Development of the diaphragm and genetic mouse models of diaphragmatic defects.код для вставкиСкачать
American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 145C:109 – 116 (2007) A R T I C L E Development of the Diaphragm and Genetic Mouse Models of Diaphragmatic Defects KATE G. ACKERMAN* AND JOHN J. GREER Improving our understanding of diaphragmatic development is essential to making progress in defining the pathogenesis and genetic etiologies of congenital diaphragmatic defects in humans. As mouse genetic technology has given us new tools to manipulate and observe development, a number of mouse models have recently emerged that provide valuable insight to this field. In this article, we review our current understanding of diaphragmatic embryogenesis including the origin of diaphragmatic tissue. We use rodent models to review the muscularization of the diaphragm and review selected genetic models of abnormal muscularization. We also review models of posterior diaphragmatic defects and discuss evidence for the pleuroperitoneal fold (PPF) tissue contributing to the diaphragm. Finally, we discuss models of anterior and central hernias. It may be simplistic to subdivide this review based on anatomic regions of the diaphragm, as evidence is emerging that defects in different regions of the diaphragm in humans and in mice may be etiologically related. However, at this time we do not have enough knowledge to make more mechanistic or genetic classifications though with time, genetic progress in the field of diaphragm development will allow us to do this. ß 2007 Wiley-Liss, Inc. KEY WORDS: CDH; diaphragm development; mouse genetic models How to cite this article: Ackerman KG, Greer JJ. 2007. Development of the diaphragm and genetic mouse models of diaphragmatic defects. Am J Med Genet Part C Semin Med Genet 145C:109–116. INTRODUCTION The diaphragm is a critical organ needed to sustain life. Abnormal development of the diaphragm in embryogenesis causes a life-threatening problem for the newborn. Our current therapies are inadequate to sustain life, or life without longterm morbidity, in many of those affected. Diaphragm defects are variable in location, size, and type. Efforts are underway to discover which genes are required for normal development of the diaphragm. One promising method of discovery comes from the evaluation of mouse models. Mice with mutations in a number of genes have a variety of diaphragmatic defects. Some of these genes have been associated with diaphragmatic defects in humans, although genetic etiologies for the common form of isolated congenital diaphragmatic hernia (CDH) are yet to be discovered. Anatomy and Embryology/Origin of Diaphragmatic Tissue The diaphragm forms as a domed structure in the human between 4 and 10 weeks of gestation and in the mouse between embryonic days 10.5 and 15.5 (gestation ¼ 18.5 days). Until recently, Kate G. Ackerman, M.D., is an Assistant Professor of Pediatrics at Harvard Medical School and a member of the Division of Genetics at Brigham and Women’s Hospital and the Division of Emergency Medicine, Department of Medicine at Children’s Hospital Boston. Dr. Ackerman is a pediatric intensivist with post-doctoral research training in mouse genetics. Dr. Ackerman’s research focuses on understanding the pathophysiology of diaphragmatic defects and pulmonary hypoplasia in humans by investigating mechanisms of development in mouse models. John J. Greer, Ph.D., is an Alberta Heritage for Medical Research Scientist and Professor of Physiology at the University of Alberta. Dr. Greer’s research is in the area of perinatal neuromuscular control and development. A major component of his research focuses on the pathogenesis and etiology of congenital diaphragmatic hernia with a particular emphasis on understanding the defect to the diaphragm. *Correspondence to: Kate G. Ackerman, M.D., Division of Genetics, Brigham and Women’s Hospital, Harvard Medical School New Research Bldg 464, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: email@example.com DOI 10.1002/ajmg.c.30128 ß 2007 Wiley-Liss, Inc. The diaphragm forms as a domed structure in the human between 4 and 10 weeks of gestation and in the mouse between embryonic days 10.5 and 15.5 (gestation ¼ 18.5 days). interpretation of diaphragm development was based on inferences derived from the anatomic appearance of the diaphragm [Irish et al., 1996; Stolar, 1997]. Several advances in technology allow us to evaluate and to alter expression patterns of genes important for development, which combined with knowledge gained from a teratogenic model of rodent CDH [Costlow and Manson, 1981] have enhanced our understanding of normal and abnormal diaphragm development. The diaphragm has a characteristic appearance which is similar in both humans and rodents (Fig. 1). Muscularization is prominent along the perimeter of the diaphragm, while the central tendon region is not muscularized and 110 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c ARTICLE Figure 1. Normal mature human (A) and mouse (B,C) diaphragms. Photo (C) shows a close up of the anterior or ventral diaphragm. Both human and mouse diaphragms have a central tendon region that is not muscularized (white arrows in photos A, B) with peripheral muscularization and a distinct crural muscular region (Cr). The inferior vena cava and esophagus pass through the diaphragm (in A, B: large black arrows—site of inferior vena cava, double arrows—site of esophagus). The pericardium is fused to the thoracic side of the human diaphragm (P). The muscle in the anterior diaphragm forms two distinct bundles (photo C, white arrows). Herniation may occur between or lateral to these bundles (in regions marked with black arrows). has a distinct shape. The central tendon remains attached to the liver by the falciform and coronary ligaments. Crural muscle in the posterior medial diaphragm surrounds the esophagus and inferior vena cava. All of the muscle of the diaphragm is skeletal muscle and is completely derived from migratory muscle precursor cell populations [Birchmeier and Brohmann, 2000]. Examination of mouse models has not supported a previous hypothesis that diaphragmatic musculature is derived from the esophageal mesentery or ingrowth of muscle from the lateral body wall. Although the diaphragm consists of different cell types (including those comprising skeletal muscle, connective tissue, mesothelium, blood vessels, and nerves), it is probably completely derived from the mesodermal tissue. While the muscle cells originate from paraxial mesoderm [Vasyutina and Birchmeier, 2006], other tissues of the diaphragm are derived from splanchnic or somatic mesoderm [Sweeney, 1998]. The potential contributions from each type of mesoderm to different regions of the diaphragm have not been clearly resolved. The splanchnic mesoderm forms the heart and the tissue surrounding the gut while the somatic mesoderm may form the pleuroperitoneal fold (PPF), a structure believed to later form the posterior diaphragm [Babiuk et al., 2003]. A sub-type of the somatic mesoderm, the septum transversum mesoderm, contributes to the anterior diaphragm [Sweeney, 1998; Babiuk et al., 2003; Liu et al., 2003; Yuan et al., 2003]. Diaphragm Muscle and Models of Muscularization Defects The muscle cells that migrate to the diaphragm are derived from cervical myotomes 3–5 (of 8), which are present by week 6 of human development. These somites are derived from paraxial mesoderm. While trunk muscles (epaxial muscles) are formed directly from dorsal myotomes (epimeres), hypaxial muscle groups are derived from the ventral myotome (hypomeres). A portion of the hypaxial muscle is migratory and it is this group that gives rise to the muscles of the diaphragm as well as the limb and the tongue. A second, non-migratory hypaxial group extends ventrally to form the intercostal and abdominal muscles including the external and internal obliques, transversus abdominis, and rectus abdominis muscles [Sweeney, 1998]. The ventral dermomyotome cells that populate the diaphragm can be identified by the expression of Pax3, a paired homeodomain transcription factor. In Pax3 mutant (Splotch) mice the diaphragm muscle does not form [Li et al., 1999]. Lbx1, a homeobox gene that controls expression of other genes required for normal muscle migration, is also expressed by these migrating muscle precursor cells and loss of Lbx1 affects the establishment of muscles formed from these precursor cells. Loss of Lbx1 decreases limb and tongue muscularization but has the most severe effect on the dorsal muscles of the hind limb. Despite expression in diaphragmatic muscle cells, loss of Lbx1 does not affect diaphragm musculature [Brohmann et al., 2000]. Thus, the transcriptional regulation of muscle migration to different regions of the body is specific. Muscle precursors must delaminate and migrate; these functions are largely controlled by scatter factor/hepatocyte growth factor (SF/HGF) and its receptor, c-Met. While c-Met is expressed in migratory and non-migratory hypaxial precursors, SF/HGF is restricted to delaminating cells. SF/HGF is expressed in the mesenchyme along the tracts of migrating muscle cells [Dietrich et al., 1999]. Embryos lacking expression of c-Met (c-Met null) have amuscular diaphragms, while embryonic lethality of SF/HGF null mice has precluded evaluation [Schmidt et al., 1995]. Mice deficient in the gene, Fog2 have lack of muscularization in the posterior diaphragm in combination with a diffuse muscular patterning defect. These mice have abnormal and restricted patterns of SF/HGF expression in the mesenchymal tissues posterior to the diaphragm in a region where migratory muscle cells temporarily reside prior to populating the diaphragm [Ackerman et al., 2005]. Gab1 is essential for c-Met signaling, and ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c Gab1 null embryos also lack the diaphragm muscle [Sachs et al., 2000]. Myogenic regulatory factors (MRFs) are necessary for development of normal skeletal muscle. These regulatory factors belong to a group of basic helix–loop– helix (bHLH) transcription factors [Ludolph and Konieczny, 1995]. This group, including MyoD, Myf-5, Mrf-4, and Myogenin, along with the Mef2 family of proteins, coordinates regulation of gene expression during myogenesis in a complex fashion [Naya et al., 1999; Berkes and Tapscott, 2005]. A number of these factors are expressed in developing diaphragm muscle and mutations in some of these genes in mice are associated with abnormal diaphragm development. For example, Myogenin null mice have amuscular diaphragms [Tseng et al., 2000], while MyoD mutant mice have thin diaphragms [Kablar et al., 2003]. Correct muscularization of the diaphragm requires complex coordination of precursor cells to delaminate, migrate, target diaphragmatic tissue, and differentiate; mutations in several genes needed for these processes have already been shown to cause aberrant muscularization. Further investigation Correct muscularization of the diaphragm requires complex coordination of precursor cells to delaminate, migrate, target diaphragmatic tissue, and differentiate; mutations in several genes needed for these processes have already been shown to cause aberrant muscularization. into mechanisms of diaphragmatic muscularization is important, as humans with CDH may also have contralateral diaphragmatic muscularization defects. Human muscularization defects (often referred to as eventrations) may be 111 diffuse or well circumscribed and may be associated with herniation of abdominal contents into the chest (‘‘sac hernia’’) (further discussed in the article in this issue by Dr. Pober). The developmental relationship between these human muscularization defect phenotypes is unknown at this time, but hopefully investigation in mouse models will help to establish a developmentally based classification (Fig. 2). Development of the Posterior Diaphragm and Models of Posterior Hernias Insight into the mechanisms of posterior diaphragm development is critical to our understanding of the most severe and common forms of CDH in humans. These hernias are often referred to as Bochdalek hernias as they were described in a publication by the professor of anatomy, Victor Alexander Bochdalek [Irish et al., 1996]. Unfortunately, the original description of probable mechanism of rupture through a Figure 2. Muscularization of the diaphragm. The diaphragm is muscularized by migratory muscle precursor cells. Photos A–D show the normal progression of muscularization in whole diaphragms during development. Mutants with lack of normal muscle migration, like c-Met null mice, have amuscular diaphragms (E). The central tendon is unmuscularized in the normal mature mouse diaphragm (F), however mutations may induce over-muscularization. The ENU-induced mutation, Overgrown, causes muscle to almost completely cover the central tendon (K. Ackerman, unpublished work). The Fog2 mutant mouse diaphragm does not develop musculature in the posterior diaphragm bilaterally (H, posterior membranous diaphragm lacking normal muscle is marked with white arrows). Figures 2A–D reprinted from J. Comp. Neurol. 391:275–292 with permission from the Wiley and Sons. 112 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c region of susceptibility in the lumbocostal triangle is probably only one of the various putative mechanisms of pathogenesis. Due to limitations in the existing anatomic classification system of CDH, different defects occurring in the lateral, posterolateral, or posteromedial regions of the diaphragm are all called Bochdalek hernias (see accompanying article by Dr Pober). These hernias are often described as occurring through the Bochdalek canal or foramen, although the interpretation of the location and boundaries of this canal varies widely between practitioners. Posterior hernias may have a sac around the herniated organs. This sac is probably parietal mesothelial tissue and/or the connective tissue components of the diaphragm. It is unknown why some posterior hernias have a sac, while some do not. Of note, virtually all anterior, central, and anterior lateral hernias in humans have a sac. The posterior diaphragm, like other regions of the diaphragm, may have muscularization defects. These can be diffused or well circumscribed and cause no herniation or massive herniation. These defects are often referred to as eventrations, although this term is confusing because it technically means herniation but is also used to describe diaphragmatic elevation associated with abnormal neurologic function, but no anatomic defect. It is unknown whether a muscularization defect without herniation is part of the same spectrum or mechanism of pathogenesis as a sac hernia (technically, a muscularization defect with large herniation) or a hernia without a sac. Evidence for these different phenotypes existing within same families or same syndromes suggests that they are developmentally related [Rodgers and Hawks, 1986; Stratton et al., 1993; Slavotinek, 2004]. ARTICLE The complete spectrum of phenotypes that occur in humans has not yet been duplicated in the mouse; however there are now an increasing number of rodent posterior hernias that may correspond to human defects (Table I) including those with mutations or deletions in the COUP-TFII (Chicken ovalbumin upstream promoter-transcription factor II, annotated as Nr2f2) [Klaassens et al., 2005; Slavotinek et al., 2006], Fog2 (Friend of Gata, 2) [Ackerman et al., 2005], and Wt1 (Wilms tumor 1) genes [Scott et al., 2005; Cho et al., 2006]. COUP-TFII is a member of the nuclear receptor super family and is essential for normal embryonic development. COUP-TFII null embryos die prior to diaphragmatic embryogenesis [Pereira et al., 1999], however a conditional deletion model has allowed for discovery of a role in posterior diaphragm development [You et al., TABLE I. Selected Genetic Mouse Models of Abnormal Diaphragm Development Model Defect c-Met Amuscular diaphragm [Babiuk and Greer, 2002] Posterior hernia (no sac) [You et al., 2005] COUP-TFII (Nkx 3.2 conditional model) Fog2 (Zfpm2 lil/lil) Gab1 Gata4 þ/ LOX MyoD Myogenin MyoR/Capsulin Pax3 (Splotch) RARa/RARb2(retinoic acid receptors) Slit3 Wt1 Posterior hernia (sac) [Ackerman et al., 2005] muscle patterning defect Amuscular diaphragm [Sachs et al., 2000] Central hernia (sac) [Jay et al., 2006] Central diaphragmatic rupture [Hornstra et al., 2003] Thin diaphragm, not functional [Kablar et al., 1997] Amuscular diaphragm [Tseng et al., 2000] Posterior hernia (? sac) [Tseng et al., 2000] Amuscular diaphragm [Li et al., 1999] Compound receptor nulls have posterior diaphragmatic hernias [Mendelsohn et al., 1994] Central midline hernia (sac) [Liu et al., 2003; Yuan et al., 2003] Posterior hernia [Clugston et al., 2006; Kreidberg et al., 1993] Human correlate Unknown Yes, cytogenetic hotspot 15q26.1-26.2 (DIH1, OMIM# 142340) (syndromic) [Klaassens et al., 2005; Slavotinek et al., 2006] Yes, de novo mutation (non-syndromic) [Ackerman et al., 2005] Unknown Suspected, cytogenetic Hotspot 8p23.1 (DIH2, OMIM#222400) [Lopez et al., 2006; Slavotinek et al., 2006] Unknown Unknown Unknown Unknown Unknown Unknown, suspected Unknown Yes, syndromic [Scott et al., 2005; Cho et al., 2006] ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c COUP-TFII is a member of the nuclear receptor super family and is essential for normal embryonic development. COUP-TFII null embryos die prior to diaphragmatic embryogenesis, however a conditional deletion model has allowed for discovery of a role in posterior diaphragm development. 2005]. A majority of embryos with deletion of COUP-TFII restricted to mesenteric tissue had posterior diaphragmatic hernia. Although COUPTFII is expressed in primordial diaphragmatic tissue, the central tendon, and developing pulmonary mesenchyme, this model (Nkx 3.2 Cre expressing cells) did not delete COUP-TFII in all of these structures [You et al., 2005], leaving open the possibility that the diaphragmatic phenotype could be more severe than what is observed in this mouse model. COUP-TFII is believed to play a role in human diaphragmatic defects associated with other multiple birth defects (syndromic CDH), as it is one of the few genes located in a recently narrowed cytogenetic hotspot for CDH on chromosome 15q26.2 (discussed further in the article in this issue written by Dr. Pober). Wt1 mutations are also associated with posterior diaphragmatic defects. These defects were first reported in 1993 [Kreidberg et al., 1993]; however early embryonic lethality prevented detailed analysis of the diaphragmatic phenotype. Recently, expression of Wt1 in primordial diaphragmatic tissue (PPF) was characterized and structure of this primordial tissue in Wt1 null embryos was found to be abnormal [Clugston et al., 2006]. Like COUPTFII and Fog2, Wt1 is also required for the development of multiple organs including the genitourinary system [Kreidberg et al., 1993], the spleen [Herzer et al., 1999], and the heart [Kreidberg et al., 1993]. Mutations and deletions of WT1 are associated with congenital anomalies in humans, including syndromes that may include diaphragmatic defect [Natoli et al., 2002; Scott et al., 2005; Cho et al., 2006] and is discussed in the accompanying article by Dr. Slavotinek. Fog2 is a transcription cofactor for the GATA family of transcription factors. Its interaction with Gata4 in vivo is necessary for normal lung, diaphragm, cardiac, and gonadal development [Crispino et al., 1999; Tevosian et al., 2002; Ackerman et al., 2006; Jay et al., 2006]. Fog2 is expressed in mesodermal tissues including the pulmonary mesenchyme and mesothelium, and the PPF tissue (Fig. 3). Fog2 null mice die of cardiac defects prior to diaphragm development, but mice carrying a homozygote hypomorphic mutation in Fog2 occasionally live until late gestation. These mice have posterior membranous diaphragms and an abnormal pattern of muscularization with no delineated central tendon. Fog2 is required for normal pulmonary development, as Fog2 null lungs explanted to grow in vitro are hypoplastic and lack normal lobar structure. Fog2 is the first Fog2 is the first gene recognized to be necessary for both primary lung development and primary diaphragm development providing evidence for the hypothesis that diaphragmatic defects may be associated with primary lung defects. gene recognized to be necessary for both primary lung development and primary diaphragm development providing evidence for the hypothesis that diaphragmatic defects may be associated with primary lung defects. A neonate who died at birth with severe pulmonary hypoplasia and a muscularization defect 113 in the eventration-sac hernia spectrum was found to have a de novo stop mutation in FOG2. In this patient, lung size was smaller than expected based on the extent of the diaphragmatic defect further supporting the hypothesis that the mutation in Fog2 caused both a failure in primary lung development and primary diaphragm development. Other genes are now known to affect both diaphragm and lung development including COUP-TFII, Gata4, and retinoic acid signaling pathway genes [Mendelsohn et al., 1994; Malpel et al., 2000; Ackerman et al., 2006; Jay et al., 2006]. It is probable that some posterior defects arise from genetic mutations that do not independently affect lung development, while others affect the development of both organs (and often the heart as well). Once more human causes are identified; it may be possible to predict risk of pulmonary and cardiac sequelae based on the specific genetic mutation. Although there is ample evidence to support the hypothesis that similar developmental genes are needed for both lung and diaphragm development and that both structures may be primarily affected in CDH, there is insufficient evidence to support the hypothesis that defects in lung development lead to secondary defects in diaphragm development. The most compelling evidence against this latter hypothesis is that mice lacking lung development (FgF10null/null) have normal diaphragms [Babiuk and Greer, 2002]. The tissues that contribute to the posterior diaphragm are referred to as the PPFs and the post hepatic mesenchymal plate tissue (PHMP). Although it is unclear whether these structures represent different sections of the same mesodermal tissue, the terms are used interchangeably by different investigators [Greer et al., 2000a; You et al., 2005] to describe tissue contributing to the diaphragm. The PPFs are largest and most evident in the embryonic thorax at the level of the forelimb just prior to diaphragm development (e11.5 in the mouse). The PPF has been shown to be the precursor tissue of the diaphragm in studies of teratogenically induced CDH [Greer et al., 2000b]. 114 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c ARTICLE Figure 3. Primordial diaphragmatic tissue and gene expression. The pleuroperitoneal fold (PPF) tissue contributes to the posterior diaphragm and is visualized in the mouse thorax at e11.5 as two triangular structures (A, black arrows). The phrenic nerves (marked with *) innervate the diaphragm and are surrounded by PPF tissue. Three-dimensional reconstruction of the PPF in a normal mouse embryo (B) and in a nitrofen exposed mouse embryo (C). The nitrofen mutant PPF lacks tissue in the posterior and caudal region of the PPF. Immunohistochemical staining of the PPFs (D,E). COUP-TFII and Pax 3 (marker for migratory muscle cells) are expressed in different cells of the PPF (D, COUP-TFII labeled green and Pax3 labeled red) while COUP-TFII and Wt1 share some co-expression in the PPF (E, COUP-TFII labeled green, Wt1 labeled red, double labeled cells appear yellow). Genetically engineered mice carrying LacZ reporter genes show Wt1 expression (F) and Fog2 expression (G) in the PPF tissue. Wt1 is expressed in the lateral PPF while Fog2 expression is diffuse. Figure 3B-E reprinted from Am. J. Pathol. 2006 169: 1541–1549 with permission from the American Society for Investigative Pathology. This model will be discussed in more detail in the article by Dr. Kling and Dr. Schnitzer, but in short, embryos exposed to the herbicide nitrofen in utero develop posterior diaphragmatic defects. Examination of the structure of the PPF in these embryos reveals tissue loss in the rostral and lateral aspects. This morphologically abnormal structure resembles those observed in vitamin A deficient embryos that are also predisposed to developing posterior hernias. It should be noted that one group has suggested that nitrofen-induced PPF defects create posteromedial hernias rather than posterolateral hernias [Fisher and Bodenstein, 2006]. This was concluded from a two-dimensional computer simulation of the muscular diaphragm from a single type of nitrofen-induced PPF defect. However, there are clear examples of defects in the lateral regions of the diaphragm in the nitrofen model [Allan and Greer, 1997]. Modeling is required that focuses on the three-dimensional mesenchymal nonmuscular substratum and takes into consideration the full range of PPF defects observed in the nitrofen model. Currently known genes expressed in PPF tissue include those restricted to migratory muscle precursor cells (Pax3), and those needed for normal diaphragmatic development (Fog2, COUP-TFII, Wt1) which are not expressed in muscle cells. There is co-localization of Wt1 and COUP-TFII protein in the PPF, especially along the dorsal mesothelial surface, and this expression does not co-localize with the muscle precursors labeled with Pax3 [Clugston et al., 2006]. Fog2 is expressed diffusely in the PPF and also does not co-localize with muscle precursor markers (Fig. 3). An interaction between Wt1 and COUPTFII has not been established, and it remains unclear how these genes might work together to promote normal diaphragmatic development. Models of Central and Anterior Hernias Diaphragmatic hernias and muscularization defects may also occur in the anterior or central diaphragm. Most anterior defects are referred to as Morgagni defects in humans as these and other anterior defects were described by Giovanni Battista Morgagni in the 1700s [Irish et al., 1996]. The musculature of the anterior or ventral and midline diaphragm is attached to the ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS): DOI 10.1002/ajmg.c sternum and xyphoid process. This anterior muscle is distinct from the peripheral muscle and forms two distinct muscle bundles. The regions medial and lateral to these muscle bundles are potential areas of weakness predisposed to herniation. This anterior diaphragmatic anatomy is shown in Figure 1. Historically, Morgagni hernias were described as occurring lateral to these anterior muscles on the right side. Hernias may also occur centrally (subxyphoid or retrosternal) or on the left side (Larrey hernia). Although they may represent different types of hernias, most anterior hernias are simply classified as being Morgagni hernias in humans [Salman et al., 1999; Loong and Kocher, 2005]. It is unknown at this time whether mouse models of anterior hernias correspond to anteriorly located human diaphragmatic defects. Mouse models of anterior and/or central diaphragmatic defect include those with mutations in Slit3, Gata4, and lysyl oxidase (LOX). Slit genes (Slit1, Slit2) are expressed prominently with the CNS, where they bind to Robo receptors to act as axonal guidance molecules. In contrast, Slit3 is expressed predominantly in other tissues including the lung and the diaphragm [Yuan et al., 2003; Yuan et al., 1999]. To investigate in vivo functions for Slit3, a null mutation was introduced into Slit3 in mice by targeted mutagenesis in embryonic stem (ES) cells. Slit3 null mice develop a central and midline diaphragmatic defect that ranges in location from being very anterior to involving a major portion of the central tendon. In these mice, a circular portion of the anterior tendon is significantly thinner than in wild-type mice and the connective tissue appears disorganized. The enlarging liver distends and ultimately herniates through the weakened area of the tendon [Liu et al., 2003; Yuan et al., 2003]. This phenotype may be similar to the very rare ‘‘septum transversum’’ type of CDH or the anterior defects associated with Pentalogy of Cantrell [Wesselhoeft and DeLuca, 1984] but a genetic association between SLIT mutations and human diaphragmatic defect has not yet been found. A similar defect occurs in approximately 30% of mice heterozygous for a mutant allele of Gata-4 [Jay et al., 2006], a transcription factor that interacts with Fog2. These mice also have herniation of the liver through the central diaphragm. It is not known whether Gata4 mediated diaphragm development is Fog2 dependent, although it is probable as Fog2– Gata4 interactions are necessary for the development of other organs including the heart, gonads, and lungs [Crispino et al., 2001; Tevosian et al., 2002; Ackerman et al., 2006]. Early embryonic lethality in Gata4 and Fog2 null mice makes comparison of phenotypes and mechanism of diaphragmatic development difficult, and further investigation is needed, perhaps utilizing conditional gene deletion models. An additional model is that of diaphragmatic rupture of the central tendon due to deficiency in the enzyme, lysyl oxidase (LOX). Lox targeted null mice die with ruptured arterial aneurysms and diaphragms secondary to defects in collagen development [Hornstra et al., 2003]. Although defects in the diffuse collagen structure may result in diaphragmatic herniation, these are currently not thought to be developmentally associated with most neonatal CDHs. CONCLUSIONS Defining the normal embryogenesis of the diaphragm is critical to advancing our understanding of diaphragmatic defects in animal models and in humans. The study of diaphragm and other organ development can be achieved using genetic models. CDH has naturally occurred and has a suspected genetic etiology in some families of monkeys, pigs, and dogs [Bush et al., 1980; Valentine et al., 1988; Ohkawa et al., 1993]; however their use for studying organ development in the laboratory is limited. Genetic mouse models provide excellent tools for doing this, and there are now multiple genes known to be important for diaphragm development as well as primary lung, and heart development. Since humans with CDH often have lung, cardiac, and 115 other defects, the discovery of genetic pathways controlling development independently in the thoracic and upper abdominal regions of the embryo or fetus will result in a better understanding of the disease. Hopefully, this will lead to the discovery of therapeutic regimens to treat co-morbid lung, cardiovascular, and gastrointestinal disease in CDH patients. REFERENCES Ackerman KG, Herron BJ, Vargas SO, Huang H, Tevosian SG, Kochilas L, Rao C, Pober BR, Babiuk RP, Epstein JA, Greer JJ, Beier DR. 2005. Fog2 is required for normal diaphragm and lung development in mice and humans. PLoS Genet 1:58–65. Ackerman KG, Wang JN, Fujiwara Y, Luo LL, Wait A, Orkin SH, Beier DR. 2006. Gata4 is necessary for normal pulmonary lobar development. 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