cells and tissues, including bones, muscles, blood vessels, and skin. Limb development in mice during the embryonic stages originates from extensions of the limb primordium (limb bud), which is composed of mesenchymal cells derived from the lateral plate mesoderm and ectoderm covering the mesenchyme. The limb bud has specialized regions such as the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA). The AER, which is located at the tip of the limb buds, maintains outgrowth of the limb bud by expressing
n
Corresponding author. Tel.: +81 3 3784 8163; fax: +81 3 3784 5555. E-mail address: [email protected] (D. Suzuki).
fibroblast growth factor 8 and 4 (Fgf8, Fgf4), which in turn keeps the underlying mesenchymal cells in the progress zone (PZ) in an undifferentiated state [1–4]. A group of cells located in the posterior mesenchyme of the limb bud, the ZPA, acts as the organizer of the anterior–posterior (AP) polarity of the limb bud; the polarizing activity of the ZPA is mediated by sonic hedgehog (Shh) [5]. The analysis of various gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH (hedgehog), TGF-β/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning [6]. After maturation of the limb buds, mesenchymal cells are recruited via migration and condensed at the position of each of the bones during the patterning stage, and then the chondrocyte progenitors are differentiated from condensing mesenchyme to proliferate and differentiate in the fixed regions during the next step [7]. Cells undergoing chondrogenesis acquire a distinct
1349-0079/$ - see front matter & 2013 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.job.2013.05.002
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spherical cell morphology and initiate expression of the transcription factors Sox9 [8], Sox5, and Sox6, which regulate the genes encoding the extracellular matrix (ECM) molecules collagen II and aggrecan [9–11]. Chondrocytes differentiate by hypertrophy, downregulate the expression of collagen II, and initiate the expression of collagen X [12], the matrix molecule bone sialoprotein (Bsp) [13], and secreted factors such as matrix metalloproteases 9 and 13 (Mmp9, Mmp13). The metalloproteases degrade the ECM during the remodeling process for hypertrophic enlargement, proper vascularization, and ossification [14,15]. Hypertrophic chondrocytes mineralize their surrounding matrix and undergo apoptosis [16]. The vascular tissue is stimulated to invade into this region, allowing the entrance of osteoclast and osteoblast precursors, which remodel the remaining hypertrophic matrix and lay down the bone tissue [17].
2. Rac1 The proteins in the mammalian family of Rho proteins were initially isolated as Ras-like small GTP-binding proteins and they were found to be composed of the Rho, Rac, and Cdc42 subfamilies [18]. These proteins act as molecular switches; they are inactive when bound to GDP, but upon exchange of GDP for GTP, they bind to and activate a number of downstream effectors that regulate multiple cellular processes such as actin dynamics, gene expression, and cell cycle progression [19,20]. The switching of Rho GTPases between these two states is regulated by three sets of proteins: guanine nucleotide exchange factors (GEFs), GTPaseactivating proteins (GAPs), and guanine nucleotide-dissociation inhibitors (GDIs). Rac proteins form a subfamily within the Rho GTPases. They occur in one of three isoforms, stimulate lamellipodium and membrane ruffle formation, and induce membrane extension during phagocytosis [21]. The three Rac isoforms have different expression patterns. Rac1 is the best-studied member of this family and is ubiquitously expressed, whereas Rac2 expression is mostly restricted to cells of hematopoietic origin, and Rac3 mRNA is most abundant in the brain [22–24]. Recently, analysis of the functions of genes in vivo by using genetically modified mice has become a routine procedure. In the case of Rac1 global knockout mice, the mice displayed the characteristic of embryonic lethality between embryonic days 8.5 and 9.5 because of an abnormal formation of the three germ layers during gastrulation [25]. Therefore, we had to create conditional knockout mice by using the Cre-loxP system, which have a deletion of Rac1 in specific tissues, for more detailed analyses of the functions of Rac1 in specific cells and organs during development [26,27]. This review focuses on the current knowledge of the roles of Rac1 during limb development and the factors that are involved in this process, according to the results of analyses using Rac1 conditional knockout mice. 2.1. The roles of Rac1 in chondrocytes Some in vitro data have shown important roles of Rac1 in the control of mesenchymal condensation, proliferation, and differentiation of chondrocytes, as well as apoptosis. Pharmacological inhibition of Rac1 expression by NSC23766 in micromass culture resulted in reduction of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6, decrease in the chondrogenic markers Col2a1 (collagen II) and Acan (aggrecan), and a decrease in the accumulation of glycosaminoglycans [28]. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased Sox9, Sox5, Sox6, Col2a1, and Acan expression, reduced cell numbers, and markedly accelerated hypertrophic differentiation and apoptosis
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[28,29]. Rac1 overexpression results in activation of the p38 MAP kinase (MAPK) pathway in ATDC5 cells. Inhibition of p38 MAPK signaling blocks the upregulation of collagen X promoter activity and activation of caspase-1 and -3 by Rac1 overexpression [29]. These results suggest that Rac1 signaling accelerates the progression of chondrocyte hypertrophy and apoptosis through a p38 MAPK-dependent mechanism (Fig. 1A). In addition, activation of Rac1 increased with maturation compared with immature primary chondrocytes. Activated Rac1 overexpression induced chondrocyte enlargement and increased matrix expression of Mmp9 and Mmp13, which are characteristic of mature chondrocytes. Conversely, Rac1 inactivation by expression of dominant negative forms of Rac1 diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes [30]. These data provide evidence that Rac1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process (Fig. 1A). Genetic deletion of Rac1 in proliferating collagen II-expressing chondrocytes using Col2-Cre transgenic mice resulted in skeletal deformities, severe kyphosis, and dwarfism (Fig. 1B) [31,32]. These Rac1-deficient mice (Rac1fl/fl; Col2-Cre) have disorganized growth plates, reduced proliferation, hypertrophic zones with chondrocytes of abnormal shape and size, reduced proliferation by expression of the cell cycle genes cyclin D1 and p57, and increased apoptosis. Moreover, phosphorylation of p38 MAPK is greatly reduced and Col10a1 (collagen X), Ibsp (bsp), and Ihh (Indian hedgehog), key regulators of cartilage development, are increased in Rac1fl/fl; Col2-Cre mice. In addition, a recent study showed the molecular pathways of reduced chondrocyte proliferation in Rac1fl/fl; Col2-Cre mice [33]. The Rac1fl/fl; Col2-Cre mouse growth plates have reduced inducible nitric oxide synthase (iNOS) and nitric oxide (NO) expression and increased activation of the transcription factor 3 (Atf3), a known suppressor of cyclin D1 expression in chondrocytes [34]. The growth plate of iNOS knockout mice showed reduced chondrocyte proliferation and expression of cyclin D1, resembling the phenotype of Rac1fl/fl; Col2-Cre growth plates, including increased Atf3. These data suggest that Rac1 is required for iNOS expression and NO production in chondrocytes. NO suppresses the expression of Atf3, which acts as a transcriptional repressor of cyclin D1, which in turn slows chondrocyte proliferation and induces premature exit of the cell cycle. There are some different results between in vitro and in vivo studies in this field. The most likely potential explanation for this discrepancy is that a standard two-dimensional cell culture system as used in vitro cannot recapitulate an in vivo model; e.g., the three-dimensional growth plate organization and tightly controlled cell-ECM interactions [32]. According to in vitro data, Rac1 regulates Sox9 expression [28,29]; however, Rac1fl/fl; Prx1Cre mice, which have Rac1 deleted in the limb mesenchyme before the initiation of Sox9 expression (more detailed information is indicated in the third main paragraph), did not show changes in the Sox9 expression pattern during mesenchymal condensation [35]. Although Rac1 overexpression in ATDC5 cells suppressed cell proliferation, Rac1fl/fl; Col2-Cre mice demonstrated reduced expressions of cyclin D1 and p57, BrdU-positive cells in the growth plates, and a shorter proliferative zone. Rac1fl/fl; Col2-Cre mice also had reduced phosphorylation of p38 MAPK and increased expression of the hypertrophic chondrocyte marker genes Col10a1 and Ibsp. Col2a1-MKK6EE mice, transgenic mice with expression of a constitutively active mutant of MKK6 (a MAPK kinase that specifically activates p38 MAPK) in chondrocytes, showed a reduced zone of hypertrophic chondrocytes and reduced expression of Ihh and Col10a1 [36]. These in vivo data imply that Rac1 inhibits chondrocyte hypertrophy by activation of p38 signaling even though in vitro data showed that Rac1 induced hypertrophy and apoptosis in chondrocytes through p38 MAPK (Fig. 1A).
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Control
Rac1 ; Col2-Cre
Fig. 1. Model of Rac1 functions during chondrocyte differentiation. (A) Chondrogenesis starts with the condensation of mesenchymal precursor cells, followed by chondrogenic differentiation within the condensations. A subset of chondroblasts then initiates proliferation, subsequently undergoes hypertrophic differentiation, and finally undergoes apoptosis. Each stage is characterized by the expression of specific marker genes shown in each of the cells. In vitro data, upper flow, indicated that Rac1 induces differentiation from the mesenchymal cell to the chondroblast and from the prehypertrophic chondrocyte to apoptosis through p38 MAPK. Rac1 inhibits chondrocyte proliferation, although differentiation of chondroblasts to proliferating chondrocytes is not clear, while p38 MAPK represses Ihh expression [54]. Activated Rac1 is increased along with chondrocyte differentiation. Rac1 upregulates chondrocyte proliferation and downregulates hypertrophy, including both prehypertrophic and hypertrophic chondrocytes, through activation of p38 MAPK; this has been indicated by in vivo data using genetically modified mice. Apoptosis in terminal differentiation of chondrocytes may be inhibited by Rac1; however, there is a possibility that the function can be changed depending on the developmental stages. Col2a1, collagen II; Acan, aggrecan; Ihh, Indian hedgehog; Col10a1, collagen X; Bsp, bone sialoprotein; Mmp13, matrix metalloproteases 13. (B) Skeletal preparations revealed that Rac1 conditional knockout mice in chondrocytes (Rac1fl/fl; Col2-Cre) have dwarfism and kyphosis. The control genotype is Rac1fl/fl or Rac1fl/+; Col2-Cre. Modified from Wang G et al. [32] (http:// dx.doi.org/10.1016/j.ydbio.2007.03.520).
Rac1fl/+; Msx2-Cre
Rac1fl/fl; Msx2-Cre
Fig. 2. Model of Rac1 functions in the apical ectodermal ridge (AER). (A) Skeletal preparations indicated that Rac1 conditional knockout in the limb bud ectoderm (Rac1fl/fl; Msx2-Cre) showed a lack of all hindlimb structures (upper panels, arrows) and exhibited truncations at various levels in the forelimb (lower panels). S: scapula; H: humerus; R: radius; U: ulna; P: phalanges. Modified from Wu X et al. [38] (http://dx.doi.org/10.1016/j.cell.2008.01.052). (B) Wnts stimulate receptors to transmit signals and activate Rac1. Activated Rac1 binds β-catenin and leads to nuclear accumulation of β-catenin, which regulates gene expression. In AER, Wnt/β-catenin signaling with Rac1 induces expression of Fgf8, Fgf4, and Bmp4. The combination of Fgf8 and Fgf4 inhibits apoptosis in the ectoderm and mesenchyme, and Fgf8, Fgf4, and Bmp4 advance the limb bud outgrowth by collaboration of the factors. In addition, this Wnt signaling may be negatively regulated by extracellular Dkk1 binding to LPR5/6 before binding to Wnts.
Taken together, despite discrepancies between in vitro and in vivo models, it is clear that Rac1 functions in chondrocyte proliferation and differentiation during endochondral bone development. 2.2. The roles of Rac1 in the limb bud ectoderm Genetic removal of Rac1 from the apical ectodermal ridge (AER) of mouse embryonic limb buds, accomplished by crossing Rac1 flox and Msx2-Cre transgenic mice, resulted in mice that had truncations at various levels in the forelimbs and that lacked all hindlimb structures (Fig. 2A) [37,38]. An increase in apoptosis was observed in both the mesenchyme and ectoderm in the forelimb of Rac1fl/−; Msx2-Cre embryos at E10.5. Moreover, the expression of Fgf8, Fgf4, and Bmp4 in the AER was markedly reduced in Rac1fl/-; Msx2-Cre
embryos at E10.5. Single conditional knockout mice, which were inactivated for each of the genes Fgf8, Fgf4, or Bmp4 by crossing Msx2-Cre mice, indicated extremely mild phenotypes in limbs compared to Rac1fl/−; Msx2-Cre limbs [37,39,40]; however, double conditional knockout mice for Fgf8 and Fgf4 (Fgf8fl/−; Fgf4fl/−; Msx2Cre) displayed severe forelimb truncations and lack of all hindlimb structures with marked increase in apoptotic cells in the limb buds of both the AER and the mesenchyme [4]. These results suggest that the absence of limb structures in Rac1fl/−; Msx2-Cre mice is a result of the failed outgrowth of the limb bud and increased apoptosis in the AER and mesenchyme caused by a marked decrease in the expression of growth and apoptosis controlling factors Fgf8, Fgf4, and Bmp4. Conversely, limb bud extension generally occurs with proper development of the AER and mesenchyme, which is based on strictly controlled apoptosis and
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Hind limb
Fore limb
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Rac1fl/fl
Rac1fl/fl; Prx1-Cre
Fig. 3. Model of Rac1 function in the limb bud mesenchyme. (A) Rac1 conditional knockout in the limb bud mesenchyme (Rac1fl/fl; Prx1-Cre) showed syndactyly in both foreand hind-limbs. Modified from Suzuki D et al. [35] (http://dx.doi.org/10.1016/j.ydbio.2009.09.014). (B) Rac1 controls apoptosis-related genes Bmp2, Bmp7, Msx1, and Msx2, which induce apoptosis in the interdigits of the mesenchyme, leading to webbing separation.
proliferation caused by regulation of the expressions of various genes such as Fgf8, Fgf4, and Bmp4, which have cooperative functions through Rac1 (Fig. 2B). Interestingly, mouse embryos lacking one copy each of Rac1 and β-catenin in the limb bud ectoderm (Rac1fl/+; β-cateninfl/+; Msx2-Cre) developed no hindlimbs and had severe truncations in the forelimbs [38]. Mice with removal of Wnt3a or β-catenin in the limb ectoderm also presented with severe limb phenotypes, including altered expression of Fgf8 and Bmp4 [41,42]. On the other hand, stabilized β-catenin in the limb ectoderm markedly increased the expression of Fgf8 and Bmp4 in the AER [42]. Moreover, mouse embryos lacking one copy of Rac1 and overexpressing one copy of Dkk1 (Dickkopf 1), which is another ligand of the Wnt receptors LRP5/6 that antagonizes the Wnt signaling pathway (Fig. 2B), in the limb bud ectoderm (Rac1fl/+; R26-Dkk1; Msx2-Cre) also displayed limb hypoplasia similar to the Rac1fl/−; Msx2-Cre and Rac1fl/+; β-cateninfl/+; and Msx2-Cre mouse limb phenotypes [38]. R26-Dkk1 mice are engineered so that Dkk1 can only be transcribed when the loxP sites, containing a stop signal, are recombined by Cre [43,44]. Heterozygous mice with a single modification of Rac1, β-catenin, or R26-Dkk1 showed no phenotype. Thus, Wnt signaling controls the limb outgrowth by the proportions of multiple regulators such as Rac1, β-catenin, and Dkk1 (Fig. 2B). These results are consistent with in vitro data that indicated that Rac1 binds β-catenin and prompts Wnt-induced βcatenin nuclear accumulation, leading to functions of Wnt signaling [38]. Overall, Rac1 plays a critical role in canonical Wnt signaling and is essential for the development of the distal ectoderm of the limb bud. 2.3. The roles of Rac1 in the limb bud mesenchyme To analyze Rac1 function in the limb bud mesenchyme, we generated Rac1 conditional knockout mice (Rac1fl/fl; Prx1-Cre), which have limb bud mesenchyme-specific inactivation of the Rac1 gene by using Prx1-Cre transgenic mice [35]. Prx1-Cre transgenic mice express Cre recombinase under the control of a Prx1 limb enhancer as the limb bud mesenchyme, not ectoderm, of fore- and hind-limbs after E9.5 and E10.5 respectively [45]. Rac1fl/fl; Prx1-Cre mice were shorter in body length and had a lower body weight compared to the controls. Rac1fl/fl; Prx1-Cre mice had short limbs and demonstrated delayed endochondral ossification in limb bones by an experiment of skeletal preparations. Although this phenotype of long bone development in limbs is similar to cartilage-specific Rac1 conditional knockout mice (Rac1fl/fl; Col2-Cre), detailed analysis of the molecular mechanism is needed in the future. The most striking feature of the fore- and
hind-limbs of Rac1fl/fl; Prx1-Cre mice was profound soft tissue syndactyly (Fig. 3A). Experiments of skeletal preparations and micro-CT analyses indicated that Rac1fl/fl; Prx1-Cre mice do not have an abnormal number of phalanges. These results introduced one possibility: a defect in apoptosis in the Rac1fl/fl; Prx1-Cre mouse interdigital regions during the process of webbing separation in limb development at embryonic stages. To determine whether Rac1fl/fl; Prx1-Cre webbing was due to a defect in apoptosis, TUNEL assays were performed on the control and Rac1fl/fl; Prx1-Cre embryos at E12.5-E14.5, and demonstrated a significant reduction in the degree of interdigital apoptosis in Rac1fl/fl; Prx1-Cre limbs. We subsequently investigated the major genes causing reduced apoptosis in knockout mice, and whole mount in situ hybridization analysis showed that expression levels of Bmp2, Bmp7, Msx1, and Msx2 were down-regulated in the Rac1fl/fl; Prx1-Cre mouse interdigital region. The implantation of BMP2- or BMP7-soaked beads in the interdigital mesenchyme promotes apoptosis in the chick limb and accelerates interdigital regression [46]. Msx1 and Msx2 expression increases in the chick limb mesenchyme in association with the cell death-inducing activity of Bmps [47]. Additionally, Bmp2fl/fl; Prx1-Cre mice had soft tissue syndactyly [48], and the interdigital webbing in Msx1−/−; Msx2−/− mice did not regress [49]. These results suggest that insufficient expression of Bmp2, Bmp7, Msx1, and Msx2 may be the root cause of the lack of interdigital apoptosis and associated syndactyly in Rac1fl/fl; Prx1-Cre mice. Our study infers that interdigit formation occurs with webbing separation that is dependent on the induction of apoptosis caused by a regulation of the expression of the apoptosis-related genes Bmp2, Bmp7, Msx1, and Msx2 through Rac1 in the limb bud mesenchyme (Fig. 3B). Interestingly, limb bud mesenchymespecific β-catenin conditional knockout mice (β-catenin fl/−; Prx1Cre) indicated more severe phenotypes in limbs compared to Rac1fl/fl; Prx1-Cre mice [50], even though there is a close resemblance between Rac1fl/−; Msx2-Cre and β-cateninfl/−; Msx2-Cre mice in the limb phenotypes and altered expressions of genes [41]. This observation implies that there may be different molecular mechanisms of intercellular signaling in limb bud mesenchymal cells compared to the relationship of Rac1 and β-catenin in the limb mesoderm.
3. Conclusion and future directions The analysis of Rac1 conditional knockout mice indicates the essential roles of Rac1 during limb development. Additionally, our group demonstrated that Cdc42, a member of the Rho family, also
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has crucial functions during limb development, including apoptosis in the interdigits, even though limb bud mesenchyme-specific Cdc42 conditional knockout mice (Cdc42fl/fl; Prx1-Cre) displayed a slightly different type of syndactyly compared to Rac1fl/fl; Prx1-Cre mice by having an altered expression pattern of Sox9 in the Cdc42fl/fl; Prx1-Cre limb bud [51]. In recent years, the significance of RAC1 and CDC42 during development, including that of the limb, has been shown in several human diseases and syndromes. Heterozygous gain-of-function mutations of ARHGAP31, a RAC1/CDC42 GTPase regulatory protein, cause Adams–Oliver syndrome (AOS), which is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD) [52]. Additionally, recessive mutations in the dedicator of cytokinesis 6 gene, DOCK6, which encodes an atypical guanidine exchange factor (GEF) known to activate RAC1 and CDC42, lead to AOS [53]. These data suggest that recessive and dominant AOS can be caused by mutations in the two modulators of the RAC1 and CDC42 GTPase activity, which makes it possible for other modulators of their signaling to be potential candidate genes in this genetically heterogeneous condition. Further analyses of the functions and interactions of Rho family-related factors and applications to diseases, including diagnosis and treatment as AOS, are expected in the near future.
Conflict of interest
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The authors declare no conflict of interest. [16]
Acknowledgments We would like to thank Prof. T. Shiroishi, Dr. M. Tamura, Dr. T. Amano (National Institute of Genetics, Shizuoka, Japan), Prof. T. Nohno (Kawasaki Medical School, Okayama, Japan), and Dr. N. Wada (Tokyo University of Science, Chiba, Japan) for their kind instructions on the experimental techniques of whole mount in situ hybridization analysis and for giving useful advice; Prof. N. Tsumaki (Kyoto University, Kyoto, Japan), Dr. S. Takeda (Keio University, Tokyo, Japan), and Dr. A. Kimura (Tokyo Medical and Dental University, Tokyo, Japan) for kindly providing the Prx1-Cre transgenic mice and for critical advice; Prof. A. Aiba (Tokyo University, Tokyo, Japan) and Dr. M. Sakahara (Japanese Foundation for Cancer Research, Tokyo, Japan) for kindly providing the Rac1 flox mice and for their valuable advice; Prof. M. Nakamura (Showa University, Tokyo, Japan) for their instructions on the experimental techniques of immunohistochemistry and cell death analysis and for giving useful advice; Dr. R. Yasuhara (Showa University, Tokyo, Japan) for giving us the proper advice on the generation of mice; and all our other colleagues for encouraging us. This work was supported in part by the Project to Establish Strategic Research Center for Innovative Dentistry from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. D. Suzuki is also grateful for the award from the Japanese Association for Oral Biology (JAOB) at the 54th Annual Meeting of JAOB. References [1]
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