Homeobox Genes in Pulmonary Vascular Development and Disease

Homeobox Genes in Pulmonary Vascular Development and Disease Peter Lloyd Jones*

Determining how the pulmonary vascular system is formed, maintained, or disrupted during development and disease represents a major challenge in contemporary lung biology. Whereas it is appreciated that cellular proliferation, differentiation, migration, and apoptosis need to be carefully controlled in order to attain pulmonary vascular homeostasis, knowledge of the underlying cellular and molecular mechanisms involved remains surprisingly limited. Because homeobox genes represent master regulators of organogenesis and tissue patterning, it is likely that these transcription factors play a critical role in the formation of blood vessels within the lung, as well as in pathologic states in which the highly ordered structure of the pulmonary vascular tree is compromised. The aim of this review is to discuss some of the known functions of homeobox genes in the vasculature, and to extrapolate these findings to their potential roles in developing and diseased pulmonary vessels. (Trends Cardiovasc Med 2003;13:336–345) © 2003, Elsevier Inc.

• Homeobox Genes Homeobox genes encode highly conserved transcription factors that control positional identity and morphogenesis throughout development (McGinnis and Krumlauf 1992). All proteins encoded by homeobox genes contain a 60 amino acid DNA-binding motif—designated the homeodomain—which folds into three  helices. This motif arises from an 180 nucleotide sequence designated the homeobox, so called because mutations in some of these genes result in homeotic

Peter Lloyd Jones is at the Department of Pediatrics, Section of Critical Care & Developmental Lung Biology, University of Colorado Health Sciences Center, Denver, Colorado, USA. * Address correspondence to: Peter Lloyd Jones, PhD, University of Colorado Health Sciences Center, Department of Pediatrics, 4200 East 9th Avenue, B-131, Denver, CO 80262, USA. Tel.: (1) 303-315-4497; fax: (1) 303-315-1326; e-mail: [email protected]. © 2003, Elsevier Inc. All rights reserved. 1050-1738/03/$-see front matter

336

transformations, in which one body structure replaces another. Since the discovery of homeobox genes in Drosophila, homologous genes have been identified in vertebrates. In mice and humans, these Hox genes are arranged as four unlinked complexes, designated Hoxa, Hoxb, Hoxc, and Hoxd, which contain a total of 39 genes that are believed to have arisen by gross duplication of an ancestral cluster (Figure 1). Thus, corresponding genes (or paralogues) located in different clusters on separate chromosomes resemble one another closely, and members of the same paralagous group often collaborate with one another during development (Chen and Capecchi 1999). Another feature of Hox genes is that those located at the 3 end of each cluster are expressed prior to those at the more 5 end, and this is also reflected in their spatial pattern of expression—that is, 3 genes are expressed in a more anterior position, whereas 5 genes are more posterior (Duboule 1998). This colinearity likely explains why the majority of Hox proteins found in the

developing lung are encoded by genes located at the more 3 end. In addition to Hox genes, at least 160 other divergent homeobox genes have been identified that lie outside Hox clusters, all of which possess a homeodomain. In general, binding of homeobox proteins to target promoter sequences, and subsequent transcriptional activation of downstream genes, represents the predominant manner in which homeobox genes control tissue patterning and cellular events required for development, such as proliferation, migration, differentiation, and survival. Given the diversity of functions specified by homeobox genes, it is surprising to learn that homeodomain interactions with DNA are highly conserved, raising the question as to how different homeobox proteins specify distinct functions. Certainly, interactions with cofactors, such as Pbx proteins, represent one way to achieve this (Mann and Chan 1996). In addition, residues outside the homeodomain play an important role in generating specificity in different tissues (Chauvet et al. 2000). Although little is known about how homeobox genes control lung-specific programs of gene expression, it is clear that they are crucial for normal lung development. • Homeobox Genes in the Developing Lung To ensure efficient gas exchange in the postnatal lung, pulmonary vessels form in parallel to the airways and as capillary networks surrounding the alveoli. Vasculogenesis and angiogenesis are the two main processes believed to give rise to the initial endothelial cell (EC) networks within the developing lung (Schachtner et al. 2000). Thereafter, the immature endothelium is invested by mesenchymal cells that differentiate into different cell types required for assembly and differentiation of the blood vessel wall, including fibroblasts and smooth muscle cells (SMCs) (Jeffery and Morrell 2002). Adding to the complexity of generating the pulmonary vasculature is reliance upon tissue interactions between the branching airway epithelium and its surrounding mesenchyme. For example, when fetal rat lung mesenchyme is cultured with fetal lung epithelium, differentiated ECs emerge from within the distal mesenchyme. However, when fetal lung mesenchyme is cultured alone, EC differentiation TCM Vol. 13, No. 8, 2003

Figure 1. Organization of Hox genes in Drosophila melanogaster and mouse. Clusters of homeotic genes involved in specifying segment identity—originally identified in Drosophila— are also present in other animals, including mice and humans. In vertebrates, Hox genes are related to the Antennapedia homeobox of Drosophila. In the mouse, there are four unlinked Hox complexes, designated Hoxa, Hoxb, Hoxc, and Hoxd located on different chromosomes. Note that Hox genes at the 3’ end are expressed in a more anterior position than are those at the 5’ end.

is suppressed (Gebb and Shannon 2000). A major factor that appears to be critical for lung tissue interactions is vascular endothelial growth factor (VEGF), which is expressed at high levels by the epithelium. For example, mice that do not express the VEGF120 isoform fail to develop a normal peripheral lung vasculature (Galambos et al. 2002). Similarly, genetic ablation of hypoxia-inducible transcription factor 2 results in diminished VEGF levels in alveolar cells, and a defect in vascularization of alveolar septa (Compernolle et al. 2002). These findings, coupled with the observation that pulmonary vascular cells of the same type exhibit a remarkable degree of structural and functional heterogeneity (Jeffery and Morrell 2002), demonstrate that programs of gene expression within the developing lung need to be precisely co-coordinated in time and space to generate the exquisite fractal patterns that define the pulmonary vasculature (Figure 2). In this regard, homeobox genes likely play a key role. A review of the prevailing literature indicates that many homeobox genes are expressed in the developing lung (Table 1). Although the precise functions of many of these genes remain to be deTCM Vol. 13, No. 8, 2003

fined, knockout experiments in mice reveal that a number of them control lung development. For example, Hoxa-1 knockout mice fail to initiate breathing (Lufkin et al. 1991), and Hoxa-3 null mice die

shortly after birth, possibly as a result of pulmonary failure (Chisaka and Capecchi 1991). Hoxa-5–deficient mice display respiratory distress at birth due to profound dysmorphogenesis of the developing lung, and decreased expression of surfactant-associated proteins (Aubin et al. 1997). Further, expression of Nkx2.1 and HNF-2 (genes that regulate the production of surfactant-associated proteins in the endoderm), is altered in Hoxa-5 null mice (Aubin et al. 1997). Because Hoxa-5 expression is confined to the mesenchyme of the respiratory tract in the developing lung (a cell population that interacts with overlying endoderm during lung morphogenesis), this study exemplifies how homeobox genes may regulate lung tissue interactions. Other gene knockout studies demonstrate that paralagous Hox genes cofunction during lung development. For example, whereas the gross appearance of Hoxa-1 or Hoxb-1 single knockout mice appears to be normal, the amount of lung tissue and number of lung lobes is reduced in Hoxa1:Hoxb-1 double knockout mice (Rossel and Capecchi 1999). In addition to transgenic studies, many of the dramatic effects that retinoic acid (RA) wields on lung morphogenesis involve Hox genes (Bogue et al. 1994, Cardoso et al. 1996, Packer et al. 2000). For instance, ex vivo treatment of embryonic

Figure 2. The pulmonary arterial system. Photomicrograph of a methylmethacrylate pulmonary arterial cast from a 140-day fetal lamb. (Reprinted with permission from Levin et al. 1976, p. 146. Copyright 1996 Lippincott Williams & Wilkins.)

337

Table 1. Examples of homeobox genes expressed during lung development and disease Gene Hoxa-1

Expression pattern, regulation, and function

Reference

Null mice are anoxic and die at birth

Lufkin et al. 1991

Co-expressed with Hoxa-3, Hoxa-5, Hoxb-3, Hoxb-4, Hoxb-6, Hoxb-7, and Hoxb-8 in branching region of embryonic day (E)11.5 mouse lung

Mollard and Dziadek 1997

Hoxa-1/b-1 double knockouts possess smaller lungs

Rossel and Capecchi 1999

Hoxa-1-regulated-62 gene represents putative target in adult lung

Shen and Gudas 2000

Transcripts expressed in normal adult lung

Golpon et al. 2001

Hoxa-2

Expression pattern suggests role in differentiation of proximal mesenchyme derivatives and vasculogenesis

Cardoso et al. 1996

Hoxa-3

Null mice die shortly after birth, conceivably of pulmonary failure

Chisaka and Capecchi 1991

Hoxa-4

Expressed between E8.5–12.5 in lung mesenchyme

Behringer et al. 1993

Expression in lung relies on a conserved retinoic acid response element (RARE) in the 5 flanking region

Packer et al. 1998

By E14.5, Hoxa-4 is restricted to proximal mesenchyme, smooth muscle cells, subepithelial fibroblasts, and alveolar cells. Expression may be determined by opposing effects of retinoic acid (RA) and transforming growth factor (TGF)-1

Packer et al. 2000

Hoxa-5

Expressed in E8–E13 lung, but not by E18

Dony and Gruss 1987

Null mice display improper lung morphogenesis, respiratory distress, and perinatal lethality. Controls tissue interactions, because loss of mesenchymal Hoxa-5 leads to surfactant deficiency and altered expression of Nkx2.1, HNF-3, and N-myc in pulmonary epithelium

Aubin et al. 1997

Induced by RA in human bronchial fibroblasts, but not in dermal fibroblasts. Basis for cell-type specificity unknown

Bernacki et al. 1992

Induction by RA relies on Hoxa-4

Packer et al. 2000

Hoxa-10

Expressed in normal human lung and primary pulmonary hypertension

Golpon et al. 2001

Hoxb-1

Expression in foregut endoderm, which gives rise to lungs, depends on a 3 RARE

Huang et al. 1998

Hoxb-2

Co-expressed with Hoxb-5 in distal lung mesenchyme at E10.5–E14.5. Hoxb-3 and b-4 detected in mesenchyme of trachea, mainstem bronchi, and distal lung. Indicates that specific combinations of Hoxb genes specify differences between proximal and distal mesenchyme

Bogue et al. 1996

In human fetal and adult lung, as well as in emphysema and primary pulmonary hypertension

Golpon et al. 2001

Co-expressed with Hoxb-4 and b-5 in E9.5 foregut

Bogue et al. 1996

Expressed in prenatal lung, involved in retinol-induced gene expression of Clara-specific secretory protein. Reduces expression of surfactantassociated protein C

Nakamura et al. 2002

Hoxb-3

Hoxb-4

Expressed in embryonic lung mesenchyme

Graham et al. 1988

Hoxb-5

In fetal mouse lung, mesodermal cells surrounding branching epithelial cell layer accumulate high levels of Hoxb-5

Krumlauf et al. 1987

In E13.5 mouse to postnatal day 2, expressed in conducting airways and surrounding mesenchyme. At E14.5, levels decrease in mesenchyme distal from airways, but persist in fibroblasts underlying conducting airways

Volpe et al. 1997

Expressed with Hoxb-6 in developing chick respiratory tract. Prior to branching, expressed around ventral-distal tip of lung bud, eventually demarcating trachea, bronchial tree, and airsacs. May specify positional identity, because Hoxb-6 through Hoxb-9 expression corresponds to morphologic subdivisions of airsacs along proximodistal axis

Sakiyama et al. 2000

Required for branching morphogenesis in cultured fetal lungs. Controls morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part by Hoxb-5

Volpe et al. 2000

At E14, primarily in mesenchymal cells. By E19, appears mainly in epithelial cells of prealveolar structures and adjacent subepithelium. Potential role in lung maturation, because expression persists in hypoplastic lung mesenchyme throughout development and in postnatal lung

Chinoy et al. 2002

(Continued)

338

TCM Vol. 13, No. 8, 2003

Table 1. Continued Gene

Expression pattern, regulation, and function

Reference

Hoxb-6

Hoxb-6 expression reduced during lung development. Potential role in distal airway branching

Cardoso et al. 1996

Divergent Homeobox Genes Barx2 Gax

Expressed in developing mouse lung buds Expressed in adult rat cardiovascular tissues, including lung Inhibits vascular smooth muscle cell (SMC) proliferation and migration

Hex

Expressed in newborn mouse lung Detected in E12.5 and E15.5 lung Expressed at E11.5, especially in distal lung mesenchyme. Suppressed by E16.5, and upregulated in adult lung. Indicates role in later stages of development and in control of gene expression in adult lung Expressed in E10.5 mouse foregut lung mesenchyme. Suggested role in controlling tissue interactions Expressed in E8.5 foregut region. During glandular development, present in lung epithelium. Expression declines at end of cannilicular stage. Suggested control epithelial/mesenchymal tissue interactions in concert with Gli1, 2 and 3 Controls surfactant protein A and B gene promoters

Hlx Irx1 and Irx2

Nkx2.1

Pitx2 Prx1 and Prx2

Six4 and Six5

Antisense oligonucleotides inhibit epithelial branching morphogenesis in embryonic mouse lung explants Activates transcription of Clara cell-specific protein Null mouse lungs consist of dilated sacs arrested at E11–E15. Reduction in bone morphogenetic protein-4 expression may account for this branching defect. Controls vascular endothelial growth factor and pulmonary epithelial surfactant expression Transactivation of Nkx2.1 gene promoter in pulmonary epithelial cells relies on interactions with Sp1 and Sp3 transcription factors Detailed examination of null mice indicates that distal lung morphogenesis is Nkx2.1 dependent Siblings with heterozygous deletion of Nkx2.1 display congenital thyroid dysfunction and recurrent acute respiratory distress Controls RA stimulation of surfactant protein B promoter activity Appears to cooperate with CAAT enhancer binding protein beta alpha in determining high- level, lung epithelial-specific expression of Clara cell secretory protein during later stages of lung development and in adult lung Sma- and Mad-related protein 3 interactions with Nkx2.1 and HNF-1 underlie the basis for TGF-dependent repression of surfactant protein-B Regulates expression of midkine Null mice develop right pulmonary isomerism Expressed in walls of developing muscularized chick pulmonary arteries. Prx1 is especially prominent at adventitial-medial boundary. Prx2 is also expressed in media. Indicates roles in assembly of the vascular wall by promoting recruitment and segregation of smoothmuscle and fibroblasts, as well as matrix synthesis and deposition Prx1-null mouse exhibits cardiovascular defects and dies of respiratory distress soon after birth. Lung phenotype not examined Prx1 and Prx2 induced in experimental pulmonary vascular disease Activate transcription of tenascin-C and extracellular matrix protein that promotes pulmonary artery SMC proliferation and survival Six4 is present in embryonic lung, whereas Six5 is expressed in adult lung

TCM Vol. 13, No. 8, 2003

FS Jones et al. 1997 Gorski et al. 1993 Perlman et al. 1999 Witzenbichler et al. 1999 Bogue et al. 1994 Keng et al. 1998 Bogue et al. 2000

Lints et al. 1996 Becker et al. 2001

Bohinski et al. 1994 Bruno et al. 1995 Minoo et al. 1995 Zhang et al. 1997 Minoo et al. 1999

Li et al. 2000 Yuan et al. 2000 Iwatani et al. 2000 Naltner et al. 2000 Cassel et al. 2002

Li et al. 2002 Reynolds et al. 2003 Lin et al. 1999 Bergwerff et al. 1998

Martin et al. 1995 FS Jones et al. 2001

Ohto et al. 1998

339

lungs with RA suppresses distal lung formation in favor of proximal tubules (Packer et al. 2000). This effect is accompanied by upregulation and redistribution of Hoxa-4 and Hoxa-5 from the proximal to the peripheral region of the lung, suggesting that these genes ordinarily function to proximalize the lung. Furthermore, the effects of RA on Hoxa-5 expression are lost in Hoxa-4 null lungs, indicating that proximalization of the lung by RA is mediated, in part, through cross-modulation of different Hox genes (Packer et al. 2000). Despite these experiments, only a handful of homeobox genes expressed in the developing lung have been linked to one or more processes considered essential for pulmonary vascular development. Nevertheless, identifying which homeobox genes are expressed in the lung and relating these findings to their known functions in other tissues provides some clues with regard to their potential roles in pulmonary vascular development. • Homeobox Genes in Vasculogenesis and Angiogenesis Vasculogenesis, or differentiation of ECs from the uncommitted distal mesoderm and organization of endothelial progenitors into a primitive vascular plexus, occurs as early as embryonic day (E)9.5 to 10 in the mouse lung (deMello et al. 1997). Although it is not clear how cells within the lung mesenchyme are selected to undergo vasculogenesis, recent evidence indicates that the divergent homeobox gene, Hex, may be involved (Bogue et al. 1994, Thomas et al. 1998). This gene is expressed in a range of multipotent progenitor lines and is generally downregulated during terminal differentiation (Pellizzari et al. 2000). In the mouse embryo, Hex first materializes in the primitive endoderm of the implanting blastocyst. Subsequently, it appears in the foregut endoderm and within the mesoderm, where it is transiently expressed in the nascent blood islands of the yolk sac, and later in the embryonic angioblasts and endocardium (Thomas et al. 1998). In the embryonic mouse lung, the highest levels of Hex are observed in the distal lung mesenchyme (Bogue et al. 1994). Taken together with the aforementioned studies, Hex appears to be well poised, both temporally and spatially, to drive vasculogenesis. In fact, a

340

comparison with flk-1 expression indicates that Hex is an early marker of endothelial precursors (Thomas et al. 1998). Unlike flk-1, however, expression of Hex in this progenitor population is more transient, being downregulated once EC differentiation commences. Furthermore, whereas flk-1–null mice fail to generate a vascular network and lack hematopoietic progenitor cells (Shalaby et al. 1995), disruption of Hex gene expression has no effect on either of these processes (Martinez Barbera et al. 2000). All the same, the overlap in flk-1 and Hex expression implicates this homeobox gene in the initial stages of EC selection and/ or differentiation within the mesoderm, an idea substantiated by the finding that expression of the Hex homologue hHex in zebrafish leads to early and ectopic expression of flk-1 (Liao et al. 2000). In addition to Hex, in situ hybridization studies suggest that Hoxa-2 may play a role in lung vasculogenesis (Cardoso et al. 1996), yet ablation of this gene has no effect on the normal lung phenotype (Gendron-MacGuire et al. 1993). Other approaches, including analysis of compound knockouts, involving one or more Hoxa-2 paralogues, will no doubt reveal whether this or other Hox genes truly contribute to pulmonary vascular development. As well as developing a distal vascular plexus by vasculogenesis, angiogenesis (i.e., sprouting of ECs from pre-existing vessels), is also believed to contribute to pulmonary vascular development by connecting the great vessels of the heart with those formed by vasculogenesis. Once again, little is known about how homeobox genes control this process in the lung, but studies in other tissues indicate that they are likely to be involved. For example, Hoxb-7, which is expressed in the fetal lung (Mollard and Dziadek 1997), upregulates a variety of angiogenic stimuli, including fibroblast growth factor (FGF)-2, VEGF, and matrix metalloproteinase (MMP)-9 (Care et al. 2001). Similarly, HOXD3 (i.e., the human homologue of mouse Hoxd-3) colocalizes with integrin v3 in angiogenic vessels formed in response to FGF-2 on a chick chorioallantoic membrane assay (Figure 3), and it also promotes the invasive phenotype of human umbilical vein and dermal microvascular ECs by stimulating expression of urokinase plasminogen activator (uPA) and integrin v3 (Bou-

dreau et al. 1997), recognized components of the angiogenic cascade. Although HOXD3 and HOXB7 participate in the invasive phase of angiogenesis, HOXB3 (a HOXD3 paralogue expressed in prenatal lung tissue (Nakamura et al. 2002) promotes capillary morphogenesis on basement membrane extracellular matrix (ECM) by controlling expression of ephrin A1 and subsequent activation of the EphA2 receptor (Myers et al. 2000). Following capillary morphogenesis, angiogenic ECs become quiescent. Appreciating that during development HOX genes at the 3 end of each HOX cluster are expressed prior to those at the more 5 end, Myers et al. (2002) hypothesized that subsequent to the initial stages of angiogenesis, maturing capillaries would begin to express more 5 HOX genes. Indeed, HOXD10 expression is greater in quiescent ECs when compared with their angiogenic counterparts. Consistent with this, expression of HOXD10 in vivo reduces the angiogenic response of ECs to FGF-2. Mechanistically, HOXD10 suppresses expression of uPA receptors and blocks FGF-2-dependent migration of ECs into fibrin gels, a process that relies on uPA receptors. Because HOXD10 appears to be expressed in adult but not fetal lung tissue (Golpon et al. 2001), it is conceivable that this gene actively maintains the fully differentiated state of ECs within the mature lung. • Homeobox Genes and Assembly of the Pulmonary Vascular Wall Homeobox genes have also been implicated in a variety of processes required for assembly and elaboration of the vascular wall, including the promotion and inhibition of SMC proliferation, differentiation, and changes in cell adhesion to the ECM. For example, Hoxb-7—which appears in the developing lung (Mollard and Dziadek 1997)—is expressed more highly in fetal than in adult vascular SMCs (Miano et al. 1996), indicating that this gene may play a role in the early expansion and differentiation of SMCs. Consistent with this, Hoxb-7 promotes proliferation, as well as inducing expression of early SMC markers, such as SM22 (Bostrom et al. 2000). In contrast to Hoxb-7, the divergent homeobox gene Gax (which is expressed in adult cardiovascular tissues including heart, lung, and blood vessels) inhibits vascuTCM Vol. 13, No. 8, 2003

Figure 3. HOXD3 (red) colocalizes with integrin v3 (green) in angiogenic capillaries on a chick chorioallantoic membrane assay. (Image courtesy of Dr. Nancy Boudreau, Department of Surgery, University of California San Francisco).

lar SMC proliferation (Gorski and Walsh 2000). Within the vasculature, Gax is expressed in quiescent SMCs and is downregulated when these cells are stimulated to proliferate (Perlman et al. 1999, Smith et al. 1997). In keeping with this growth-suppressive role, overexpression of Gax inhibits SMC proliferation by activating the cell cycle inhibitor protein p21 (Smith et al. 1997). Gax also attenuates SMC motility by downregulating integrins v3 and v5 in a manner that is dependent on the cell cycle (Witzenbichler et al. 1999). Prx1 and Barx2 also encode divergent homeobox genes that are believed to promote SMC differentiation (Hautmann et al. 1997, Herring et al. 2001, Meech et al. 2003), and both of these genes are expressed in the developing lung (Bergwerff et al. 1998, FS Jones et al. 1997). Mechanistically, Prx1 and Barx2 have been shown to control smooth musclespecific gene expression in vitro by enhancing binding of serum response factor (SRF) to CarG elements, a DNA motif present in many smooth musclespecific gene promoters (Herring et al. 2001, Meech et al. 2003). Furthermore, the Barx2 gene promoter contains an SRF-binding serum response element, which is essential for its activity (Meech et al. 2003), indicating that this gene may be autoregulated. However, whereas Barx2 is expressed in the differentiated adult aorta (Herring et al. 2001), Prx1 only appears in developing and injured blood vessels, when SMC differentiation is being actively suppressed in favor of proliferation (Bergwerrf et al. 1998, FS Jones et al. 2001). It is possible, nonetheless, that Prx1 and SRF cofunction TCM Vol. 13, No. 8, 2003

in other cell types within the blood vessel wall, (i.e., activated adventitial fibroblasts), that express smooth muscle markers during development and in pathologic remodeling. In addition to arteries, homeobox genes appear to play a role in assembly of pulmonary veins. HOXA3-null mice

die shortly after birth, and frequently have heart and blood vessel defects, including hypertrophy of the atria and enlargement of the pulmonary veins (Chisaka and Capecchi 1991). This finding is intriguing, given that the wall of pulmonary veins not only possesses EC lining and smooth muscle tunica media,

Figure 4. Hypothetic schema showing the regulation and functions of Prx1 in vascular smooth muscle cells (SMCs) and adventitial fibroblasts within the injured pulmonary arterial wall. Following injury, activated metalloproteinases (MMP) proteolyze fibrillar type I collagen, converting it to a monomeric form. Re-adhesion of SMCs or fibroblasts to this modified extracellular matrix substrate leads to phosphorylation of focal adhesion kinase (FAK), and subsequently induction of Prx1. In turn, Prx1 interacts with the TN-C gene promoter, activating transcription leading to the synthesis and secretion of tenascin-C (TN-C) protein. Following secretion into the extracellular space, TN-C interacts with SMC and fibroblast cell-surface receptors that promote proliferation and migration.

341

but also an outer layer of cardiac cells (Millino et al. 2000). Considering the phenotype of Hoxa-3 null mice, it plausible that this gene functions to limit cardiac cell migration during pulmonary venous development. • Homeobox Genes and Lymphangiogenesis In addition to pulmonary arteries, veins, and a bronchial circulation, a lymphatic pulmonary vasculature collects extravasated immune cells, and keeps the airways dry by collecting fluid leaked from blood vessels. Identifying factors that regulate lymphangiogenesis is a crucial, yet understudied, aspect of lung biology. A recent study (Wigle et al. 2002) reveals that the Prox1 homeobox gene is essential for this process. Prox1 is expressed in a subpopulation of ECs that, after budding from veins, give rise to the mammalian lymphatic system. In Prox1null embryos, budding is arrested at E11.5, resulting in embryos that are capable of undergoing vasculogenesis and angiogenesis, but fail in lymphangiogenesis. Further, ECs that ordinarily express lymphatic markers—including VEGFR3 and lymphatic endotheliumspecific hyaluronan receptor-1 (LYVE-1)— are absent in these mice, being replaced by markers that characterize the blood vascular phenotype (Wigle et al. 2002). Thus, Prox1 appears to be required for maintenance of budding of venous ECs and differentiation toward the lymphatic phenotype. In light of this work, it will be important to determine when and where Prox1 is expressed in the developing lung lymphatics, and whether this gene is dysregulated in lung lymphatic diseases, such as familial pulmonary lymphangiectasia. • Homeobox Genes in Pulmonary Vascular Disease Loss or disruption of pulmonary vascular form and function underscores a number of diseases, including emphysema, bronchopulmonary dysplasia, and pulmonary hypertension. As with pulmonary vascular development, formation of occlusive vascular lesions in the lung also depends on migration and proliferation of ECs, SMCs, and fibroblasts, as well as inappropriate expression of smooth muscle markers, and excessive catabolism and

342

synthesis of specific ECM proteins (Jeffery and Morrell 2002). It is likely, therefore, that many of the homeobox genes involved in pulmonary vascular development are re-expressed in pulmonary vascular disease. In keeping with this, a comparison of HOX gene expression in human fetal lungs versus tissue derived from patients with primary pulmonary hypertension and emphysema indicates that this is the case for certain HOX genes, including HOXA5 and HOXB2 (Golpon et al. 2001). On the other hand, the tissue distribution of some of these HOX genes appears to differ between fetal and remodeled adult lung. For example, whereas HOXA5 expression is confined to the mesenchyme of the respiratory tract in the developing lung (Aubin et al. 1997), this gene has been reported to be expressed by ECs in hyperproliferative, occlusive plexiform lesions in adult patients with primary pulmonary hypertension (Golpon et al. 2001). It is possible, however, that HOXA5 is also expressed in muscle cells that are known to contribute to these lesions (PL Jones et al. 1997a). Clearly, additional studies are needed to determine the precise cellular origin and role of HOXA5 in primary pulmonary hypertension, and how this gene may confer different functions in distinct tissues. Recent work in our laboratory indicates that the paired-related homeobox genes, Prx1 and Prx2, contribute to pulmonary vascular disease by promoting expression of tenascin-C (TN-C) (FS Jones et al. 2001), an ECM glycoprotein that supports SMC proliferation and migration (PL Jones et al. 1997b, McKean et al. 2003). TN-C is expressed in the developing vasculature, but is suppressed in normal, quiescent pulmonary vessels. In hypertensive vessels, however, TN-C reappears at the adventitial-medial boundary, and later throughout the vascular wall, where it surrounds proliferating and migrating SMCs and fibroblasts (Jones and Rabinovitch 1996, PL Jones et al. 1997a). At a functional level, TN-C promotes proliferation and survival of pulmonary artery SMCs via its ability to cross-modulate the activity of receptor tyrosine kinases. In terms of its effects on migration, TN-C destablizes focal adhesions, thereby facilitating cell detachment from the underlying ECM (Chung et al. 1996). Given the importance of these functions in vascular development

and disease, it is of interest to determine how this ECM protein is regulated. Multiple factors induce TN-C, including ECM-degrading MMPs. For example, inhibition of MMP activity suppresses TN-C expression and pulmonary artery SMC proliferation, and reduces the severity of vascular lesions (Cowan et al. 2000, PL Jones et al. 1997b). These studies indicate that MMPs lie upstream in an adhesion-dependent signaling pathway that controls TN-C. Consistent with this, the TN-C gene promoter contains an ECM-responsive element that is silenced on native type I collagen (an 21 integrin ligand), but is activated on the MMPproteolyzed or denatured type I collagen (which represents an v3 integrin ligand) (PL Jones et al. 1999). This ECM/ 3 integrin-responsive element in the TN-C gene promoter was subsequently shown to harbor a homeodomain binding site, suggesting that induction of TN-C in response to changes in SMC adhesion might depend on homeobox proteins (FS Jones et al. 2001), and in this regard, Prx transcription factors represent ideal candidates. For example, like TN-C, Prx1 and Prx2 are expressed during embryogenesis, predominantly in mesenchymespecific patterns (Leussink et al. 1995). In the developing cardiovascular system, Prx1 and Prx2 expression overlaps with TN-C in the endocardial cushions and valves, the epicardium, and the wall of the great arteries and veins (Bergwerff et al. 1998, reviewed in Jones and Jones 2000). Further, Prx1 and Prx2 gene knockouts exhibit skeletal defects and vascular anomalies, including a twisted aorta and elongated ductus arteriosus (Bergwerff et al. 2000), defects that conceivably arise due to dysregulated TN-C production. In further support of a link between Prx proteins and TN-C expression, examination of hypertensive adult rat lungs showed that Prx1 and Prx2 are re-expressed at the adventitial-medial boundary of remodeling pulmonary arteries, and later within the arterial wall, co-localizing with TN-C and proliferating SMCs (FS Jones et al. 2001). Moreover, identical to TN-C, Prx1 and Prx2 expression is also suppressed and induced by native and denatured type I collagen, respectively, and overexpression of Prx1 promotes TN-C gene transcription and SMC proliferation (FS Jones et al. 2001). Whether Prx1 promotes SMC proliferation by inducing TN-C remains to be determined. TCM Vol. 13, No. 8, 2003

Because expression of Prx1 and TN-C both depend on changes in cell adhesion to the ECM via integrins, we hypothesized that activation of focal adhesion kinase (FAK)—an integrin-activated nonreceptor tyrosine kinase that is required for SMC and fibroblast migration— supports Prx1-dependent induction of TN-C. In keeping with this, migrationdefective fibroblasts and E8.5 embryos devoid of FAK express reduced levels of Prx1 and TN-C when compared with their wild-type counterparts, whereas overexpression of Prx1 in FAK-null cells restored TN-C expression and cell migration (McKean et al. 2003). Taken together with our previous studies, a tenable working hypothesis is that during vascular development and following injury, MMP-dependent activation of FAK stimulates TN-C production via Prx1, thereby contributing to the formation of a TNC-rich ECM that supports SMC and fibroblast proliferation and migration (Figure 4). In addition to promoting Prx1 and TN-C expression, FAK is also required for capillary morphogenesis (Ilic et al. 2003). Because TN-C also favors this process (Castellon et al. 2002), it is possible that FAK-dependent induction of TN-C via Prx1 promotes capillary formation. This idea is especially appealing given that Prx1-null mice are cyanotic and die soon after birth from respiratory distress (Martin et al. 1995). Whether TN-C and capillary formation are affected in the developing lungs of Prx1 knockout mice awaits further investigation. • Conclusion Although an increasing body of evidence implicates homeobox genes in vascular biology, it is imperative that these functions be tested in the lung. In addition, although a number of homeobox gene knockouts cause lung defects, the pulmonary vasculature of many of these mice has not been scrutinized. Also, it will be necessary to identify cofactors that modulate homeobox gene function, and to determine how these contribute to pulmonary vascular-specific gene expression. Irrespective of the outcome, homeobox gene research in the lung is still in its infancy, providing fertile ground for determining how these master control genes impact pulmonary vascular development and disease. TCM Vol. 13, No. 8, 2003

• Acknowledgments This research was supported by grants awarded to P.L.J. (National Institutes of Health [NIH] 1 RO1 HL68798-01 and NIH 2 P50 HL57144-06), and by The American Physiological Society Giles Filley Memorial Award. The author would like to thank Drs. Nancy Boudreau, Neil Davie, Sarah A. Gebb, Raphael Nemenoff, and Kurt Stenmark for their helpful comments.

References Aubin J, Lemieux M, Tremblay M, et al.: 1997. Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev Biol 192:432–445. Becker MB, Zulch A, Bosse A, Gruss P: 2001. Irx1 and Irx2 expression in early lung development. Mech Dev 106:155–158. Behringer RR, Crotty DA, Tennyson VM, et al.: 1993. Sequences 5 of the homeobox of the Hox-1.4 gene direct tissue-specific expression of lacZ during mouse development. Development 117:823–833. Bergwerff M, Gittenberger-de Groot AC, DeRuiter MC, et al.: 1998. Patterns of paired-related homeobox genes Prx1 and Prx2 suggest involvement in matrix modulation in the developing chick vascular system. Dev Dyn 213:59–70. Bergwerff M, Gittenberger-de Groot AC, Wisse LJ, et al.: 2000. Loss of function of the Prx1 and Prx2 homeobox genes alters architecture of the great elastic arteries and ductus arteriosus. Virchows Arch 436:12–19. Bernacki SH, Nervi C, Vollberg TM, Jetten AM: 1992. Homeobox 1.3 expression: induction by retinoic acid in human bronchial fibroblasts. Am J Respir Cell Mol Biol 7:3–9. Bogue CW, Gross I, Vasavada H, et al.: 1994. Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression. Am J Physiol 266:L448–L454. Bogue CW, Lou LJ, Vasavada H, et al.: 1996. Expression of Hoxb genes in the developing mouse foregut and lung. Am J Respir Cell Mol Biol 15:163–171. Bogue CW, Ganea GR, Sturm E, et al.: 2000. Hex expression suggests a role in the development and function of organs derived from foregut endoderm. Dev Dyn 219:84–89. Bohinski RJ, Di Lauro R, Whitsett JA: 1994. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organspecific gene expression along the foregut axis. Mol Cell Biol 14:5671–5681.

Bostrom K, Tintut Y, Kao SC, et al.: 2000. HOXB7 overexpression promotes differentiation of C3H10T1/2 cells to smooth muscle cells. J Cell Biochem 78:210–221. Boudreau N, Andrews C, Srebrow A, et al.: 1997. Induction of the angiogenic phenotype by HoxD3. J Cell Biol 139:257–264. Bruno MD, Bohinski RJ, Huelsman KM, et al.: 1995. Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1. J Biol Chem 270:6531–6536. Cardoso WV, Mitsialis SA, Brody JS, Williams MC: 1996. Retinoic acid alters the expression of pattern-related genes in the developing rat lung. Dev Dyn 207:47–59. Care A, Felicetti F, Meccia E, et al.: 2001. HOXB7: a key factor for tumor-associated angiogenic switch. Cancer Res 61:6532–6539. Cassel TN, Berg T, Suske G, Nord M: 2002. Synergistic transactivation of the differentiation-dependent lung gene Clara cell secretory protein (secretoglobin 1a1) by the basic region leucine zipper factor CCAAT/enhancerbinding protein alpha and the homeodomain factor Nkx2.1/thyroid transcription factor1. J Biol Chem 277:36,970–36,977. Castellon R, Caballero S, Hamdi HK, et al.: 2002. Effects of tenascin-C on normal and diabetic retinal endothelial cells in culture. Invest Ophthalmol Vis Sci 43:2758–2766. Chauvet S, Merabet S, Bilder D, et al.: 2000. Distinct hox protein sequences determine specificity in different tissues. Proc Natl Acad Sci USA 97:4064–4069. Chen F, Capecchi MR: 1999. Paralogous mouse Hox genes, Hoxa9, Hoxb9, and Hoxd9, function together to control development of the mammary gland in response to pregnancy. Proc Natl Acad Sci USA 96:541–546. Chinoy MR, Nielsen HC, Volpe MV: 2002. Mesenchymal nuclear transcription factors in nitrofen-induced hypoplastic lung. J Surg Res 108:203–211. Chisaka O, Capecchi MR: 1991. Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5. Nature 350:473–479. Chung CY, Murphy-Ullrich JE, Erickson HP: 1996. Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II. Mol Biol Cell 7:883–892. Compernolle V, Brusselmans K, Acker T, et al.: 2002. Loss of HIF-2 and inhbition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 8: 702–710. Cowan KN, Jones PL, Rabinovitch M: 2000. Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C

343

antisense prevents progression, of vascular disease. J Clin Invest 105:21–34. deMello DE, Sawyer D, Galvin N, et al.: 1997. Early fetal development of lung vasculature. Am J Respir Cell Mol Biol 16:568–581. Dony C, Gruss P: 1987. Specific expression of the Hox 1.3 homeo box gene in murine embryonic structures originating from or induced by the mesoderm. Embo J 6:2965– 2975. Duboule D. 1998: Vertebrate hox gene regulation: clustering and/or colinearity? Curr Opin Genet Dev 8:514–518. Galambos C, Ng Y-S, Ali A, et al.: 2002. Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. Am J Respir Cell Mol Biol 27:194–203. Gebb SA, Shannon JM: 2000. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev Dyn 217:159–169. Gendron-MacGuire M, Mallo M, Zhang M, Gridley T: 1993. Hoxa-2 mutant mice exhibit homeotic transformation of skeletal elements derived from cranial neural crest. Cell 75:1317–1331. Golpon HA, Geraci MW, Moore MD, et al.: 2001. HOX genes in human lung: altered expression in primary pulmonary hypertension and emphysema. Am J Pathol 158:955–966. Gorski DH, Walsh K: 2000. The role of homeobox genes in vascular remodeling and angiogenesis. Circ Res 87:865–872. Gorski DH, LePage DF, Patel CV, et al.: 1993. Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells. Mol Cell Biol 13:3722–3733. Graham A, Papalopulu N, Lorimer J, et al.: 1988. Characterization of a murine homeo box gene, Hox-2.6, related to the Drosophila Deformed gene. Genes Dev 2:1424–1438. Hautmann MB, Thompson MM, Swartz EA, et al.: 1997. Angiotensin II-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox. Circ Res 81:600–610. Herring BP, Kriegel AM, Hoggatt AM. 2001: Identification of Barx2b, a serum response factor-associated homeodomain protein. J Biol Chem 276:14,482–14,489. Huang D, Chen SW, Langston AW, Gudas LJ: 1998. A conserved retinoic acid responsive element in the murine Hoxb-1 gene is required for expression in the developing gut. Development 125:3235–3246. Ilic D, Kovacic B, McDonagh S, et al.: 2003. Focal adhesion kinase is required for blood vessel morphogenesis. Circ Res 92:300–307. Iwatani N, Mabe H, Devriendt K, et al.: 2000. Deletion of NKX2.1 gene encoding thyroid

344

transcription factor-1 in two siblings with hypothyroidism and respiratory failure. J Pediatr 137:272–276. Jeffery TK, Morrell NW. 2002: Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Prog Cardiovasc Dis 45:173–202. Jones FS, Jones PL: 2000. The tenascin family of ECM glycoproteins: structure, function and regulation during embryonic development and tissue remodeling. Dev Dyn 218:235–259. Jones FS, Kioussi C, Copertino DW, et al.: 1997. Barx2, a new homeobox gene of the Bar class, is expressed in neural and craniofacial structures during development. Proc Natl Acad Sci USA 94:2632–2637. Jones FS, Meech R, Edelman DB, et al.: 2001. Prx1 controls vascular smooth muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary vascular disease. Circ Res 89:131–138. Jones PL, Rabinovitch M: 1996. Tenascin-C is induced with progressive pulmonary vascular disease in rats and is functionally related to increased smooth muscle cell proliferation. Circ Res 79:1131–1142. Jones PL, Cowan KN, Rabinovitch M: 1997a. Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease. Am J Pathol 150:1349–1360. Jones PL, Crack J, Rabinovitch M: 1997b. Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 139: 279–293. Jones PL, Jones FS, Zhou B, Rabinovitch M: 1999. Induction of vascular smooth muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a beta3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element. J Cell Sci 112(Pt 4):435–445. Keng VW, Fujimori KE, Myint Z, et al.: 1998. Expression of Hex mRNA in early murine postimplantation embryo development. FEBS Lett 426:183–186. Krumlauf R, Holland PW, McVey JH, Hogan BL: 1987. Developmental and spatial patterns of expression of the mouse homeobox gene, Hox 2.1. Development 99:603–617. Leussink B, Brouwer A, el Khattabi M, et al.: 1995. Expression patterns of the pairedrelated homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain. Mech Dev 52: 51–64. Levin DL, Rudolph AM, Heymann MA, et al.: 1976. Morphological development of the pulmonary vascular bed in fetal lambs. Circulation 53(1):144–151.

Li C, Ling X, Yuan B, Minoo P: 2000. A novel DNA element mediates transcription of Nkx2.1 by Sp1 and Sp3 in pulmonary epithelial cells. Biochim Biophys Acta 1490:213–224. Li C, Zhu NL, Tan RC, et al.: 2002. Transforming growth factor-beta inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors. J Biol Chem 277:38,399–38,408. Liao W, Ho CY, Yan YL, et al.: 2000. Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish. Development 127:4303–4313. Lin CR, Kioussi C, O'Connell S, et al.: 1999. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis. Nature 401:279–282. Lints TJ, Hartley L, Parsons LM, Harvey RP: 1996. Mesoderm-specific expression of the divergent homeobox gene Hlx during murine embryogenesis. Dev Dyn 205:457–470. Lufkin T, Dierich A, LeMeur M, et al.: 1991. Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression. Cell 66:1105–1119. Mann RS, Chan SK. 1996: Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins. Trends Genet 12:258–262. Martin JF, Bradley A, Olson EN: 1995. The paired-like homeobox gene MHox is required for early events of skeletogenesis in multiple lineages. Genes Dev 9:1237–1249. Martinez Barbera JP, Clements M, Thomas P, et al.: 2000. The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Development 127:2433–2445. McGinnis W, Krumlauf R: 1992. Homeobox genes and axial patterning. Cell 68:283–302. McKean DM, Sisbarro L, Ilic D, et al.: 2003. FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration. J Cell Biol 161:393–402. Meech R, Makarenkova H, Edelman DB, Jones FS: 2003. The homeodomain protein Barx2 promotes myogenic differentiation and is regulated by myogenic regulatory factors. J Biol Chem 278:8269–8278. Miano JM, Firulli AB, Olson EN, et al.: 1996. Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells. Proc Natl Acad Sci USA 93:900–905. Millino C, Sarinella F, Tiveron C, et al.: 2000. Cardiac and smooth muscle cell contribution to the formation of the murine pulmonary veins. Dev Dyn 218:414–425. Minoo P, Hamdan H, Bu D, et al.: 1995. TTF1 regulates lung epithelial morphogenesis. Dev Biol 172:694–698.

TCM Vol. 13, No. 8, 2003

Minoo P, Su G, Drum H, et al.: 1999. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(/) mouse embryos. Dev Biol 209:60–71. Mollard R, Dziadek M: 1997. Homeobox genes from clusters A and B demonstrate characteristics of temporal colinearity and differential restrictions in spatial expression domains in the branching mouse lung. Int J Dev Biol 41:655–666. Myers C, Charboneau A, Boudreau N: 2000. Homeobox B3 promotes capillary morphogenesis and angiogenesis. J Cell Biol 148: 343–351. Myers C, Charboneau A, Cheung I, et al.: 2002. Sustained expression of homeobox D10 inhibits angiogenesis. Am J Pathol 161:2099–2109. Nakamura N, Yoshimi T, Miura T: 2002. Increased gene expression of lung marker proteins in the homeobox B3-overexpressed fetal lung cell line M3E3/C3. Cell Growth and Differentiation 13:195–203. Naltner A, Ghaffari M, Whitsett JA, Yan C: 2000. Retinoic acid stimulation of the human surfactant protein B promoter is thyroid transcription factor 1 site-dependent. J Biol Chem 275:56–62.

Sakiyama J, Yokouchi Y, Kuroiwa A: 2000. Coordinated expression of Hoxb genes and signaling molecules during development of the chick respiratory tract. Dev Biol 227: 12–27.

Volpe MV, Martin A, Vosatka RJ, et al.: 1997. Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate. Histochem Cell Biol 108:495–504.

Schachtner SK, Wang Y, Scott Baldwin H: 2000. Qualitative and quantitative analysis of embryonic pulmonary vessel formation. Am J Respir Cell Mol Biol 22:157–165.

Volpe MV, Vosatka RJ, Nielsen HC: 2000. Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung. Biochim Biophys Acta 1475:337–345.

Shalaby F, Rossant J, Yamaguchi TP, et al.: 1995. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376:62–66. Shen J, Gudas LJ: 2000. Molecular cloning of a novel retinoic acid-responsive gene, HA1R-62, which is also up-regulated in Hoxa-1-overexpressing cells. Cell Growth Differ 11:11–17. Smith RC, Branellec D, Gorski DH, et al.: 1997. p21CIP1-mediated inhibition of cell proliferation by overexpression of the gax homeodomain gene. Genes Dev 11:1674– 1689. Thomas PQ, Brown A, Beddington RS: 1998. Hex: a homeobox gene revealing periimplantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 125:85–94.

Wigle JT, Harvey N, Detmar M, et al.: 2002. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J 21:1505–1513. Witzenbichler B, Kureishi Y, Luo Z, et al.: 1999. Regulation of smooth muscle cell migration and integrin expression by the Gax transcription factor. J Clin Invest 104:1469–1480. Yuan B, Li C, Kimura S, et al.: 2000. Inhibition of distal lung morphogenesis in Nkx2.1(/) embryos. Dev Dyn 217:180–190. Zhang L, Whitsett JA, Stripp BR: 1997. Regulation of Clara cell secretory protein gene transcription by thyroid transcription factor-1. Biochim Biophys Acta 1350:359–367.

PII S1050-1738(03)00162-2

TCM

Ohto H, Takizawa T, Saito T, et al.: 1998. Tissue and developmental distribution of six family gene products. Int J Dev Biol 42: 141–148. Packer AI, Crotty DA, Elwell VA, Wolgemuth DJ: 1998. Expression of the murine Hoxa4 gene requires both autoregulation and a conserved retinoic acid response element. Development. 125:1991–1998. Packer AI, Mailutha KG, Ambrozewicz LA, Wolgemuth DJ: 2000. Regulation of the Hoxa4 and Hoxa5 genes in the embryonic mouse lung by retinoic acid and TGFbeta1: implications for lung development and patterning. Dev Dyn 217:62–74. Pellizzari L, D’Elia A, Rustighi A, et al.: 2000. Expression and function of the homeodomain-containing protein Hex in thyroid cells. Nucleic Acids Res 28:2503–2511. Perlman H, Luo Z, Krasinski K, et al.: 1999. Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis. Gene Ther 6:758–763. Reynolds PR, Mucenski ML, Whitsett JA: 2003. Thyroid transcription factor (TTF) -1 regulates the expression of midkine (MK) during lung morphogenesis. Dev Dyn 227: 227–237. Rossel M, Capecchi MR: 1999. Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development. Development 126:5027–5040.

345

Post-Doctoral Fellow, Cardiovascular Research Saint Louis University, a Catholic Jesuit Institution dedicated to education, research and health care, has a Post-Doctoral Position available July 1, 2003 in the Department of Surgery’s Theodore Cooper Surgical Research Institute. Applicants are being sought with demonstrated research experience in Cardiovascular research and with evidence for obtaining extramural funding in areas of hypoxia/reoxygenation injury, signal transduction in primary and secondary myocyte cell cultures, and acute or chronic models of heart disease. The successful candidate will be expected to collaborate with School of Medicine basic science departments that have similar interests. Development and completion of an independent research program as evidenced by publication in a peer-reviewed journal will be expected as well as participation in departmental teaching objectives. Secondary appointments in the School of Medicine basic science departments may be available. Applicants should submit a current curriculum vita, a statement of current and future research plans, and the names and contact information of three references. Contact: Gregory S. Smith, Ph.D. Director, Theodore Cooper Surgical Research Institute Saint Louis University, School of Medicine 1402 South Grand Blvd. Saint Louis, MO 63104 Phone 314-577-8561 FAX 314-268-5180 Saint Louis University is an equal opportunity, affirmative action employer. Women and minorities are encouraged to apply.

TCM Vol. 13, No. 8, 2003