Mouse House THE ANATOMICAL RECORD PART A 284A:424 – 430 (2005) Ventral Abdominal Wall Dysmorphogenesis of Msx1/Msx2 Double-Mutant Mice HIDENAO OGI, 1,2 KENTARO SUZUKI, 1 YUKIKO OGINO, 1 MIKA KAMIMURA, 1 MAMI MIYADO, 1 XU YING,1 ZUNYI ZHANG,3 MASANORI SHINOHARA,2 YIPING CHEN, 3 AND GEN YAMADA1* 1 Center for Animal Resources and Development, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan 2 Department of Oral and Maxillofacial Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 3 Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana ABSTRACT Msx1 and Msx2 genes encode the homeodomain transcription factors. Several gene knockout mice and expression studies suggest that they possess functionally redundant roles in embryogenesis. In this study, we revealed that Msx1 and Msx2 were expressed during ventral body wall formation in an overlapping manner. Msx1/Msx2 double-mutant mice displayed embryonic abdominal wall defects with disorganized muscle layers and connective tissues. These ﬁndings indicate that Msx1 and Msx2 play roles in concert during embryonic ventral abdominal wall formation. © 2005 Wiley-Liss, Inc. Key words: Msx gene; homeobox gene; body wall; congenital hernia; embryogenesis; mouse During mouse development, the ventral body wall consisting of thin epithelial membrane and loose mesenchyme is formed after the primary body wall develops. The generation of the mature secondary ventral body wall is observed after 12.0 days postcoitum (dpc). Around these stages, the entire ventral body wall remains open and visceral organs are exposed. Later in development, embryonic body wall mesenchyme is mainly contributed by entering cells derived from somites. Somite-derived cells enter into primary body wall developing to distal (ventral) side of embryos forming the secondary body wall. The secondary body wall of the abdomen includes skin, muscle, and abdominal band derivatives (Christ et al., 1983; Kaufman, 1999; Brewer and Williams, 2004b). Up to now, several works by gene knockout mice have been issued to explain the basis of the pathogenesis of body wall defects related with some congenital human birth defects. In particular, mutations identiﬁed in some of the genes for transcription factors, cell signaling molecules, and proteases are associated with various degrees of body wall developmental abnormalities (Hasty et al., 1993; Nabeshima et al., 1993; Suzuki et al., 1996; Zhang et al., 1996; Eggenschwiler et al., 1997; Roebroek et al., 1998; Tremblay et al., 1998; Manley et al., 2001; Dunker and © 2005 WILEY-LISS, INC. Krieglstein, 2002; Brewer and Williams, 2004a, 2004b). However, comprehensive and integrated understanding of molecular genetic cascades for the development of body walls awaits further analyses. The vertebrate Msx homeobox gene family contains three members, two of which (Msx1 and Msx2) have been well studied with respect to their expression patterns and Grant sponsor: Grant-in-Aid for Scientiﬁc Research on Priority Areas (1,2), General Promotion of Cancer Research in Japan (11177101), Mechanisms of Sex Differentiation (16086208); Grant sponsor: the 21st Century COE Research Program and Child Health and Development (14C-1), the Ministry of Health, Labor and Welfare. *Correspondence to: Gen Yamada, Center for Animal Resources and Development, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan. Fax: 81-96-373-6560. E-mail: firstname.lastname@example.org Received 31 August 2004; Accepted 20 December 2004 DOI 10.1002/ar.a.20180 Published online 31 March 2005 in Wiley InterScience (www.interscience.wiley.com). VENTRAL ABDOMINAL WALL DYSMORPHOGENESIS functional properties. These genes encode closely related homeoproteins that function as transcriptional repressors through interactions with components of the core transcription complex as well as other homeoproteins (Bendall and Abate-Shen, 2000). Both Msx1 and Msx2 are expressed in an overlapping manner spatially and temporally during development in discrete regions of the facial primordia, limbs, neural tube, and other embryonic regions (Davidson, 1995; Satokata et al., 2000; Zhang et al., 2002; Alappat et al., 2003; Cheng et al., 2004). It has also been demonstrated that Msx genes can regulate cellular proliferation and differentiation (Pavlova et al., 1994; Chen et al., 1996, 2000; Ferrari et al., 1998; Dodig et al., 1999). In this study, we analyzed Msx1/Msx2 double-mutant embryos and revealed their abnormal abdominal wall development with aberrantly aligned connective tissues and body wall muscles. MATERIALS AND METHODS Msx1/Msx2 Double-Mutant Embryos Mice carrying a targeted deletion of the Msx1 gene or the Msx2 gene were described previously (Satokata and Maas, 1994; Satokata et al., 2000). Homozygous mutant embryos were obtained by heterozygote mating. Msx1/ Msx2 double-mutant embryos were obtained by double heterozygote mating. The genotype of each embryo was identiﬁed by PCR as previously described (Satokata and Maas, 1994; Satokata et al., 2000). Laboratory animal care and animal use for the present study were performed under the guidance of animal researches of the Tulane and Kumamoto University. In Situ Hybridization Whole-mount in situ hybridization for gene expression analysis was performed with digoxigenin-labeled probes by standard procedures (Wilkinson, 1992) with probes for Msx1 and Msx2 (Zhang et al., 2003). Histological Analysis Embryos were ﬁxed in 4% paraformaldehyde. They were dehydrated in graded ethanol, embedded in parafﬁn, and sectioned at a thickness of 5.0 m. Sections were stained with hematoxylin and eosin (H&E) by standard procedures. Connective tissue was stained with Masson’s trichrome technique (Muto Pure Chemicals). RESULTS Msx1 and Msx2 Are Expressed in Primary Body Wall During Development Several expression and gene knockout mice studies suggest that Msx1 and Msx2 possess functionally redundant roles in embryogenesis (Satokata et al., 2000; Alappat et al., 2003; Zhang et al., 2003). As for possibly interacting genes with Msx genes, Bmp genes have been suggested as interacting in genetic cascades with Msx genes. In fact, orchestration of regulatory genes involved in Bmp signaling has been reported for organogenesis such as tooth, palate, and heart development (Chen et al., 1996; Bei and Maas, 1998; Zhang et al., 2002; Brugger et al., 2004). Bmp genes are expressed in developing body wall and they are required for normal body wall development (Suzuki et al., 1996; Funayama et al., 1999; Sudo et al., 2001). Hence, we analyzed the spatial and temporal expression pattern of 425 Msx1 and Msx2 during abdominal body formation and performed histological studies on abdominal body walls of Msx1/Msx2 double-mutant embryos. In early embryogenesis, ventral body wall develops ﬁrst as the primary body wall. In mouse embryos, the primary body wall lacks somite derived tissues before 12.0 dpc, but the secondary body wall is formed afterwards by somite derived cells after 12.0 dpc. Primary ventral body wall develops distally, which engulfs visceral organs in the coelom accompanied with the growth of the visceral organs (Kaufman, 1999; Gilbert, 2003; Brewer and Williams, 2004a, 2004b). The Msx1 and Msx2 were expressed in the developing primary body wall at 9.5 dpc (Fig. 1A and B). They were mainly expressed in the developing somatopleure in earlier stages such as at 8.0 dpc (data not shown). Both Msx1 and Msx2 were expressed in overlapping manner at the level of the trunk in the developing primary body wall mesenchyme as shown by the abdominal transverse sections (Fig. 1C and D). Msx1 was expressed in the restricted region in the body wall anteroposteriory at 11.0 dpc (Fig. 1E). This region of the body wall corresponded to the periumbilical region surrounding the umbilical cord. In contrast, Msx2 was expressed broader than Msx1 anteroposteriory at 11.0 dpc (Fig. 1F). The level of Msx1 was drastically reduced at 12.5 dpc embryos and only faint signals were observed around the umbilical ring (data not shown). In contrast, the signal of Msx2 was decreased rapidly after 11.0 dpc (data not shown). The above expression patterns prompted us to examine the phenotypes of the body wall formation in the corresponding gene knockout mice. Dysmorphogenesis of Abdominal Body Wall in Msx1/Msx2 Double-Mutant Embryos The developmental time course of the abdominal body wall formation of the Msx1/Msx2 double-mutant embryos were examined histologically. The ventral body wall develops to the midline and most of the visceral organs, including stomach, were engulfed in the coelom at 12.5 dpc in wild-type embryos (Fig. 2A and C). Prominent hypoplasia of the abdominal body wall was observed in Msx1/Msx2 double-mutant embryos. Abdominal body wall elongation and subsequent engulfment of the visceral organs were not observed particularly in the distal abdominal wall region of the double mutant embryos (Fig. 2B and D). The developing abdominal mesenchymal tissues were observed in distal regions of the body wall in wildtype mice (Fig. 2C, white arrowheads). In contrast, the developing abdominal wall was markedly thinner and hypoplastic and its distal (ventral) development was defective with only thin amnionic membrane locating in the ventral side of Msx1/Msx2 double-mutant embryos (Fig. 2D). In normal embryogenesis, three muscle layers develop composed by transversus abdominis muscle, internal oblique muscle, external oblique muscle within the abdominal muscle layers at 14.5 dpc (Fig. 2G, black arrowheads). In contrast to such distally elongated muscle development, misarranged muscular tissues were observed in lateral abdominal regions of the Msx1/Msx2 double-mutant embryos (Fig. 2H, black arrowhead). Msx1/Msx2 double-mutant embryonic body wall did not reach to the ventral (distal) side (Fig. 2F). The collagen ﬁbers in the connective tissues of the abdominal regions were arranged in a regular parallel pattern in the wildtype specimens (Fig. 2I). In contrast, Msx1/Msx2 double- 426 OGI ET AL. Fig. 1. Gene expression pattern of Msx1 and Msx2. Whole-mount in situ hybridization for gene expression (A–F) and their expression at the abdominal level (C and D). Msx1 and Msx2 were expressed in overlapping manner in the primary body wall mesenchyme including abdominal region at 9.5 dpc (A–D, arrowheads). The region of Msx1 expression was expressed narrower anteroposteriory than that of Msx2 as indicated by white arrowheads at 11.0 dpc (E and F arrowheads). FL, fore limb; UC, umbilical cord; T, tail; HL, hind limb; GT, genital tubercle. Scale bar ⫽ 500 m (A, B, E, and F); 100 m (C and D). mutant embryos displayed spongy and highly disorganized alignment of collagen ﬁbers in the connective tissue of the abdominal body wall (Fig. 2J). Cutaneous structures including cutaneous maximus muscle and connective tissue of the dermis displayed mostly normal architecture in Msx1/Msx2 double-mutant embryos (Fig. 2E and F, black arrow). Null mutant of each gene did not display malformations (data not shown). In sum, Msx1/Msx2 doublemutant embryos displayed abnormal development of the connective tissues and muscular tissues in abdominal body wall resulting in severe hernia of the visceral organs. formation of the muscles takes place in several characteristic steps as shown by chick embryologic studies (Christ et al., 1983). The ventral somatic buds which consist of myotome and dermomyotome cells enter the somatopleure of chick embryos (Christ et al., 1983). Thus, in contrast to limb muscle precursors, muscle precursors for the abdominal muscles are basically considered not to migrate to their target site in chick embryos. The murine embryonic body wall of the abdomen is composed by mesenchymal connective tissues and somite derived muscles at 14.5 dpc (Christ et al., 1983; Kaufman, 1999; Gilbert, 2003; Brewer and Williams, 2004a, 2004b). Several mutant mice with body wall abnormalities have been extensively analyzed (Qu et al., 1997; Manley et al., 2001; Dunker and Krieglstein, 2002; Brewer and Williams, 2004a, 2004b). However, the molecular mechanism of mammalian body wall formation and pathogenesis of body wall abnormalities are still poorly understood. Msx genes have been reported to play roles during various organogenesis (Bei and Maas, 1998; Satokata et al., 2000; Zhang et al., 2003). In the current report, histological description of the abdominal body wall defects of DISCUSSION In early embryogenesis, ventral body wall develops ﬁrst as the primary body wall consisting of thin epithelia and loose mesenchymes. In the abdominal region of the embryonic body wall, somite-derived cells participate in many embryonic structures. Somites form various muscles and abdominal wall and the dermis of the dorsal skin. In mouse embryos, the primary body wall lacks somitederived tissues before 12.0 dpc, but the secondary body wall is formed afterwards by somite derived cells. The VENTRAL ABDOMINAL WALL DYSMORPHOGENESIS 427 Fig. 2. Lateral view of the embryos (A and B) and transverse sections with H&E staining (C and D) of the abdominal wall at 12.5 dpc. Masson staining of abdominal wall (E and F) and photographs of higher magniﬁcations at 14.5 dpc (G–J). Compared to the wild-type embryos (E, G, I), body wall hypoplasia including disorganized muscle layers (G and H, black arrowheads) and connective tissues (I and J) were observed in Msx1/Msx2 double-mutant embryos. Cutaneous structures were not markedly disorganized (E and F, arrows). V, ventral side; D, dorsal side; am, amnion; eo, external oblique muscles; io, internal oblique muscles; ta, transversus abdominis muscles; St, stomach. Scale bar ⫽ 60 m (A and B); 200 m (C and D); 500 m (E and F); 250 m (G and H); 20 m (I and J). Msx1/Msx2 double-mutant mice was presented. The phenotypes reﬂect spatial and temporal expression of Msx1 and Msx2 genes. Msx1 and Msx2 genes are known to encode closely related homeodomain containing transcriptional repressors (Bendall and Abate-Shen, 2000). Several abdominal body wall abnormalities have been reported in gene knockout mice for homeobox-containing genes. Alx4, a member of the aristaless family of homeobox genes, has been reported as displaying abdominal body wall defects in its mutants. Possible interactions between Alx genes and Msx genes have yet been unelucidated. However, recent double-mutant analysis for Alx4 and Msx2 genes suggested their interaction in organogenesis such as during heart, lung, and diaphragm formation (Antonopoulou et al., 2004). Alx4 is expressed in an overlapping manner in the prospective ventral body wall region with Msx1 and Msx2 genes from 9.0 until 11.5 dpc (Qu et al., 1997). Ventral abdominal wall is markedly thinned both in Alx4 (Qu et al., 1997) and Msx1/Msx2 double knockout mutants. While the phenotypes of Msx1/Msx2 double-mutant 428 OGI ET AL. mice became visible from 12.5 dpc, ventral body wall defects of Alx4 mutant mice become ﬁrst apparent approximately at 15.5 dpc (Qu et al., 1997). Mechanisms for different abdominal body wall phenotype onset in both mutants await further analysis. Hox gene knockout mice also displayed various degrees of body wall abnormalities. Both Hoxb2 mutant mice and Hoxb4 mutant mice have defects in primary body wall development for the chest regions (Manley et al., 2001). It has been known that Hox genes could be regulated by retinoic acid (RA) indicated by various experimental systems (Kessel and Gruss, 1990; Yashiro et al., 2004). As for another retinoic acid-inducible transcription factor, Ap-2, the AP-2␣ knockout mice also exhibit severe ventral body wall closure defects (Schorle et al., 1996; Zhang et al., 1996; Brewer and Williams, 2004b). These results raise questions about possible regulatory cascades among such genes for ventral body wall formation. Msx genes may modulate the regulation of type I collagen possibly affecting the formation of extracellular matrix (ECM) development (Dodig et al., 1996; Alappat et al., 2003). Tgf-␤ genes are members of the Tgf-␤ super family and they elicit cellular responses via signaling through speciﬁc type I and type II serine/threonine kinase receptors. TGF-␤s are known to promote matrix deposition by increasing the expression of ﬁbronectin and collagens and also by upregulating inhibitors of matrix proteases (Grotendorst, 1997; Hocevar and Howe, 2000). Tgf-␤2/Tgf-␤3 double-mutant mice display severe ventral body wall malformations (Dunker and Krieglstein, 2002). The body wall muscles of these mutants did not reach to the ventral side and their thickness was reduced (Dunker and Krieglstein, 2002). Previous reports and the current analysis indicate that both Msx1/Msx2 double-mutant embryos and Tgf-␤2/ Tgf-␤3 double-mutant embryos exhibit spongy connective tissues with loosely organized pores and mesh-like collagen ﬁber bundles. Further analyses including marker analysis on the relationships with Msx genes and Tgf-␤ genes are required. Three layers of developing muscles, including transversus abdominis muscle, internal oblique muscle, and external oblique muscle, are derived from the lateral side of dermomyotome, which has been suggested to secure the strength of the body wall around 14.5 dpc (Kaufman, 1999; Sadler, 2000; Gilbert, 2003). Various developmental studies have suggested a possible involvement of regulatory factors, including secretory signals regulating dermomyotome formation from the lateral plate (Cossu et al., 1996; Pourquie et al., 1996; Dietrich et al., 1998; Alvares et al., 2003). Although this study does not provide information about the involvement of Msx genes related with regulatory mechanisms, these possibilities may be worth pursuing with marker studies. It was shown that Bmp4 was expressed in the developing primary body wall and to regulate MyoD expression and the generation of hypaxial myotomes (Pourquie et al., 1996). Various reports on other organogeneses suggested relationships between Msx1, Msx2, and Bmp4 genes (Brugger et al., 2004). Msx1 and Msx2 have also been suggested as factors necessary for proper Bmp4 expression in organogenesis (Semba et al., 2000; Zhang et al., 2002). These data might imply a possibility of the perturbed signaling including genes, e.g., Bmps during the abnormal primary body wall formation in Msx1/Msx2 double mutants. Aberrant muscle formation reported in this study could also be derived from the Msx1 and Msx2 expression in the developing dermomyotome. The current in situ gene expression analysis did not detect Msx1 or Msx2 expression in the developing dermomyotome per se in contrast to the previous report utilizing LacZ knock-in mice (Houzelstein et al., 1999). Such discrepancy about the expression levels may be due to the sensitivity of detection. The possible involvement of Msx1 and Msx2 genes in this differentiation process should be analyzed further. The Pax3 gene could also be listed as a candidate interacting with Msx genes during body wall formation. It has also functions during abdominal body wall and limb muscle formation (Tremblay et al., 1998). The Pax3 gene is an early marker for the entire paraxial mesoderm and its dorsal derivative, the dermomyotome. Later, its expression becomes restricted to the lateral dermomyotome and to the migratory muscle precursors giving rise to the hypaxial musculature (Dietrich et al., 1998). Pax3 expression may be involved as a trigger for the myogenic program and induces expression of MyoD in paraxial mesoderm explants (Epstein et al., 1995; Maroto et al., 1997; Bendall et al., 1999). Ectopic coexpression of Msx1 and Pax3 neutralizes the effect on MyoD and MSX1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3 (Bendall et al., 1999). The regulation of Pax3 could affect Msx2 expression in cardiac neural crest (Kwang et al., 2002). In the mutant mice with disrupted Pax3 function (Sploch), the three layers of abdominal muscles were present laterally, but they displayed a disorganized array of ﬁbers, and their development did not progress ventrally (Tremblay et al., 1998). In human, absence of abdominal muscles generally results in syndromes with exposed visceral organs and it has been speculated as induced by multifactorial genetic elements. The majority of human body wall malformations are non-syndromic and their mechanism of pathogenesis is unclear. Hence, comprehension of normal and abnormal development of body wall formation depends still largely on genetic studies utilizing various gene knockout mouse models. The current study offered histological data of the abdominal dysmorphology of Msx1/Msx2 double-mutant embryos. Involvement of Msx1/Msx2 for human birth defects as part of the plausible genetic elements should be further pursued. ACKNOWLEDGMENTS The authors thank Drs. 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