Development of Vessels, Airways and Cartilage Rings: the Role of T-Box Genes

Development of Vessels, Airways and Cartilage Rings: The role of T-box genes

Ripla Arora

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY 2012

ABSTRACT

Development of Vessels, Airways and Cartilage Rings: The role of T-box genes

Ripla Arora

Tbx4 and Tbx5 are two closely related genes that belong to the T-box family of transcription factor genes. Loss of Tbx4 results in absence of chorio-allantoic fusion and a failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5.

Using a candidate gene approach we identified a number of genes downstream of Tbx4 in the allantois including, extracellular matrix molecules Vcan, Has2, Itgα5; transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2. In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and an inhibitor of the canonical Wnt signaling pathway phenocopies the

Tbx4 mutant phenotype in wildtype allantoises. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical

Wnt signaling pathway in the process of allantois-derived blood vessel formation.

Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing respiratory system. Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Although many genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. We investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4

and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes in the epithelium, Bmp4 and Spry2, is downregulated. Lung branching undergoes arrest ex vivo when

Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous;Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal-bronchial cartilage rings. Sox9, a master regulator of cartilage formation, is expressed in the trachea but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction.

Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal-bronchial cartilage development.

Table of contents

Chapter 1. Introduction to T-box genes, the murine allantois and the respiratory system 1

1.1 T-Box Genes 1 History 1 Defining features of the family 1 Phylogenetic analysis 2 Role in development 3 Tbx4 and Tbx5: expression and function 4

1.2 The murine allantois: a model system for the study of blood vessel formation 6 Developmental origin and vascular development of the allantois 6 Yolk sac vasculogenesis and dorsal aorta formation 8 Building the allantois vascular network: cellular and extracellular components 8 Building the allantois vascular network: signaling pathways and transcription factors 11 Methods for allantois culture ex vivo 17 Use of adherent allantois cultures to study gene function in blood vessel formation 20 Applications in disease and therapeutics 23 Conclusion 24 Known role of T-box genes in vascular development 24

1.3 Development of the respiratory system 25 Specification of the respiratory system 26 Lung branching and morphogenesis 27 Vascular development in the branching lung 28 Innervation of the branching lung 29 Signaling pathways implicated in lung branching 30 Extracellular matrix in lung branching 33 Development of the trachea 33 Known role of T-box genes in development of the lung and trachea 35

Chapter 2. Canonical Wnt signaling acts downstream of Tbx4 in development of the allantois vasculature and the chorio-allantoic placenta 48

Introduction 48

Materials and methods 48 Mice 48 Histology and vessel counts in the chorio-allantoic plate 49 In situ hybridization and immunohistochemistry 49 Ex vivo allantois culture 49

Results 50 Tbx4 mutant allantoises fail to form a vascular plexus ex vivo 50 Tbx4 is upstream of ECM molecules, signaling molecules and transcription factors 51 Agonists of the canonical Wnt signaling pathway rescue and an antagonist phenocopies the Tbx4 mutant phenotype ex vivo 53 Tbx4 and Wnt2 genetically interact during the formation of the chorio-allantoic placenta 54

Discussion 55 Chapter 3. Multiple Roles and interactions of Tbx4 and Tbx5 in development of the respiratory system 64

Introduction 64 Materials and methods 65 Mouse strains, crosses and embryo collection 65 Tamoxifen injections 65 In situ hybridization, immunohistochemistry and histology 65 Alcian blue staining 66 Foregut and lung bud culture 66 Lung branching analysis 67

Results 67 Expression of Tbx4 and Tbx5 in the developing lung and trachea 67 Efficiency of recombination of conditional alleles 68 Early loss of Tbx5, but not Tbx4, leads to a unilateral loss of lung bud specification and absence of tracheal specification 69 Lung branching is severely affected by the loss of Tbx4 and Tbx5 70 Loss of Tbx4 and Tbx5 affects the Fgf10 signaling pathway and Wnt2 expression 72 Tbx4 and Tbx5 interact with Fgf10 during lung development 73 Loss of Tbx4 and Tbx5 causes tracheal-bronchial ring defects 74 Tbx4 and Tbx5 do not interact with Fgf10 during trachea development 75

Discussion 75 Loss of Tbx5 affects specification of the lung buds and trachea 75 Tbx4 and Tbx5 interactions in lung development 76 Tbx4 and Tbx5 interactions in tracheal-bronchial cartilage development 78

Chapter 4. T-box gene function in organogenesis 96

T-box genes lie upstream of Fgf signaling 96 T-box genes interact with canonical Wnt signaling 97 T-box genes regulate expression of major components of extracellular matrix 98 Future perspectives 99 References 102

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List of tables and figures

Figure 1.1 Phylogenetic tree of T-box genes of zebrafish, Xenopus, chicken, mouse and human 37 Figure 1.2 Diagrammatic representation of the developing mouse embryo from the bud stage to the 22-26 somite stage highlighting the development of the allantois and its vasculature 39

Figure 1.3 Diagrammatic representation of vasculogenesis 41 Figure 1.4 Three methods for allantois culture 42 Figure 1.5 Signaling pathways involved in primary lung budding 43 Table 1.1 Human syndromes caused by mutations in specific T-box genes and their respective mouse mutant phenotypes 44 Table 1.2 Gene expression related to blood vessel formation and corresponding mutant phenotype in the allantois or placenta 45 Table 1.3 Gene function identified using allantois culture as a model system 47 Figure 2.1 Tbx4 mutant allantoises fail to form a vascular plexus ex vivo 59 Figure 2.2 Identification of genes downstream of Tbx4 in the allantois using a candidate gene approach and ISH or IHC 60 Figure 2.3 Wnt signaling is essential for vessel formation in allantoises ex vivo 61 Figure 2.4 Genetic interactions between Tbx4 and Wnt2 in formation of the chorio-allantoic placenta 62 Figure 3.1 Expression of Tbx4 and Tbx5 in the developing lung and trachea 81 Figure 3.2 Analysis of excision efficiency of Tbx4fl and Tbx5fl 83 Figure 3.3 Early loss of Tbx5 leads to aberrant lung bud and trachea specification 84 Figure 3.4 Loss of Tbx4 and Tbx5 causes reduced lung branching and lethality at birth 85 Figure 3.5 Loss of Tbx4 and Tbx5 has multiple affects on branching morphogenesis 87 Figure 3.6 Loss of Tbx4 and Tbx5 leads to branching arrest ex vivo 88 Figure 3.7 Loss of Tbx4 and Tbx5 affects the Fgf10 signaling pathway and Wnt2 expression 89 Figure 3.8 Tbx4 and Tbx5 interact with Fgf10 but FGF10 fails to rescue Tbx4 and Tbx5-deficient lungs 90

Figure 3.9 Loss of Tbx4 and Tbx5 disrupts tracheal-bronchial cartilage and smooth muscle formation 91 Figure 3.10 Lack of genetic interactions between Tbx4, Tbx5 and Fgf10 in trachea formation 93 Figure 3.11 Model for the role of Tbx4 and Tbx5 in lung and trachea development 94 Table 3.1 Different allelic combinations and the descriptive nomenclature 95

Acknowledgements

I owe my deepest gratitude to Ginny for being such an amazing mentor. She has always been very encouraging, patient, kind and understanding. She let me work independently and guided me whenever I found myself lost. She taught me a lot about scientific analysis, techniques, lab management and scientific writing. She has always been available, whether to show her embryology magic or just to lend a listening ear to my crazy ideas. Thank you so much for everything.

It has been a pleasure working in such an intellectual scientific environment with wonderful lab mates. I would like to thank Naiche for teaching me a lot of what I know today, especially tedious allantois dissections. I have enjoyed our long scientific discussions and learnt a lot from them. Thank you Elana for being my dancing and cooking partner. The first few years of being in a new place would not have been fun if it wasn’t for you. The lab would have been a dull place without my dear friend Danny. I have enjoyed our scientific arguments, volleyball and your awesome mimicry skills. Andy, thank you for being very helpful especially with reading and critiquing my write ups. You always make sure to give credit, whether it is a small contribution or a good idea. Nataki, thank you for being my colleague, my friend and my counselor. Thank you for understanding who I am and always being by my side. Thank you

Salma for being so caring and for the yummy delicious food. Thank you Lana for teaching me

EndNote. Chelsea, thank you for being an amazing student and a good friend. Working on the placentas was a lot more fun because of you. Thank you Kathy, Jessica and Fariha for making the lab environment so cheerful and fun.

I would like to thank Gerard and Frank for critiquing and providing helpful suggestions throughout the course of this work.

I would like to thank members of the Bestor lab and Costantini lab for scientific and non- scientific discussions in joint lab meetings and during lunch hours.

I would like to thank all the members of the Genetics Department for providing an inspiring and intellectual environment. I would like to thank everyone in the Genetics Office for being very helpful through the years.

I would like to thank all my friends especially Parag, Sudha, Paromita, Shahrnaz and

Milo for being my home away from home. I would also like to thank my parents, my brother and my sister for their constant support and encouragement.

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Preface

T-box genes are a family of transcription factor genes, important for normal embryonic development in different organisms. Tbx4 and Tbx5, the most closely related pair of T-box genes, show distinct and overlapping areas of expression during embryogenesis. Here we explore the function of these genes in areas of unique expression, Tbx4 in the developing murine allantois, Tbx5 in the early foregut; and in areas of common expression, Tbx4 and Tbx5 in the developing lung and trachea. Chapter 1 outlines the discovery of T-box genes, highlights the salient features of this family of genes as transcription factors, and their general role in development, in particular Tbx4 and Tbx5. Upon targeted mutagenesis, loss of Tbx4 produces an allantois and hindlimb phenotype but the mechanisms by which Tbx4 acts in the allantois is not clear. The second part of Chapter 1 introduces the murine allantois and provides an extensive review of literature described under the subheading “The murine allantois: a model system for the study of blood vessel formation”. This section has been submitted as an invited review to the journal Blood. The final section in Chapter 1 introduces the respiratory system, an organ system that expresses both Tbx4 and Tbx5. In Chapter 2, a battery of genes affected by loss of Tbx4 expression in the allantois were discovered using a candidate gene approach. Further, the role of canonical Wnt signaling was characterized downstream of Tbx4 in the development of the allantois and placental vasculature. Although Tbx5 function has been extensively studied in the heart, neither Tbx4 nor Tbx5 function has been examined in the developing murine respiratory system. Chapter 3 characterizes the expression pattern of Tbx4 and Tbx5 in the primordia of the respiratory system, the developing lung and developing trachea. Subsequently, the function of both these genes and their interactions were analyzed in the specification of the lung/trachea primordia, lung branching morphogenesis and tracheal/bronchial cartilage formation. Chapter 2

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and Chapter 3 have been submitted in manuscript format to the journals PLoS ONE and PLoS

Genetics respectively.1

1 All experiments in these manuscripts were planned and carried out by Ripla Arora

Chapter 1. Introduction to T-box genes, the murine allantois and the respiratory system

1.1 T-Box Genes

History

A mutation in the Brachyury or T locus, first described in 1927, was one of the earliest recognized developmental genes in mice. Embryological defects caused by mutations at this locus cause embryonic death in homozygotes and defects in tail development in heterozygotes

(hence the locus name Brachyury which means short tail, symbolized as T for tail). In 1990, the gene itself was cloned and found to be a novel transcription factor, and soon the existence of a family of T-related genes was demonstrated by the discovery of genes in both Drosophila and mice, with sequence homology to T. The conserved DNA binding domain that defined the family was named the T-box, giving the family its name. T-box genes have now been found in virtually all metazoan species as divergent as the roundworm, Caenorhabditis elegans, and Homo sapiens

(Papaioannou, 2001).

Defining features of the family

The T-box family of genes encodes putative transcription factors. The defining feature of the family is a 180-190 amino acid long T-box domain, which has been shown to have DNA binding activity. This domain is conserved amongst all the T-box family members. DNA binding activity, along with the nuclear localization of the gene products, suggests that these proteins act as transcriptional regulators of other genes (Papaioannou, 2001; Naiche et al., 2005). T-box proteins have, in fact, been shown to act as both repressors and activators of transcription. For example, Tbx6 is known to activate the notch ligand Dll1 in the paraxial mesoderm (White and

Chapman, 2005); on the other hand Tbx2 is known to repress heart chamber differentiation- specific genes including Nppa, Cx40 and Cx43 (Christoffels et al., 2004; Harrelson and

Papaioannou, 2006).

T-box proteins can act as homodimers or heterodimers with different members of the family and can also interact with transcription factors of other families. The polypeptide encoded outside of the T-box region varies between different T-box genes and hence imparts specificity of function, for example, by promoting interactions with a different set of proteins or transcription factors. Tbx20 interacts with GATA5 but not the related transcription factor

GATA4 (Stennard et al., 2003). LMP4 binds both of the most closely related vertebrate T-box proteins Tbx4 and Tbx5, but interacts with each via a different LIM domain repeat (Krause et al.,

2004).

The DNA sequence which the T-box binds, the T-box binding element (TBE) or T-site, was first defined for Brachyury as the sequence with the highest affinity (Kispert and Herrmann,

1993). Brachyury binds this palindromic sequence as a dimer, with each monomer of Brachyury binding half of the sequence of the T-site (5’-AGGTGTGAAATT-3’). All the T-box proteins tested are capable of binding the T-half site as monomers. In addition to the T-half site, specific binding elements for other T-box genes have also been identified, which are similar but not identical to the T-half site. The binding specificity of the TBE provides another example of how the T-box genes are similar yet distinct in function (Naiche et al., 2005).

Phylogenetic analysis

Phylogenetic analysis of the T-box gene family reveals that this is an ancient gene family. Its initial expansion from a single progenitor sequence appears to have occurred at the outset of metazoan evolution and further expansions have occurred by gene duplication along individual

3 evolutionary lineages. Together with gene expression studies, phylogenetic comparisons also provide evidence for the existence of T-box gene subfamilies whose more recently duplicated members retain similar or overlapping patterns of gene expression, possibly correlated with conserved function. For example, the Tbx2 and Tbx3 genes are members of the ancient vertebrate

Tbx2 subfamily that expanded prior to the divergence of bony fish and tetrapods. These two genes have expression patterns that are broadly similar, both within and between species, although temporal and spatial differences in expression may reflect divergence of function that could have occurred since the separation of the genes (Epstein et al., 2008).

Phylogenetics also allows the identification of what are likely to be orthologs of the same gene in different species. Orthologs are defined as direct descendant from a single ancestral gene that was present in the genome of the common ancestor of the species under analysis, for example the Brachyury orthologs found in many species (Figure 1.1, T subfamily). Analysis of the T-box family tree can direct the search for new T-box genes by predicting the existence of orthologs in species where they have yet to be discovered, thus hastening gene discovery

(Epstein et al., 2008).

Role in development

Although several T-box genes are known to be expressed in adult tissues, their widespread expression in embryonic tissues, particularly in areas of inductive tissue interactions emphasizes a major role for these genes in development. The best way to study T-box gene function is by finding or creating loss of function mutations in a gene in order to study the effects of its disruption. In addition to the well studied Brachyury mutations in the mouse which affect development of posterior structures including the tail (Yanagisawa et al., 1981), mutant alleles of the Drosophila ortholog, Trg (Singer et al., 1996), and mutant alleles of the zebrafish Brachyury

4 ortholog, no tail, also show effects on the development of posterior structures (Schulte-Merker et al., 1994), illustrating comparable functions of orthologs in widely divergent species.

Tbx4 and Tbx5: expression and function

Tbx4 and Tbx5 belong to the Tbx2 subfamily and are the most closely related T-box genes with

94% homology in their T-box amino acid sequence (Epstein et al., 2008). In the developing chick embryo Tbx4 expression has been detected in the hind limb, lung bud/trachea, genital papilla and the notochord (Gibson-Brown et al., 1998). In the developing mouse embryo Tbx4 is expressed in the allantois, hindlimb, genital papilla, lung mesenchyme, trachea mesenchyme, mandible mesenchyme (Chapman et al., 1996), a small domain in the proximal core of the forelimb (Naiche et al., 2011) and both male and female germ cells (Douglas et al., 2011). In zebrafish Tbx4 expression has been reported in the developing and adult pelvic fin (Tamura et al., 1999) which is the homolog for the hindlimb in the chick and the mouse. Tbx5 expression in the chick has been identified in the heart atrium, ventricle, genital papilla, forelimb, lung bud/trachea, notochord and optic vesicle (Gibson-Brown et al., 1998). In the developing mouse embryo Tbx5 expression has been detected in the forelimb, heart atrium and ventricle, genital papilla, lung mesenchyme, trachea mesenchyme, mandible mesenchyme and neural retina of the optic vesicle (Chapman et al., 1996). In the zebrafish, Tbx5 expression has been detected in the retina, the pectoral fin buds, which are the homologues for forelimbs (Tamura et al., 1999) and the developing heart atrium and sinus venosus (Begemann and Ingham, 2000).

In the chick Tbx4 function has been studied in the developing lung and trachea. Ectopic expression of Tbx4 on the border of trachea and esophagus causes tracheoesophageal fistula.

Furthermore, ectopic Tbx4 expression induces Nkx2.1 expression, Fgf10 expression and lung bud formation in the esophagus suggesting that Tbx4 has the ability to induce formation of the

5 respiratory primordia on its own (Sakiyama et al., 2003). Homozygous loss of Tbx4 in mice is embryonic lethal due to lack of chorio-allantoic fusion and absence of placenta formation.

Additionally, the Tbx4 null mutant allantois shows apoptosis and a failure of differentiated endothelial cells (ECs) to form umbilical vessels. The hind limb buds in Tbx4 null mutant embryos appear but then fail to outgrow and lose Fgf10 expression (Naiche and Papaioannou,

2003). Later in development, once hindlimb outgrowth has successfully occurred, Tbx4 is important for formation of core tissue (Naiche and Papaioannou, 2007b), limb muscle and tendon formation (Hasson et al., 2010) but not for hindlimb identity or outgrowth. In humans heterozygous loss of Tbx4 causes autosomal dominant Small Patella Syndrome

(OMIM#147891), characterized by patellar aplasia or hypoplasia and anomalies of the pelvis and feet (Bongers et al., 2004). In adult mice Tbx5 heterozygous mutation produces enlarged and dilated atria and ventricles in the heart and subtle forelimb skeletal defects (Bruneau et al., 2001).

Tbx5 homozygous null mutants die during mid-gestation due to impaired cardiac differentiation once the linear heart tube is formed (Bruneau et al., 2001). Additionally, these mutant embryos fail to activate Fgf10 expression resulting in failure of formation of forelimb buds (Agarwal et al., 2003). Tissue-specific ablation of Tbx5 using Prx1-Cre leads to a complete absence of forelimb formation (Rallis et al., 2003). In humans heterozygous loss of Tbx5 results in the Holt

Oram Syndrome (OMIM #142900). Individuals suffering from this syndrome show upper limb malformations and congenital heart defects. The Tbx5 heterozygous mutant mice are a good model for the Holt Oram Syndrome as they have many phenotypic manifestations similar to the humans suffering from this syndrome.

These mutant phenotypes, as well as the phenotypes of all other known mutations in T- box family genes, indicate critical roles in the specification and differentiation of tissues and

6 structures during embryonic development. A list of T-box genes, loss of which results in a human phenotype, along with phenotypes of the corresponding mouse orthologs is presented in

Table 1.1. However, most of the T-box gene mutations in mouse are embryonic lethal when homozygous and mutant embryos die during mid-gestation, hence making it difficult to study the role of these genes in organ development beyond mid-gestation. Efforts to make conditional mutations and reporter genes are currently underway, which will enhance our understanding of the role of these genes in later stages of development and in the adult.

1.2 The murine allantois: a model system for the study of blood vessel formation

The allantois is the embryonic precursor of the umbilical cord in mammals and is one of several embryonic regions, including the yolk sac and dorsal aorta, that undergoes vasculogenesis, the de novo formation of blood vessels. Despite its importance in establishing the chorioallantoic placenta and umbilical circulation, the allantois is frequently, and somewhat inexplicably, overlooked in embryological studies. Nonetheless, the allantois is a highly vascularized tissue and recent studies demonstrate the importance to allantois function of vasculogenesis, remodeling of the vasculature, and angiogenesis as the allantois invades the chorion. Thus, the allantois has the potential to be a good system to understand the physiological process of blood vessel formation.

Developmental origin and vascular development of the allantois

The allantois of the mouse emerges from the posterior end of the embryo at the neural plate stage of development as a bud of mesoderm derived from the primitive streak (Inman and Downs,

2006a; Inman and Downs, 2007). As development proceeds, the allantois grows towards the chorion through the exocoelomic cavity. Its distal end undergoes a process of cavitation

7 whereby the extracellular spaces increase in volume and accumulate hyaluronic acid (HA)

(Brown and Papaioannou, 1993). Contact of the allantois with the chorion occurs at the 4-6 somite pair (s) stage, followed by chorio-allantoic fusion and spreading of the allantois on the surface of the chorion to form the chorio-allantoic plate. The chorion forms the placental villi and the vasculature of the allantois grows into these villi to form the labyrinthine layer of the placenta (Watson and Cross, 2005) (Figure 1.2).

The growing allantois, which consists of an outer layer of mesothelium enveloping a mesenchymal core, increases in size both by cell proliferation and by addition of cells from the primitive streak (Inman and Downs, 2007). De novo endothelial cell (EC) differentiation starts in the distal allantois as early as the headfold (HF) stage, and is characterized by expression of the molecular EC markers Flk1, Pecam and VE-Cadherin (VE-cad) (Drake and Fleming, 2000).

Starting at the 1s stage, the ECs coalesce in a distal to proximal direction to form vessels in a process termed vasculogenesis (Figure 1.3). These vessels form the primary vascular plexus, evident at the 4s stage, which unites with the embryonic and yolk sac circulation at the base of the allantois. The primary vascular plexus remodels to form the umbilical artery and vein

(Inman and Downs, 2007) which then invade the chorion by sprouting angiogenesis (Rossant and Howard, 2002). It is still an open question whether or not the allantois contributes to the definitive hematopoietic cell lineage. Cells of the allantois do express markers of hematopoietic cell progenitors and can differentiate to form cells of the definitive erythroid and myeloid lineages in vitro (Zeigler et al., 2006; Corbel et al., 2007), but whether this occurs in vivo is unknown.

Yolk sac vasculogenesis and dorsal aorta formation

The visceral yolk sac (VYS), consisting of a mesodermal and an endodermal layer, is another site of vasculogenesis and a major site of fetal hematopoiesis. Clusters of cells within the mesoderm form the yolk sac blood islands which contain hemangioblasts, common precursors of

ECs and hematopoietic cells. By E8.5, the blood islands contain a single layer of ECs that surround the hematopoietic cells. The ECs then connect to form a continuous primary vascular plexus which is remodeled into large and small vitelline vessels (Palis et al., 1995). Unlike the allantois, where ECs migrate through the extracellular matrix of a mesenchymal structure to form the primary vascular plexus, the yolk sac vascular plexus forms within a bilaminar epithelial structure, between the endoderm and mesothelium.

Within the body of the embryo, angioblasts, the precursors of ECs, appear in the lateral plate mesoderm ventral to the somites and migrate medially to fuse together forming the paired dorsal aortae. The dorsal aortae connect anteriorly with the aortic arteries exiting the primitive heart tube and fuse posteriorly to form a single dorsal aorta in the midline of the embryo at the level of the forelimbs (Lanzer and Topol, 2002). The lateral plate mesoderm, similar to the VYS but unlike the allantois, is dependent on endodermal signaling for formation of blood vessels.

Hematopoiesis takes place in the dorsal aorta from hemogenic endothelium lining the blood vessels (Dieterlen-Lievre and Jaffredo, 2009).

Building the allantois vascular network: cellular and extracellular components

Although the genetic control of vasculogenesis and angiogenesis has been well reviewed in the embryo and yolk sac (Patan, 2000; Rossant and Howard, 2002; Patan, 2004; Kowanetz and

Ferrara, 2006; Ribatti et al., 2009; De Val, 2011; Patel-Hett and D'Amore, 2011), similar attention has not been given to the allantois. Here we compile information for the allantois about

9 genes known to be important for differentiation of vascular components and for vascular network formation, and examine what is known from mutant analysis about their function in allantois vessel formation (Table 1.2).

Vegf signaling and specification of ECs

The vascular endothelial growth factor (Vegf) signaling pathway lays the foundation for formation of ECs from mesoderm and also plays a major role in angiogenesis. Vegf and its tyrosine kinase receptors, Flk1 (also known as Kdr and Vegfr2) and Flt1 (also known as Vegfr1), are critical for development of the vascular system. Flk1 is the earliest marker of EC development (Rossant and Howard, 2002). In the early yolk sac, Flk1 marks the hemangioblasts but later in development its expression is restricted to the EC lineage (Yamashita et al., 2000). In the allantois, Flk1 is expressed at the HF stages in cells that will give rise to the vascular network

(Drake and Fleming, 2000). Both Flk1 and Flt1 are expressed in the allantoises of 9-16s stage embryos in putative angioblasts and nascent blood vessels (Downs et al., 2001). Vegf mRNA expression has been shown to be confined to the mesothelium of the allantois (Dumont et al.,

1995) whereas protein expression is localized to both mesothelial and core cells (Downs et al.,

2001). Thus, similar to other sites in the embryonic vasculature, Vegf expression in the allantois is associated with active Flk1 expression and is suggestive of Vegf’s role in the induction and maintenance of EC fate. Later in development both Flk1 and Flt1 are expressed in the labyrinthine vasculature of the placenta and associated labyrinthine trophoblasts, whereas the labyrinthine stromal cells express Vegf (Dumont et al., 1995).

Loss of one allele of Vegf by targeted mutagenesis results in lethality and affects vasculogenesis and angiogenesis in both embryo and yolk sac (Carmeliet et al., 1996; Ferrara et al., 1996). Although the development of the allantois vasculature was not studied in these

10 mutants, it was suggested that the endothelium lining the fetal placental vasculature degenerated

(Ferrara et al., 1996). Targeted mutation of the Flk1 gene resulting in a null allele causes embryonic lethality at E9 with both embryo and allantois showing a lack of mature ECs (Shalaby et al., 1995). On the other hand, Flt1 null mutants show a disorganized embryonic and yolk sac vasculature and possibly an overgrowth of ECs, suggesting an inhibitory role for this receptor in vascular development (Fong et al., 1999). However, the allantois endothelium was not examined in Flt1 mutants. In addition, Vegf coreceptors, neuropilin 1 (Np1) and neuropilin 2 (Np2) are important for vascular development. Embryos double mutant for Np1 and Np2 die around E 8.5 and show an absence of vasculature in the yolk sac and embryo. Here again, the allantois was not examined in the double mutants and the expression of Np1 and Np2 in the allantois has not been determined (Takashima et al., 2002).

The supporting mural cells of the allantois

Mural cells, which include pericytes and smooth muscle cells (SMC), support the developing vasculature (Figure 1.3) and are characterized by the expression of platelet derived growth factor receptor β (PDGFRβ). The ligand for this receptor, PDGFB, is secreted by immature ECs and promotes the differentiation of the surrounding mesenchyme and the recruitment of mural cells

(Betsholtz et al., 2001). Both PDGFRβ and PDGFB null mutants show defective smooth muscle recruitment in the embryo and the placenta, with disorganized and dilated vasculature (Ohlsson et al., 1999).

Extracellular matrix in the allantois

Extracellular matrix (ECM) is essential during vessel formation for the adhesion, migration, proliferation and vascular organization of ECs (Figure 1.3) (Hynes et al., 2002; Stupack and

Cheresh, 2002; Stupack and Cheresh, 2004; Hynes, 2007; Somanath et al., 2009). A number of

ECM components are known to be important for development of the vasculature but only a handful have been implicated in allantois vasculogenesis. Fibronectin (Fn) and its binding partner α5 integrin (Itgα5) are both necessary for EC tube formation. Fn and Itgα5 are both expressed in the allantois and mutations in either affects chorio-allantoic fusion (George et al.,

1993; Francis et al., 2002) although the vasculature of mutant allantoises has not been analyzed.

Embryonic stem cell-derived embryoid bodies (EBs) have been used as a model to study blood vessel formation. EBs lacking Fn or Itgα5 differentiate to form ECs but these fail to form tubular structures (Francis et al., 2002).

In addition to matrix proteins, the proteoglycan HA is important for angiogenesis and, depending on the context, can act as a pro- or anti-angiogenic agent (West et al., 1985; Tempel et al., 2000). HA is present in the allantois (Fenderson et al., 1993). It is synthesized by hyaluronic acid synthase 2 (Has2), which is expressed in the allantois (Tien and Spicer, 2005).

Mutation in Has2 produces defects in endocardial cushion formation in the heart, but the allantois vasculature has not been analyzed (Yamamura et al., 1997; Camenisch et al., 2000).

Another category of ECM molecules important for vascular morphogenesis is the metalloproteases, which includes MMPs, TIMPs, ADAMs and the ADAMTS family of proteins.

In this category, only ADAMTS9 is known to be expressed in the allantois (Jungers et al., 2005).

ADAMTS9 is an EC-autonomous inhibitor of angiogenesis and also a tumor suppressor.

ADAMTS9 null mutants die prior to gastrulation hence its role in vasculogenesis has not been analyzed (Koo et al., 2010).

Building the allantois vascular network: signaling pathways and transcription factors

Signaling pathways involved in vascular remodeling

The angiopoietin-Tie signaling pathway has been implicated in branching, remodeling and stabilization of the vascular plexus. The ligands angiopoietin1 (Ang1) and angiopoietin 2 (Ang2) and the endothelial-specific receptor tyrosine kinases, Tie1 and Tie2 (also known as Tek) interact to remodel the primary vascular plexus. Both Tie1 and Tie2 are expressed in allantoises at the

9-16s stage (Downs et al., 2001). Ang2 expression has been reported in the allantois at approximately the 30s stage (Yuan et al., 2000) but has not been investigated earlier; expression of Ang1 has not been investigated in the allantois. Both Tie2 (Sato et al., 1995) and Ang1 (Suri et al., 1996) homozygous null mutants show dilation of blood vessels, defects in vascular remodeling, vessel sprouting and branching, suggesting a role for these genes in EC interactions with the surrounding mesenchyme and ECM. Tie1 null mutants die at birth due to hemorrhage and lack of vascular integrity (Sato et al., 1995). Ang2 acts as a negative regulator of Ang1-Tie2 interactions. Ectopic expression of Ang2 under the control of the Tie2 promoter results in an angiogenic remodeling phenotype similar to the lack of Tie2 or Ang1 (Maisonpierre et al., 1997).

The allantois vasculature has not been analyzed in any of these mutants although the umbilical vessels in the Ang1 nulls reportedly show a lack of complexity (Suri et al., 1996).

The Tgfβ signaling pathway has also been implicated in vascular remodeling. Tgfβ and its downstream effectors Activin receptor like kinase 1 (Alk1) and Alk5 (also known as TβR1) are all involved in angiogenesis (Rossant and Howard, 2002). Tgfβ1 is expressed in the allantois

(Akhurst et al., 1990) and null mutants for Tgfβ show defects in vascular network formation in the yolk sac and allantois. Some of the mutants also show an absence of chorio-allantoic fusion

(Dickson et al., 1995). ECs derived from Alk5 null mutant embryos show defective migration in in vitro assays. Additionally, Alk5 mutants show defects in infiltration and sprouting of the allantois vasculature into the labyrinthine layer of the placenta (Larsson et al., 2001).

Signaling pathways involved in differentiation between arteries and veins

Differentiation of blood vessels into arteries or veins was once assumed to be the result of physical differences such as oxygenation, blood pressure and shear forces but over the last decade it has been recognized that artery and vein distinction is inherently programmed. Two types of molecules, the ephrins and Notch family molecules, have been identified as molecular markers for this differentiation process (Rossant and Howard, 2002). EphrinB2 an Eph family ligand is expressed in the arterial system and conversely the receptor Eph-B4 is expressed in the venous system (Wang et al., 1998). Deficiency of either of these molecules produces a similar phenotype of loss of angiogenesis in both arteries and veins, presumably due to a lack of interaction and hence lack of differentiation of the capillary beds (Wang et al., 1998; Gerety et al., 1999). Similar to the embryonic and yolk sac vasculature, the ligand ephrinB2 is expressed in the umbilical artery and the receptor Eph-B4 is expressed in the umbilical vein (Wang et al.,

1998).

In addition, it has been shown that Notch family members Notch1, Notch3, Notch4,

Delta-like 4 (Dll4), Jagged1 (Jag1) and Jagged2 are all expressed in arteries (Villa et al., 2001).

Notch signaling pathway lies upstream of ephrin signaling and has been implicated to drive arterial differentiation (Lin et al., 2007). Notch2 is expressed in the allantois; the death of

Notch2 null mutants at E10.5 has been ascribed to apoptosis in the embryo, although the allantois and placental vasculature were not analyzed (Hamada et al., 1999) and could play a contributing role. Loss of one allele of Dll4 by targeted mutagenesis results in lethal haploinsufficiency.

Heterozygous mutants display degenerating vasculature in the placental labyrinth (Gale et al.,

2004). Mutation in Jag1 leads to yolk sac and embryonic vasculature defects, but the allantois and placenta have not been analyzed in these mutants (Xue et al., 1999). The basic helix loop

14 helix transcription factors Hey1 and Hey2 (also called Hes) are activated downstream of Notch signaling and act as transcriptional repressors. Both Hey1 and Hey2 are expressed in the allantois from E8.5 (Leimeister et al., 1999). Loss of both Hey1 and Hey2 leads to a vascular phenotype similar to the ephrinB2 ligand or Eph-B4 receptor null mutants where there is a defect in the remodeling of the vasculature. Indeed, Hey1 and Hey2 double mutants show a loss of ephrinB expression in vascular tissues. The allantois of double mutants attaches to the chorion and although the chorionic vessels form, they fail to invade the placental labyrinth (Fischer et al., 2004).

Dependence of vascular network formation on hedgehog signaling

Flk1-positive angioblasts of the VYS organize into vascular channels in vitro only in the presence of a visceral endodermal layer, suggesting that signals from the endoderm are essential for vascular network formation. On the other hand, the yolk sac mesodermal layer differentiates into Flk1-positive angioblasts upon culture in the presence or absence of the adjacent endodermal layer (Palis et al., 1995). In the absence of endoderm, Ihh, one of the ligands for the hedgehog (Hh) signaling pathway, can induce a vasculogenic program in anterior epiblast cells in vitro (Dyer et al., 2001). Furthermore, EBs lacking Ihh or smoothened (Smo), the receptor for

Hh ligands, form Pecam-positive ECs that fail to form endothelial tubes (Byrd et al., 2002).

Additionally, Hh signaling from the endoderm is essential to promote vascular network formation in the embryo (Byrd and Grabel, 2004) highlighting the role of Hh signaling in blood vessel formation in different vasculogenic systems. A Smo null mutation, which results in abrogation of all Hh signaling, provides evidence that Hh signaling has different functions in development of the dorsal aorta, yolk sac and allantoic vasculature: In the anterior region of mutant embryos, Pecam-positive ECs fail to connect and cavitate to form the dorsal aorta (Vokes

15 et al., 2004); in the yolk sac, Pecam-positive ECs connect to form a primary vascular plexus but fail to remodel into a mature vascular network (Astorga and Carlsson, 2007); in the allantois, there is no apparent effect on vessel formation. Thus, the loss of Hh signaling affects vasculogenesis in the dorsal aorta, angiogenesis in the yolk sac, but neither of these processes in the allantois.

Hh signaling regulates vessel formation by regulation of the transcription factor Foxf1 and its downstream effector, Bmp4, in the yolk sac and developing embryo. However, Foxf1 expression is independent of Hh signaling in the posterior end of the primitive streak and the allantois, perhaps explaining why loss of Hh signaling does not affect allantois vasculogenesis.

Allantois vascular development is nonetheless dependent on Foxf1 as mutations in Foxf1 produce vascular phenotypes in dorsal aorta, yolk sac and allantois (Astorga and Carlsson, 2007).

Wnt and Bmp signaling in blood vessel formation

The canonical Wnt family member, Wnt2 plays important roles in EC proliferation and vascular network formation in two in vitro systems, hepatic sinusoidal ECs (Klein et al., 2008) and EBs

(Wang et al., 2007). Wnt2 is expressed in the allantois throughout its growth and vascularization and Wnt2 null mutants show placental defects during late gestation (Monkley et al., 1996).

Endothelial-specific loss of β-catenin, the downstream effector of canonical Wnt signaling, affects vessel diameter in the yolk sac, umbilical vessels and the cephalic plexus. Additionally, the placentas from these mutants show a reduced number of fetal blood vessels in the labyrinthine layer (Cattelino et al., 2003). Rspo3, a secreted protein that can activate the canonical Wnt signaling pathway, is important for activation of Vegf (Kazanskaya et al., 2008).

Rspo3 is expressed in the allantois and its mutation leads to failure of the allantoic vessels to

16 invade the placenta and to vascular defects in the yolk sac (Aoki et al., 2007; Kazanskaya et al.,

2008).

Non-canonical Wnt5a and Wnt11 affect EC proliferation, migration and network formation in vitro (Masckauchan et al., 2006; Cheng et al., 2008; Stefater et al., 2011). Both

Wnt5a and Wnt11 expression have been observed in the allantois (Kispert et al., 1996;

Yamaguchi et al., 1999; Majumdar et al., 2003). Mutation of Wnt5a leads to a shortening of the

A-P axis of the embryo, although it does not affect development of the allantois (Yamaguchi et al., 1999). Most Wnt11 null mutants are embryonic lethal before midgestation, but the cause of death has not been determined (Majumdar et al., 2003) and, thus, Wnt11 might affect the development of allantois or placental vasculature.

Bmp4 is implicated in induction of mesoderm and differentiation of mesoderm into ECs

(Moser and Patterson, 2005). Loss of Bmp4 leads to a deficiency in the amount of mesoderm resulting in aberrant yolk sac blood island formation and a small or absent allantois (Winnier et al., 1995). Mutation of Smad1, a downstream effector of BMP signaling, results in defective chorio-allantoic fusion, but the vasculature of the allantois has not been analyzed (Tremblay et al., 2001).

Transcription factors important for blood vessel formation

A number of transcription factors are involved in EC and blood vessel formation in the embryo and yolk sac. A few have been investigated in the allantois including Etv2, Snai1 and dHand.

Etv2 (also known as ER71 or Etsrp71) is expressed in the allantois at E7.5 and E8.25. Etv2 is important for EC identity; Etv2 homozygous null mutants are devoid of ECs in the embryo, yolk sac, amnion and the allantois (Lee et al., 2008). Additionally, in these mutants the placental labyrinthine layer is reduced in thickness and devoid of any vasculature. The zinc finger

17 transcriptional repressor, Snai1, is important for mesoderm formation and its epiblast-specific deletion results in vascular defects throughout the embryo. ECs differentiate but fail to coalesce into a primary vascular plexus in the embryo or the allantois (Lomeli et al., 2009). dHAND, a basic helix loop helix transcription factor, is expressed in the developing vascular SMCs and their precursors. Lack of dHAND results in disorganized vasculature in the yolk sac and embryo, possibly due to regulation of Np1 by dHAND (Yamagishi et al., 2000). dHAND is expressed in the allantois although the allantoises and the placentas in dHAND mutants have not been analyzed (Charite et al., 2000).

T and Tbx4, two members of the T-box family of transcription factor genes have been implicated in the process of vasculogenesis in the developing allantois. Both of these genes are expressed in the allantois starting E7.5. T is expressed until the 4-6s stage, whereas Tbx4 is expressed at least until the formation of umbilical vessels at E10.5 (Inman and Downs, 2006b;

Naiche et al., 2011). Loss of function mutation in either of these genes leads to absence of chorio-allantoic fusion and a lack of vascular plexus formation, although the ECs differentiate and express Pecam (Naiche and Papaioannou, 2003; Inman and Downs, 2006a)

Methods for allantois culture ex vivo

The allantois has many advantages as a system to study blood vessel development. First, it can be isolated from the embryo and cultured ex vivo. Because the allantois contains mesodermal cells capable of differentiating into ECs in response to signals provided by the mesothelium, mesenchyme and surrounding ECM, also present in the explant, it is a self-contained system with the potential to undergo vasculogenesis or angiogenesis in vitro. When isolated early (HF-1s stage), explants undergo vasculogenesis and when isolated later (4-6s stage), they undergo angiogenesis (Downs et al., 1998). Secondly, allantois explants can be imaged using time lapse

18 microscopy to follow the sequence of events that occur during vessel formation in vitro (Perryn et al., 2008). Finally, cultured allantois explants can be immunostained for markers of vessel formation and can be sectioned for histological analysis (Downs et al., 2001; Downs, 2006).

Several ex vivo culture methods have been developed to analyze different aspects of blood vessel formation as discussed below.

Adherent allantois culture

The most commonly used method to culture the allantois is in serum-containing media on tissue culture plastic or glass coated with poly-D-lysine or fibronectin for up to 72 hours (Downs et al.,

1998; Downs et al., 2001; Perryn et al., 2008). After 4-6 hours, the distal tip of the allantois attaches to the plastic or glass and mesenchymal cells begin to spread. By 12 hours the entire allantois is firmly attached. As occurs in vivo, the explant begins vascularization at the distal end of the allantois. The vascular plexus expands on top of and in communication with the mesenchymal layer of cells. By 18-20 hours of culture the explant adopts a circular shape with the vascular plexus covering the entire area except at the periphery, which is populated only by mesenchymal cells (Downs et al., 2001) (Figure 1.4). As shown histologically, vessels of the plexus form lumens in vitro (Argraves et al., 2002). Explants show expression of all EC markers seen in vivo, namely Flk1, Flt-1, Tie1, Tie2, Pecam and VE-Cad. The mesothelium of allantois explants can be identified with VCAM-1 staining and is present on top of the vascular plexus

(Downs et al., 2001; Downs, 2006).

Allantois suspension culture

An alternative culture method that better preserves the 3-dimensional (3D) structure of the allantois is suspension culture in rolling tubes. With this method, isolated allantoises round up and develop a vascularized core surrounded by an outer layer of mesothelium. These 3D

19 structures can be sectioned to study histomorphology and gene expression patterns in detail

(Downs et al., 2001; Downs, 2006).

Allantois hanging drop culture

Another method that preserves 3D structure is the hanging drop culture that generates allantoic spheroids of mean diameter 440µm. These spheroids contain a 3D network of Pecam-positive endothelial tubes and an outer layer that stains for SMC markers α smooth muscle actin (αSMA),

SM22α and smooth muscle myosin. Upon addition of VEGF to the culture media, vessels in the core fuse to form uniluminal vascular spheroids characterized by an inner Pecam-positive EC layer and a closely associated outer layer of αSMA-positive cells. This culture system provides a means to study interactions between ECs and SMCs (Gentile et al., 2008). Furthermore, when two uniluminal spheroids are placed in contact with one another in the presence of VEGF, the spheroids fuse to form a single large spheroid modeling the fusion of the paired dorsal aortae in the developing embryo (Fleming et al., 2010).

Allantois culture for the derivation of hematopoietic cells

Tal1(Drake and Fleming, 2000) and CD41 (Corbel et al., 2007), markers for hematopoietic progenitors, and Runx1 (Zeigler et al., 2006), a marker for the definitive hematopoietic lineage, are all expressed in the pre-fusion allantois. Despite the presence of these markers, it is not certain whether the allantois gives rise to hematopoietic cells in vivo. However, the allantois does have the potential to form hematopoietic cells in vitro. When multiple allantoises are cultured together for 2-5 days, dissociated to a single cell suspension and seeded in media containing methylcellulose and cytokines, the cells show myeloid and erythroid differentiation in a colony forming assay (Zeigler et al., 2006; Corbel et al., 2007).

Use of adherent allantois cultures to study gene function in blood vessel formation

VE-Cad function and signaling in blood vessel formation

VE-Cad is specifically expressed by ECs at cell-cell adherens junctions. Embryos that lack VE-

Cad gene function show impaired vascular development; mutants die at E9.5 due to a failure of vessel remodeling and maturation (Gory-Faure et al., 1999). Although initial EC differentiation occurs, the mutant phenotype is complex and which subsequent step of vasculogenesis is affected is controversial (Carmeliet et al., 1999; Gory-Faure et al., 1999). One interpretation is that ECs in the allantois and yolk sac fail to form a vascular plexus; the alternative interpretation is that plexus formation proceeds normally but subsequent stabilization of the plexus fails, leading to its disintegration (Carmeliet et al., 1999; Crosby et al., 2005).

Allantois cultures with or without a VE-Cad blocking antibody (Ab) were used in two studies to address these alternative hypotheses. Culture of wild type allantoises in the presence of VE-Cad Ab results in a phenotype identical to culture of VE-Cad null mutant allantoises, that is, the presence of Pecam-positive ECs but absence of a vascular plexus (Crosby et al., 2005). In the first study, early wild type allantoises were cultured with VE-Cad Ab either during the early phase of culture followed by culture in the absence of VE-Cad Ab or vice versa. When the VE-

Cad Ab was added during the initial phase of culture, vascular plexus formation was not affected. When the antibody was added in the later phase, the plexus showed disconnected clusters of ECs. These results were interpreted as a disassembly of the vascular plexus which would suggest a role for VE-Cad in plexus stabilization, rather than plexus formation (Crosby et al., 2005). However, an alternate explanation is that loss of VE-Cad function affects EC motility.

In a second study, this possibility was examined using time lapse imaging. Wild type allantoises were cultured in the presence of Cy3-CD34, an antibody which immunolabels ECs without affecting vascular network formation allowing tracking of individual ECs during the

21 period of vessel formation (Perryn et al., 2008). Using this method, EC movements can be studied in the presence of independent movement of the allantois mesenchyme and mesothelium, mimicking the in vivo scenario of blood vessel development. The major findings of this study were that new interconnections of ECs are formed a) by gradual enlargement of an avascular area and b) by vasculogenic sprouting involving initiation and extension of a vascular cord until it meets another cord. As a new sprout extends and interacts with the surrounding matrix, new

ECs are added to the sprout from the EC cluster at the site of sprout initiation. Treatment with a

VE-Cad Ab does not prevent sprout initiation but prevents addition of ECs to the extending sprout. Without the addition of ECs, the sprouts regress and fail to form new interconnections to make a vascular plexus. In addition, movement of the ECs, independent of the movement of the mesenchymal or mesothelial layers, was reduced by 50% in the presence of VE-Cad Ab (Perryn et al., 2008). Allantois culture in conjunction with live imaging has thus helped resolve the function of VE-Cad as being important to EC-autonomous motility, contribution of ECs to sprout formation and vascular plexus formation. However, an additional role for VE-Cad in vascular plexus stability has not been ruled out.

Role of lipids in angiogenesis

Sphingosine-1-phosphate (S1P) is a phospholipid generated by the phosphorylation of sphingosine by sphingosine kinase (SK). S1P binds to one of its receptors S1P1-S1P5 and activates Ras and ERK signaling to promote cell survival. S1P plays a role in different processes important for blood vessel formation including EC migration, chemotaxis, cytoskeletal reorganization and adherens junction assembly but being a lipid its precise role cannot be determined using in vivo mutagenesis. S1P1 regulates vessel stabilization by recruitment of mural cells in vivo (Liu et al., 2000). S1P1, S1P3 and SK2 are expressed in the allantois and

22 embryo during vasculogenesis. In serum-deficient conditions, cultures of early allantoises (E7.8) form Pecam-positive ECs but fail to form a vascular plexus while allantoises cultured from later embryos (E8.5) show limited plexus forming ability. S1P rescues vascular plexus formation, by promoting expansion of the vascular network and the mesothelial layer in serum-deficient allantois cultures. S1P treatment of allantoises cultured with or without serum, did not alter the number of ECs or mesodermal cells. Thus, using the allantois as a model system revealed that

S1P does not affect endothelial, angioblast or mesenchymal cell number but instead affects the degree of expansion of the vascular network, suggesting a potential role in EC migration in vasculogenesis (Argraves et al., 2004).

Autotaxin (ATX) a protein involved in lipid metabolism converts lysophosphatidyl choline to lysophosphatidic acid (LPA) and sphingophosphocholine to S1P in vitro. Embryos homozygous for a mutation in the ATX gene Enpp2 show severe vascular defects while heterozygous adult mice show decreased circulating levels of LPA, suggesting ATX might function through the production of LPA in vivo. Vascular plexi of wild type allantois explants readily disintegrate when serum is removed from the culture media. This disintegration can be prevented by addition of ATX or LPA. Thus, these molecules are essential for maintenance of the vascular plexus (Tanaka et al., 2006).

Other gene functions established using allantois culture system

Deficiency of RA-GEF1, a guanine nucleotide exchange factor for the small GTPase Rap1, leads to impaired vascular development and decreased plexus formation in allantois cultures as measured by vessel sprout formation (Kanemura et al., 2009). ECs from RA-GEF1 null allantois explants show a decrease in VE-Cad expression and a lack of GTP bound RAP1. VE-Cad expression is rescued when the cells of the mutant allantois are transfected with a constitutively

23 active, GTP-bound form of RAP1 and cultured with mouse stroma cells. Additionally, when mutant allantoises are isolated, transfected with constitutively active RAP1 and cultured in collagen gels, an increase in vessel sprout formation is noted. Thus the allantois culture system was used to show that RA-GEF1 regulates vascular network formation by controlling RAP1 activation and VE-Cad expression in ECs (Kanemura et al., 2009).

Other gene functions established using the adherent allantois culture system are a role of

PDGFB in vascular outgrowth (French et al., 2008), BMP signaling in SMC differentiation

(Astorga and Carlsson, 2007), histone deacetylase (HDAC) in commitment of progenitor cells to an EC fate (Rossig et al., 2005), VE-PTP in remodeling of vasculature (Baumer et al., 2006),

Gas1 in EC survival (Spagnuolo et al., 2004), PI3K and ARAP3 in sprout formation

(Gambardella et al., 2010) (Table 1.3).

Applications in disease and therapeutics

Apart from its importance in normal embryonic development, blood vessel formation plays a key role in many pathological conditions such as cancer, diabetic retinopathy, rheumatoid arthritis, obesity, age related macular degeneration and neurological disorders such as Parkinson’s and

Alzheimer’s disease (Aisha et al., 2010). Thus systems that model blood vessel formation are valuable not only for understanding basic mechanisms of vessel formation but also for screening drugs that influence vessel formation. Although in vitro model systems are available that utilize cultured EC lines, these models have limitations. First, they may have acquired properties that facilitate perpetuation during derivation in vitro that could influence their responses to drugs.

Further, being homogenous EC cultures, they lack the presence of any mural cells or ECM that provides a tissue-type environment. Allantois cultures, on the other hand, have the advantages of being primary cultures that contain all the cellular and extracellular components that support

24 vessel formation. Thus the effect of drugs or small molecules on all the processes of vessel formation – EC proliferation, EC migration, endothelial tube formation and formation of an EC vascular network – can be determined in one system that closely mimics in vivo vessel formation. Additionally this culture system is robust and reproducible. Methods for quantification of different parameters in this system are now available (Zudaire et al., 2011).

Because there is no standard model that can mimic an in vivo disease situation completely, the combined use of EC cell lines and allantois explants provides for a good drug screening strategy to study the effects of the drugs on blood vessel formation.

Conclusion

In conclusion, the use of the allantois to study blood vessel formation is in its infancy although the studies reviewed here amply illustrate its usefulness. The allantois culture methodology allows teasing out function when a vascular defect results in early lethality and can be used to distinguish if a vascular phenotype is primary or secondary to the loss of gene function elsewhere in the embryo. Finally, the allantois explant culture provides an additional in vitro system for drug screening and establishing drug efficacy as pro- or anti-angiogenic. Thus, the allantois is emerging as an important tool to study vasculogenesis and angiogenesis in development and disease.

Known role of T-box genes in vascular development

There are a few examples of T-box transcription factors being involved in vessel development.

Tbx1 is required for the appropriate patterning of the pharyngeal arch arteries, aortic arch arteries and the proximal coronary arteries (Vitelli et al., 2002a; Theveniau-Ruissy et al., 2008). Upon myocardium injury in zebrafish, epicardial cells begin to express Tbx18 along with increased epicardial cell invasion into the wound site and new vessel formation of the newly formed tissue

(Lepilina et al., 2006). This suggests a potential role for Tbx18 for epicardial cell EMT or subsequent migration during coronary vasculogenesis. Tbx5 may also regulate vessel formation by affecting migration of epicardial cells (Hatcher et al., 2004). Tbx5 may also have a cytoplasmic localization in cells of the coronary vasculature (Bimber et al., 2007) further suggesting a potential role in vasculogenesis. Mutation in mouse Tbx3 results in reduced yolk sac vasculature and high levels of apoptosis in the yolk sac endodermal layer (Davenport et al.,

2003).

Loss of Tbx4 does not affect EC differentiation in the allantois, but does affect the ability of these ECs to form vessels and a vascular plexus (Naiche and Papaioannou, 2003). Although

Tbx4 leads to a vascular phenotype in the allantois, it is not expressed in the allantoic ECs or the

ECs of the umbilical vessels (Naiche et al., 2011). The allantois adherent cultures provide for a good system to characterize the Tbx4 mutant phenotype in detail and to understand the role of

Tbx4 in vascular development of the allantois.

1.3 Development of the respiratory system

The mammalian respiratory system, comprising of the lung and trachea, originates from the ventral aspect of the foregut tube. During the course of development the lung epithelium follows a stereotypic pattern of branching morphogenesis in close proximity to a branching vascular network. The branching epithelium ultimately differentiates into alveolar epithelial cells adjacent to a capillary network and together these form the functional units of the respiratory system, allowing for exchange of gases within the blood circulation. Multiple signaling pathways including the Fgfs, Wnts, Retinoic acid and the Bmps have been implicated in different aspects of development of the respiratory system

Specification of the respiratory system

The endodermal foregut tube is patterned by cues from the lateral plate mesoderm leading to specification of specialized endodermal organs. Expression of transcription factors or markers characteristic of specific organs are observed along the anterior posterior axis of the foregut tube before formation of each organ. Pax8 and Nkx2.1 specifically mark the thyroid endoderm,

Albumin marks liver endoderm, Pdx1 marks the pancreatic endoderm and Somatostatin marks the intestinal endoderm (Serls et al., 2005). Nkx2.1 has been identified as the earliest marker of lung endoderm specification and is expressed at embryonic day (E) 9.0 (19-24 somites) in the mouse (Figure 1.5). Outgrowth and branching of these Nkx2.1 expressing cells require fibroblast growth factor (FGF) signaling. FGF signaling centers from the heart and/or from the splanchic mesenchyme influence lung formation from foregut endoderm. The dose and timing of exposure of endodermal precursors to FGF signaling from the cardiac mesenchyme influences the formation of pulmonary lineages. Cells receiving less FGF stimulation form other organs including the gastrointestinal tract, liver and pancreas (Zaret, 2002). Recent evidence using in vitro foregut culture suggests that induction of mesenchymal Fgf10 in the prospective lung field is dependent on retinoic acid (RA) signaling pathway and this control of RA is selective for the mid-foregut region and occurs within a defined developmental window before primary budding

(Desai et al., 2004). In addition, using this system it has been shown that RA acts as a major regulatory signal integrating Wnt and Tgfβ pathways in the control of Fgf10 induction in the foregut mesoderm (Chen et al., 2010). Also, combined loss of Wnt2 and Wnt2b, which are expressed in the mesoderm surrounding the anterior foregut, or of β-catenin in the endoderm, leads to loss of Nkx2.1 expression and failure of lung specification (Goss et al., 2009; Harris-

Johnson et al., 2009; Domyan and Sun, 2011). Thus a number of different genes and signaling

27 pathways have been implicated as important for specification of the lung/trachea primordia

(Figure 1.5).

After specification, at E9.25 (25-28 somites) the primary lung buds appear as ventral outpouchings of the foregut (Cardoso and Lu, 2006). Whether there is a single bud, which later subdivides and develops the two lung buds or whether the endodermal lung primordia originate from primarily paired primordia is controversial. Recent evidence using scanning electron microscopy in the chick supports the latter hypothesis (Metzger et al., 2011). After the lung buds appear they grow in a ventro-posterior direction to eventually fuse together at E9.5 (~30 somites) and continue to elongate until E11.5.

Lung branching and morphogenesis

In the mouse, the left lung bud remains a single lobe and the right lung bud branches to form 4 lobes - cranial, medial, caudal and accessory (Cardoso and Lu, 2006). The lobes undergo a stereotypic pattern of branching between E11.5 and E16.5, the pseudoglandular phase. Timed analysis of branch formation in the lungs suggests that the branching pattern can be broken down into three distinct modes that are used repeatedly in different combinations. The first mode is called domain branching involving new bud formation at specific distances from the tip of a stalk and at positions around the circumference that are either dorsal/ventral or medial/lateral relative to the axis of the parent stalk. The other two modes relate to the bifurcation of the tip and are either planar or orthogonal bifurcations depending on the axis along which the two new buds form (Metzger et al., 2008). From E16.5 to E17.5, the canalicular stage, the terminal buds become narrower and from E18.5 to post natal day 5, the saccular stage, the precursors of the alveoli form (Maeda et al., 2007).

Vascular development in the branching lung

The adult lung has a dual vascular system, the bronchial and the pulmonary system. The bronchial system delivers oxygen to all non respiratory structures of the lung, whereas the pulmonary system transports deoxygenated blood to the alveoli where gas exchange occurs. The pulmonary arteries arise from the pulmonary trunk of the heart and closely follow the bronchial tree, giving rise to the alveolar capillary plexus. These capillaries drain into the pulmonary veins, which connect back to the left atrium of the heart. A detailed analysis of the pulmonary vasculature from the earliest embryonic stage suggests that the vasculature is a part of the embryonic circulation from the moment the lung starts to develop. Hence the presence of blood vessels is potentially important for the development of the lung. For example, the ECs of the splanchic mesoderm may be involved in the prepatterning and induction of the presumptive lung region as has been shown for liver and the pancreas (Parera et al., 2005).

Distal angiogenesis has been proposed as a model for pulmonary vasculogenesis, that is, lung vasculature develops from preexisting vessels (Parera et al., 2005). The tip zone, which is the distal part of the branching airway that lacks the layer of SMCs, is covered by a network of capillaries. This network expands as the lung bud grows and finally leads to the alveolar capillary plexus. The epithelial-endothelial interactions induce angiogenesis at the tip zone, which ensures that the vascular network proceeds alongside epithelial branching. New vessels undergo vascular remodeling, implying that some vessels grow and fuse with neighboring vessels, whereas others remain small or degenerate (Parera et al., 2005). Once the vascular network is set up it is maintained by the presence of vascular SMCs that surround the blood vessels. The mesothelium of the lung contributes to mesenchymal cells within the lung and in particular to the SMCs of the pulmonary vasculature (Que et al., 2008).

Innervation of the branching lung

The innervations and the airway smooth muscle (ASM) have recently been recognized as integral components of the developing lung: both are present in the epithelial tubules of the embryonic lung bud shortly after it evaginates from the foregut. Whereas, the ASM is functionally mature shortly after it is laid down, the growth and maturation of the innervations largely follows the morphological stages of development. The neural tissue and the ASM are integral part of the lung, and persist in a dynamic state throughout gestation and into postnatal life and late adulthood (Harding et al., 2003).

Two large nerve trunks lying in the outermost surface of the airway wall span the complete length of the bronchial tree. They give rise to a network of bundles which cover the

ASM from the trachea to the growing tips of the airways. The maturation of nerve trunks and ganglia occurs as the bronchial tree expands throughout gestation until the end of the canalicular stage (Tollet et al., 2001).

Little is known about the structural relationship between nerves and smooth muscle during early lung development. Neural stimulation leads to contraction of ASM in first trimester fetal pigs. The ASM is functionally mature as it responds by contracting to multiple agonists such as acetylcholine, histamine, and substance P, and by relaxing to β-adrenoreceptor agents

(Sparrow et al., 1994). ASM may function as a mechanical prerequisite for lung growth by generating a positive pressure in the liquid-filled tubules. Studies have shown that a maintained pressure in the lung liquid is necessary for normal lung growth (Harding et al., 2003). ASM tone and spontaneous contractions are implicated in lung growth possibly by inducing the expression of developmentally important genes and growth factors (Tollet et al., 2001).

Signaling pathways implicated in lung branching

Given that lung branching follows a stereotypic pattern, it is hypothesized that specific gene expression patterns would allow branching routines to occur at defined intervals. An example that supports this hypothesis is that loss of either Fog2 or Gata4 leads to a loss of the accessory lobe in the right lung (Ackerman et al., 2005; Ackerman et al., 2007). Additionally, hedgehog interacting protein 1 (hip1) null mutants display one right and one left lobe suggesting that they fail to complete the first step in branching morphogenesis – the lobation of the right lung into four lobes (Chuang et al., 2003).

The stereotypic pattern of lung branching has only been described recently (Metzger et al., 2008). Thus, most of the genes important for lung branching have been studied in the context of how these genes signal to facilitate growth of a proximal lung bud and branching of the distal tip. Genes involved in different signaling pathways, including Fgf, Wnt, Bmp, Shh and retinoic acid synthesis have been shown to play important roles in lung branch formation.

Fgf signaling in lung branching

Fgf10 which is expressed in the mesenchyme is primarily responsible for branching of the epithelial tips (Weaver et al., 2000). Fgf10 mutants form the lung buds but fail to undergo branching and the lung buds degenerate with time (Min et al., 1998; Sekine et al., 1999).

Mutation in the IIIb isoform of Fgfr2, which is the primary receptor for Fgf10 in the lung, results in a very similar phenotype as a mutation in Fgf10 where null mutants show a complete absence of lungs at birth (Arman et al., 1999). Fgf9, which is expressed in the early epithelium and the mesothelium, promotes expression of Fgf10 and Wnt2 in the distal mesoderm and is thought to be the Fgf that regulates lung mesenchyme (Yin et al., 2008). Recently it has been shown that the mesothelium-derived Fgf9 is responsible for maintaining lung mesenchyme via the control of

31 mesenchymal proliferation and that epithelium-derived Fgf9 affects epithelial branching (Yin et al., 2011).

Bmp signaling in lung branching

Inhibition of epithelial Bmp4 signaling by overexpression of Xnoggin leads to a decrease in lung size and irregularly shaped lung lobes (Weaver et al., 1999). Over-expression of Bmp4 also results in smaller lungs with reduced branching and distended terminal buds (Bellusci et al.,

1996). Abrogation of lung epithelial-specific Smad1 – a downstream effector of Bmp signaling

– results in retardation of lung branching morphogenesis and neonatal respiratory failure (Shu et al., 2002; Xu et al., 2011).

Wnt signaling in lung branching

Wnt7b is expressed in the airway epithelium and Wnt7b mutants show lung hypoplasia due to defects in mesenchymal proliferation. Additionally, these mutants also show defects in smooth muscle formation in the pulmonary vasculature leading to rupture of major vessels or hemorrhage and death at birth (Shu et al., 2002). Wnt2 and Wnt2b mutants show a complete absence of lung specification. Wnt2 mutants alone, unlike Wnt7b mutants show defects in differentiation of the airway smooth muscle but not vascular smooth muscle (Goss et al., 2011).

Mesenchyme specific loss of β-catenin leads to reduced branching and partial right isomerism.

Reduced branching in these mutants is due to decreased Fgf10 expression and decreased Fgf10 signaling in the epithelium. Loss of β-catenin in the mesenchyme also affects the amplification of the para bronchial SMCs and the differentiation of the ECs (De Langhe et al., 2008).

Additionally treatment of in vitro lung cultures with Dikkopf1 (Dkk-1) an inhibitor of canonical

Wnt signaling, impairs branching due to enlarged terminal buds, decreased αSMA expression and defective pulmonary vasculature along with decreased fibronectin deposition (De Langhe et

32 al., 2005). Targeted disruption of Wnt5a results in excess branching due to increased Fgf10 expression and decreased Shh expression (Li et al., 2002). Conversely overexpression of Wnt5a leads to reduced branching and dilated distal airways (Li et al., 2005).

Shh signaling in lung branching

Shh and different components of the Shh signaling pathway play an essential role in lung development and branching (Rutter and Post, 2005). Shh null mutant mice have a rudimentary lung sac due to complete absence of branching and delocalized Fgf10 expression throughout the branching tip instead of just the distal tips (Pepicelli et al., 1998). Additionally, conditional inactivation of Shh in lung epithelial cells leads to the formation of hypoplastic lungs with reduced branching of the peripheral tubules (Miller et al., 2004). Hip1 null mutants show a lack of secondary branching in the right lobe and subsequently show reduced airway branching and increased mesenchyme. In addition, these mutants show increased Hh signaling and show a decrease in the expression of Fgf10 at the distal tips. This phenotype can be partially rescued by removing one allele of Patched (Ptc), the receptor for Hh signaling (Chuang et al., 2003). Gli1,

Gli2 and Gli3 are target proteins of the Shh signaling pathway. Gli1 null mice are viable, Gli3 null mice have smaller lungs with a reduction in lung width, Gli2 null mice have hypoplastic lungs with a single lobe in the right lung. Gli1;Gli2 double null embryos have lungs that are smaller than Gli2 null mutant embryos. The Gli2;Gli3 double null mice fail to show any lung formation. Shh;Gli3 double null mice have a pulmonary phenotype that is less severe than the

Shh null mutant mice alone. The levels of a truncated repressor form of Gli3 (Gli3R), are much higher in Shh null lungs than in wild-type lungs. The reduction in phenotypic severity of the

Shh-/-;Gli3-/- lungs can thus be explained as in the double nulls, the Gli3R level would no longer increase but would be absent (Rutter and Post, 2005).

Extracellular matrix in lung branching

Laminin is a large trimeric glycoprotein that is a major component of basement membranes.

Addition of anti-laminin antibodies to embryonic lung cultures markedly diminishes tube formation and terminal branching as well as mitosis, in epithelial cells (McGowan, 1992). Lungs from mice lacking the integrin α3 subunit fail to branch into smaller bronchioles. Deleting both integrin α3 and α6 subunits results in marked lung hypoplasia. Integrin α8 null mice develop fusion of the medial and caudal lobes of the right lung as well as subtle abnormalities in airway division. Additionally, specific ablation of the nidogen binding site in the laminin γ1 chain causes abnormal lung development. In vitro organ culture suggests a role for fibronectin in mediating lung branching morphogenesis (Pozzi and Zent, 2011). A monoclonal antibody directed against E-cadherin impairs tube formation and branching in embryonic mouse lung organ cultures (McGowan, 1992).

Heparan sulfate proteoglycans play a crucial role in lung development. As discussed previously, Fgf10 signaling is absolutely essential for lung branching morphogenesis. Fgf10 interactions with its receptor Fgfr2 are highly dependent on the presence of heparin sulphate

(HS) proteoglycans, the position of sulfation (O or N) and the degree of sulfation. Disturbing the sulfation pattern results in alterations in lung branching patterns and gene expression in vitro

(Izvolsky et al., 2003b) which cannot be rescued using exogenous excess Fgf10 (Izvolsky et al.,

2003a).

Development of the trachea

The dorsal and ventral aspects of the foregut differentiate into esophagus and trachea respectively. The point of fusion of the elongating lung buds is thought to be the origin of the tracheal tube (Spooner and Wessells, 1970). The primitive foregut is divided into trachea and

34 esophagus by a septum called the tracheoesophageal septum. Initially forerunners of the septum appear in the lateral walls of the foregut, the so-called lateral ridges, which then fuse in the midline. This process of fusion starts caudally, moves cranially and ends in the region of the pharynx. Recent evidence in the chick argues against the presence of the tracheoesophageal septum due to absence of any signs of lateral ridges or of fusion of lateral wall components. The alternate hypothesis is that the trachea arises on the floor of an undivided foregut chamber that is first seen after the formation of the liver diverticulum caudally and the pharyngeal pouches cranially. This chamber becomes smaller as the larynx folds, the lung anlage and the esophagus appears, followed by the appearance of the trachea anlage. The trachea elongates by caudal growth probably along with the cranial movement of the tracheoesophageal fold (Metzger et al.,

2011).

Failure of separation of the trachea and the esophagus leads to a condition called tracheoesophagal fistula (TEF). Recent studies suggest patterning of the foregut endoderm along the dorsal/ventral axis is marked by greater Sox2 expression dorsally in the future esophagus while Nkx2.1 expression marks the future trachea ventrally (Domyan et al., 2011).

This patterning in turn depends on signals from the surrounding mesenchyme including Fgfs,

Wnts and Bmps. In the mouse, deficiency of Noggin, Bmp receptor (Bmpr) 1 and Bmpr2, Shh,

Gli2 and Gli3, Foxf1 and Sox2 leads to TEF (Litingtung et al., 1998; Motoyama et al., 1998;

Mahlapuu et al., 2001; Que et al., 2007; Shaw-Smith, 2010; Domyan et al., 2011). In humans, single gene mutations in SOX2 and GLI3 are associated with TEF (Scott, 2009).

After E11.5, mesenchyme surrounding the dorsal aspect of the trachea differentiates into the trachealis smooth muscle. Mesenchyme surrounding the ventral aspect of the trachea and lateral aspect of the main stem bronchi leading to the lungs, segments and differentiates into C

35 shaped rings composed of chondrocytes. Defects in smooth muscle or cartilage formation in the trachea result in tracheomalacia – lack of rigidity in the trachea and tracheal stenosis – narrowing of the trachea (Chen and Holinger, 1994). Additionally in human patients with Apert syndrome the trachea is characterized by a continuous sleeve of cartilage (Moloney et al., 1996). Ventral tracheal cartilage is formed by migration of cells that undergo mesenchymal condensations, resulting in an increase in the number of cells per unit area without an increase in cell volume

(DeLise et al., 2000). Sox9 is as an important regulator of mesenchymal condensations and chondrocyte differentiation (de Crombrugghe et al., 2001; Akiyama et al., 2002). In chondrocyte cell aggregate cultures it has been shown that in addition to Sox9, FGF2, Igf1, Tgf2 and Bmp2 enhance the process of chondrocyte formation (DeLise et al., 2000; Hardingham et al., 2006).

Mutations in a number of genes including Shh, Sox2, retinoic acid synthesis genes and Fgf signaling pathway genes affect cartilage ring formation (Mendelsohn et al., 1994; Pepicelli et al.,

1998; Vermot et al., 2003; Miller et al., 2004; Que et al., 2009; Tiozzo et al., 2009; Park et al.,

2010). Mutation in Fgfr2c produces a similar phenotype in mice and has been used as a model for Apert syndrome (Tiozzo et al., 2009).

Fgf10 mutants form a partial tracheal tube in spite of the failure of lung formation (Min et al., 1998; Sekine et al., 1999). Recent evidence shows that loss of Fgf10 leads to defects in tracheal ring formation and that overexpression of Fgf10 only between E11.5 and E13.5 disrupts tracheal rings by altering the periodic expression of Shh in the trachea (Sala et al., 2011).

Known role of T-box genes in development of the lung and trachea

All Tbx2 subfamily genes, Tbx2, Tbx3, Tbx4 and Tbx5 are expressed in the developing chick lung buds and trachea between stages 15-21 (Gibson-Brown et al., 1998). In the mouse, Tbx1 is expressed in the lung epithelium at E12.5, Tbx2 and Tbx3 are expressed in the lung mesenchyme

36 at E11.5 and Tbx4 and Tbx5 are expressed in both the lung and trachea mesenchyme at E12.5 and later (Chapman et al., 1996), although their role in the development of the respiratory system is largely unknown. Tbx1 homozygous null mutants die at birth due to severe heart defects and the lungs are never fully inflated (Jerome and Papaioannou, 2001) but lung development has not been further investigated. In Tbx4 homozygous mutants, lung buds form but the embryos die at

E10.5 due to failure of allantois development and the subsequent lack of chorio-allantoic fusion leading to placental insufficiency (Naiche and Papaioannou, 2003). Tbx5 mutants die around

E10 due to defects in heart development (Bruneau et al., 2001); lung development has not been investigated. Antisense oligonucleotide depletion of both Tbx4 and Tbx5, but not Tbx2 and

Tbx3, in lung organ cultures results in inhibition of branching and loss of Fgf10 expression

(Cebra-Thomas et al., 2003) suggesting a role for these T-box transcription factors in lung branching. In the chick embryo, interference with Tbx4 function leads to a reduction in Fgf10 expression in lung mesenchyme and inhibits lung bud formation (Figure 1.5). Ectopic expression of Tbx4 leads to ectopic expression of Fgf10 and Nkx2.1 and ectopic lung bud formation in the esophagus. Additionally, ectopic expression of Tbx4 at the boundary between the trachea and the esophagus can lead to lack of separation of these two structures, resulting in a

TEF (Sakiyama et al., 2003). In humans, Tbx5 mutations lead to Holt Oram syndrome characterized by heart and forelimb abnormalities. Amongst Holt Oram patients, a single de- novo mutation has been linked to right lung agenesis (Tseng et al., 2007). No known mutations in Tbx4 have been shown to be associated with lung defects in humans.

Although conditional alleles for Tbx4 and Tbx5 are available in mice (Rallis et al., 2003;

Naiche and Papaioannou, 2007b), a role for these genes in vivo in the development of the respiratory system has not been previously studied.

Figure 1.1 Phylogenetic tree of T-box genes of zebrafish, Xenopus, chicken, mouse and human. The T-box domain amino acid sequences of T-box genes identified in Homo sapiens

(Hs), Mus musculus (Mm), Xenopus Laevis (Xl), Gallus gallus (Gg), and Danio rerio (Dr); of the Drosophila melanogaster gene omb; and of Caenorhabditis elegans tbx11 were used to generate a phylogenetic tree. The five subfamilies are delineated by brackets to the right. Cetbx11 was outgrouped to root the tree. The following sequences were excluded from the tree due to insufficient sequence data: Drtbax, XlTbx4 and GgTbr2 (Epstein et al., 2008).

Figure 1.2 Diagrammatic representation of the developing mouse embryo from the bud stage to the 22-26 somite stage highlighting the development of the allantois and its vasculature. The age in embryonic days and the stage of development are indicated across the top as are the developmental processes taking place in the allantois. The allantois first appears as a mesodermal bud emerging from the posterior end of the primitive streak at E7.5. The bud grows and expands across the exocoelomic cavity and fuses with the chorion to form the chorioallantoic placenta. The processes in allantois vessel development at each stage are indicated across the bottom of the figure. ECs differentiate and migrate to form a primary vascular plexus. The plexus connects with the dorsal aortae of the embryo prior to chorio-allantoic fusion. The primary plexus remodels to form the central vessel; later the umbilical

artery and the umbilical vein form at E9.5. After chorio-allantoic fusion, the allantoic vessels invade the chorion and form the fetal vascular component of the labyrinthine layer of the placenta. epc, ectoplacental cone; exc, exocoelomic cavity; m, mesothelium; c, allantois core; DA, dorsal aorta; UA, umbilical artery; UV, umbilical vein; CAP, chorio-allantoic plate; lab, placental labyrinth.

Figure 1.3 Diagrammatic representation of vasculogenesis. Endothelial cell (EC) precursors called angioblasts form by de novo differentiation of mesenchyme. These ECs migrate through the extracellular matrix to first aggregate and then to extend and form endothelial tubes. Endothelial tubes secrete signaling molecules to promote the differentiation of mural cells from the surrounding mesenchyme. Mural cells are recruited to the interconnecting endothelial tubes also called the primary vascular plexus. The primary vascular plexus is then remodeled into the big and small vessels of the mature vascular network.

Figure 1.4 Three methods for allantois culture. Allantoises can be dissected from embryos from the HF to 4 somite stage, allowed to attach to glass, plastic or filters for adherent cultures (top row), or they can be kept in suspension in rolling cultures (middle row) or hanging drops (bottom row). The asterisk indicates the proximal end of the allantois, closest to the embryo. In adherent cultures the distal tip of the allantois attaches to the dish within 6 hours and a vascular plexus forms after 24 hours of culture as indicated by staining with Flk1. Rolling cultures result in development of spheroids with 3 dimensional (3D) morphology, which can be stained whole mount as shown for VCAM (Downs et al., 2001). Hanging drop cultures of allantoises also form 3D structures with a vascular plexus after 18 hours of culture. Image shows fluorescent staining of a whole mount allantois spheroid with α-SMA (red) and Pecam (green). After treatment with VEGF, the ball of vascular plexus remodels into a single layer of Pecam-positive ECs surrounded by α- SMA positive SMCs as shown in an optical section (Gentile et al., 2008).

Figure 1.5 Signaling pathways involved in primary lung budding. Foregut mesoderm is shown in gray, endoderm in blue, and the endoderm of the prospective trachea and lung in red. Lung budding (red) results from mesodermal induction of Fgf10 and from activation of Fgfr2b signaling in the endoderm (indicated by a yellow bracket). Retinoic acid (RA) and Tbx genes (TBX4 in chicks) regulate Fgf10 expression. Wnt2 and Wnt2b expressed in the mesenchyme are required for lung/trachea specification. Gli2 and Gli3 are both required for primary lung bud formation. Bmp4 is expressed in the ventral mesoderm at the lung field {Modified from (Cardoso and Lu, 2006)}.

Table 1.1 Human syndromes caused by mutations in specific T-box genes and their respective mouse mutant phenotypes {Modified from (Naiche et al., 2005)}

Human gene ; mouse gene Human syndrome Mouse heterozygous Mouse homozygous phenotype phenotype TBX1 ; Tbx1 DiGeorge, craniofacial, Viable; thymus and vascular Neonatal lethal; craniofacial, glandular, vascular and heart abnormalities glandular, vascular and heart abnormalities abnormalities TBX3 ; Tbx3 Ulnar-mammary: hypoplastic Hypoplastic mammary glands, Embryonic lethal, yolk sac, mammary glands, abnormal abnormal external genitalia limb, heart and mammary external genitalia, limb gland defects abnormalities TBX4 ; Tbx4 Small Patella Reduced allantois growth rate Embryonic lethal, allantois and limb defects TBX5 ; Tbx5 Holt-Oram: heart and hand Heart abnormalities, reduced Embryonic lethal, severe heart abnormalities viability malformations TBX19 ; Tbx19 Recessive isolated ACTH Normal ACTH deficiency, pigment deficiency defects TBX22 ; Tbx22 X-linked cleft palate with Normal Post natal lethal; submucosal ankyloglossia cleft palate, ankyloglossia and choanal atresia

Table 1.2 Gene expression related to blood vessel formation and corresponding mutant phenotype in the allantois or placenta

Category Gene name Expression in the allantois or placenta Phenotype in allantois or placenta Age Area of expression Vegf signaling VEGF 5-9s, E12.5 Mt, labyrinthine stromal Degenerating endothelium in cells placenta Flk1 E7.5, 9-16s, E12.5 EC, placental vasculature Lack of mature ECs in the allantois Flt1 9-16s, E12.5 EC, placental vasculature Nd EC Pecam E8.0 onwards EC None (viable) VE-Cadherin E8.0 onwards EC No allantois plexus Hematopoietic Tal1 E7.0-8.0 EPC Absence of blood Runx1 E8.0-8.5 Hematopoietic Nd precursors SMC SM22α E8.25 SMC None (viable) αSMA E8.25 SMC Nd ECM Fibronectin E8.25 Mes Nd Itgα5 E8.25 Mes Nd Collagenα1(I) E8.25 Mes Nd Has2 E8.25 Mes Nd Vcan E8.25 Mes Nd ADAMTS9 E7.5 Mes Nd Remodeling of Np1 Nd Nd vascular Np2 Nd Nd plexus Tie1 9-16s, E12.5 EC, placental vasculature Nd Tie2 9-16s, E12.5 EC, placental vasculature Nd Ang1 Nd Nd Lack of complexity in umbilical vessels Ang2 Nd Nd Nd TGFβ E7.5-8.5 Nd Defective chorio-allantoic fusion, lack of Flk1 expression in allantois Alk1 Nd Nd Nd Alk5 Nd EC & SMC of placenta Defective sprouting of the allantois into the placenta, lack of SMCs around allantoic vessels

Artery vs Vein ephrinB2 E9.5 Vascular plexus, UA None EphrinB4 E9.0 EC/UV Nd Notch2 E8.25 Mes Nd Dll4 E8.25 Arterial EC Degenerating vasculature in placenta Jag1 E8.25 Mes Nd Hey1 (Hey) E8.5, E9.0 Mes Vasculature in CAP but not Hey2 (Hes) placenta Signaling Wnt2 E8.0-9.5 Mes Late gestation hemorrhage and molecules tissue disruption in placenta

Wnt5a E7.0,8.0 Mes No affect on allantois Wnt11 E7.0-8.0 Mes Nd Rspo3 E8.0, 8.5 mes Vessels fail to invade placenta Bmp4 E8.25 Mes Small or no allantois Smad1 E7.25 Mes No chorio-allantoic fusion Transcription Etv2 (ER71, E7.5, E8.25 Mes Lack of EC formation factors Etsrp) Foxf1 E8.25 Mes Absence of vasculature in allantois Snai1 E8.25 Mes ECs fail to form plexus dHAND E8.25 Mes Nd T E7.5-E8.5 Mes ECs fail to form plexus, lack of chorio-allantoic fusion Tbx4 E7.5-E10.5 Mes ECs fail to form plexus, lack of chorio-allantoic fusion

CAP, chorio-allantoic placenta; EC, EC; ECM, extracellular matrix; EPC, endothelial precursor cells; Mt, mesothelium; Mes, mesenchyme; Nd, not defined, SMC, smooth muscle cell; UA, umbilical artery; UV, umbilical vein.

4 46

Table 1.3 Gene function identified using allantois culture as a model system

Category Gene name Type of LOF/GOF Effect on allantois culture Inferred function molecule (method) VE-Cad VE-cadherin Cell-cell LOF Disconnected ECs in EC motility; addition of function and adhesion (null mutant and plexus ECs to new sprouts; signaling VE-Cad Ab) plexus stabilization VE-PTP Phosphatase LOF Excess EC coverage and Remodeling of plexus to (null mutant) simple monolayer provide complexity architecture of ECs Gas1 Cell cycle LOF Apoptosis of EC survival (null mutant) mesenchymal cells Lipid Sphingosine-1- Lipid GOF Promotes plexus formation Promotes vasculogenesis function phosphate (added to culture) in serum-deprived cultures and vascular outgrowth Autotaxin Lipid GOF Prevents plexus Plexus stabilization synthesis (added to culture) disintegration in serum- deprived conditions Lysophosphatidic Lipid GOF Prevents allantois culture Plexus stabilization acid (added to culture) disintegration in serum deprived conditions PI3K PI3K Kinase LOF Decrease in sprout Angiogenesis signaling (inhibitor) formation Arap3 GTPase LOF Decrease in sprout Angiogenesis (null mutant) formation RA-GEF1 GTPase LOF Decrease in sprout Controls activation of (null mutant) formation RAP1 and induction of VE-Cad PDGFBB Growth GOF Promotes plexus formation EC survival or factor (GF in culture) in low serum conditions maintenance HDAC Deacetylase LOF Decrease in ECs Increased commitment (inhibitor) of progenitors to ECs Bmp4 Signaling LOF Decrease in plexus Recruitment of mural (inhibitor) formation cells that promote plexus stabilty LOF, loss of function; GOF, gain of function; GF, growth factor.

Chapter 2. Canonical Wnt signaling acts downstream of Tbx4 in development of the allantois vasculature and the chorio-allantoic placenta

Introduction

Mutation of the T-box transcription factor gene Tbx4 results in abnormal vascular development in the allantois, loss of cavitation, apoptosis and lack of chorio-allantoic fusion leading to embryonic death at E10.5 (Naiche and Papaioannou, 2003). Tbx2, another T-box gene expressed in the allantois and Vcam, a cell adhesion molecule are absent while Bmp4 expression is unaffected in Tbx4 mutant allantoises. Pecam, a marker of ECs, is abundantly expressed in cells of Tbx4 mutant allantoises but these ECs do not coalesce into a primary vascular plexus (Naiche and Papaioannou, 2003). Comparison of Tbx4 RNA and Pecam protein localization as well as lineage tracing using a Tbx4-cre allele suggest that the ECs of the umbilical vessels do not and never have expressed Tbx4 (Naiche et al., 2011). In spite of this, Tbx4 null mutants show a defect in allantois EC organization suggesting that Tbx4 plays a non-cell-autonomous role in formation of the vascular plexus. We took a candidate gene approach to find Tbx4 target genes expressed in the mesenchyme that could explain this non-cell-autonomous effect.

Materials and methods

Mice

Mice carrying a Tbx4 null allele, Tbx4tm1.2Pa (Naiche and Papaioannou, 2003), hereafter referred to as Tbx4-, a Wnt2 null allele, Wnt2tm1.1(rtTA)Eem (Goss et al., 2009), hereafter referred to as Wnt2-, a Tbx4-cre allele, an insertion into the endogenous Tbx4 locus resulting in a bicistronic allele that expresses both cre and Tbx4 and has been shown to be expressed in all areas of Tbx4 expression

(Naiche et al., 2011) and a β-catenin conditional allele (Brault et al., 2001), hereafter referred to as βcatenincond, were genotyped as described previously.

Histology and vessel counts in the chorio-allantoic plate

Embryos and placentas were collected from timed matings and dissected out of the decidua; yolk sacs were removed for genotyping and the placentas were fixed in Bouin’s fixative (Sigma).

After dehydration in ethanol, placentas were embedded in paraffin wax, sectioned at 10 µm and stained with hematoxylin and eosin (H&E). From cross sections through the center of the placenta, every 5th section (for a total of 15-20 sections/placenta) was used to quantitate vasculature by counting the number of vessels per section present in the chorio-allantoic plate

(CAP). The total number of vessels per placenta was used as a representation of amount of vasculature in the CAP. Three to five placentas were analyzed for each genotype.

In situ hybridization and immunohistochemistry

Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) were performed according to standard protocols (Davis, 1993; Wilkinson and Nieto, 1993). Primary antibodies used include anti-Flk1 (R&D), anti-Jag1 (R&D) and anti-Fibronectin (SCBT); secondary antibodies were peroxidase-conjugated and Cy3-conjugated donkey IgG (Jackson

Immunochemicals). For each ISH or IHC, 2 to 7 mutant embryos were analyzed.

Ex vivo allantois culture

Allantoises were dissected from embryos at different stages in DMEM-Hepes (Invitrogen) with

5% FBS and transferred to 24 well dishes containing 0.5 ml of rat serum (Pel-Freeze Biologicals) and DMEM (Invitrogen) in a 1:1 ratio. For rescue experiments, lithium chloride (LiCl) was added to the media at a final concentration of 5 mM. Wnt2 producing CHO cells were used to

50 make Wnt2 conditioned media and vector-transfected CHO cells were used to make control conditioned media (Klein et al., 2008). For conditioned media rescue experiments, allantoises were cultured in 50% rat serum, 25% control or Wnt2-conditioned media and 25% DMEM. For inhibition of canonical Wnt signaling, 100 µM IWR1 (Cayman Chemicals) prepared in DMSO or DMSO alone was added to cultures and allantoises were cultured for 36-40 hours. For antibody staining, the cultures were fixed overnight in 4% PFA, washed in PBS with 0.5%

Triton, and treated for IHC for Flk1. For each experiment n was ≥2.

Results

Tbx4 mutant allantoises fail to form a vascular plexus ex vivo

To investigate their vessel forming potential, Tbx4 mutant and control allantoises were cultured ex vivo for 24 hours starting from the HF stage to the 6 somite stage. Wild-type or Tbx4 heterozygous allantoises from the earliest stages give rise to clusters of ECs, whereas explants from later stages spread out and give rise to a network of interconnected ETs or a plexus (Downs et al., 2001; Crosby et al., 2005), as shown by Flk1 antibody staining (Figure 2.1A-F). Tbx4 mutant allantoises, on the other hand, had clusters of ECs or ETs but failed to form a vascular plexus of interconnected ETs even from the later explants (Figure 2.1G-I). Methylene blue staining for cells other than ECs in allantois cultures (Figure 2.1B,E,H) shows the extent of allantois outgrowth. The proportion of explants of each genotype that proceeded to form EC clusters, ETs or a vascular plexus is shown in Figure 2.1J. Since, Tbx4 mutant allantoises show increased apoptosis (Naiche and Papaioannou, 2003), we tested if decreased cell numbers affected plexus formation. Control allantoises isolated at 2-4 somites when split in half still formed a vascular plexus upon culture, although the plexus was smaller than that from whole allantoises suggesting that reduced cell number was not the cause of lack of plexus formation in

Tbx4 mutants. As Tbx4 is not expressed in ECs of the allantois and cells derived from Tbx4 expressing cells never contribute to the endothelium of the umbilical vessels (Naiche et al.,

2011), these data suggest that Tbx4 plays a non-cell-autonomous role in the development of allantois vasculature. Thus, Tbx4 could either regulate the developing mesenchyme surrounding the ECs or the ECM through which the ECs migrate in order to form ETs and a primary plexus.

Tbx4 is upstream of ECM molecules, signaling molecules and transcription factors

In order to explore the role of Tbx4 in the development of the allantois we took a candidate gene approach and analyzed the expression of various ECM markers, signaling molecules and transcription factors. Candidates were chosen if their loss either leads to chorio-allantoic fusion defects or results in an embryonic vascular phenotype similar to the Tbx4 mutant allantois vascular phenotype.

To examine specific components of the ECM that might be affected in Tbx4 mutant allantoises, we analyzed expression of Versican (Vcan), a chondroitin sulphate proteoglycan and a binding partner for hyaluronic acid (HA) (Yamamura et al., 1997); and Hyaluronic acid synthase2 (Has2) (Camenisch et al., 2000) because HA is involved in the process of allantois cavitation (Fenderson et al., 1993) which is disrupted in Tbx4 mutants (Naiche and Papaioannou,

2003). Both Vcan and Has2 were expressed in wild-type allantoises but were absent from Tbx4 mutants (Figure 2.2A,B). We also analyzed the expression of α5 integrin (Itgα5) and

Fibronectin (Fn), ECM genes known to be essential for vascular development. EBs lacking either of these components form Pecam-positive ECs but fail to form ETs (Francis et al., 2002).

Expression of Itgα5 was absent (Figure 2.2C) whereas Fn and Collagen αI (1) (Colα1(I)) were expressed normally in the Tbx4 mutants (Figure 2.2D,E).

Bmp signaling is important for allantois development specifically for the process of chorio-allantoic fusion and this process is affected in Bmp2 mutants (Zhang and Bradley, 1996) and double mutants for Bmp7 and Bmp5 (Solloway and Robertson, 1999). Expression of both

Bmp2 and Bmp7 was absent in Tbx4 mutant allantoises (Figure 2.2F,G). Wnt2, one of the canonical Wnt family members, is expressed in the allantois and Wnt2 mutants show placental defects (Monkley et al., 1996). Wnt2 is expressed throughout the process of growth and vascularization of the allantois starting at the LHF stage (Monkley et al., 1996). Wnt2 expression was absent in Tbx4 mutant allantoises (Figure 2.2H). On the other hand, non-canonical Wnt5a was expressed normally in mutant allantoises (Figure 2.2I). Notch signaling components Notch2

(Hamada et al., 1999) and Delta like 4 (Dll4) (Gale et al., 2004) are expressed in the vasculature of the placenta and mutations in these genes result in a poorly-vascularized allantois and labyrinth. Mutation in Jagged1 (Jag1), one of the ligands for Notch receptors, leads to yolk sac and embryonic vasculature defects (Xue et al., 1999), but the allantois and placenta have not been analyzed in these mutants. Dll4 was expressed in the mutant allantoises but Dll4-positive

ECs formed clusters and failed to form vessels (Figure 2.2J). Jag1 was expressed in control allantoises but this expression was absent in Tbx4 mutant allantoises (Figure 2.2K). Similarly,

Notch2 (Figure 2.2L) was not detected in Tbx4 mutant allantoises.

Epiblast-specific deletion of transcription factor Snai1 leads to a vascular phenotype in which the ECs of the embryo and the allantois express Pecam but fail to coalesce to form ETs

(Lomeli et al., 2009) similar to Tbx4 mutant allantoises. Twist, another transcription factor, has been shown to be important for Snai1 expression (Leptin, 1991). Both Snai1 and Twist expression was absent in the Tbx4 mutant allantoises (Figure 2.2M, N).

Agonists of the canonical Wnt signaling pathway rescue and an antagonist phenocopies the Tbx4 mutant phenotype ex vivo

Wnt2, the canonical Wnt known to be expressed in the allantois was absent in Tbx4 mutant allantoises. Therefore, we examined the contribution of the canonical Wnt pathway to the vessel-forming potential of the allantois under the control of Tbx4.

The canonical Wnt signaling pathway was activated in culture using LiCl, an inhibitor of

GSK3-β and thus an activator of β-catenin-mediated transcription (Rao et al., 2005). When treated with LiCl, Tbx4 mutant allantois explants formed ETs which interconnected and formed a vascular plexus ex vivo (Figure 2.3B) unlike mutant allantoises cultured in the absence of LiCl, which never formed a vascular plexus (Figure 2.3A). The total number of interconnections of the ETs in the vascular network was compared in rescued and control mutant allantoises as a measure of the extent of plexus formation (Figure 2.3C). The number of interconnections as shown by notched box plots was significantly different between control and LiCl treated mutants

(Mann Whitney U test p=0.0016) (McGill et al., 1978). Similar rescue of ET and plexus formation was seen when Wnt2 conditioned media was added to the mutant allantois cultures

(control mutants, n=2, values 1, 10; mutants with Wnt2 conditioned media, n=2, values 54, 63)

(Figure 2.3D, E).

IWR1 a small molecule inhibitor was used to block the canonical Wnt signaling pathway at a concentration found to be non-toxic to cells in organ culture (Karner et al., 2010).

Allantoises from wildtype embryos were isolated at EHF-LHF (Figure 2.3F,G,H) and LHF-1 somite stages (Figure 2.3I,J,K) and IWR1 in DMSO or DMSO alone was added to the cultures.

In the presence of inhibitor, wildtype allantoises failed to form a vascular plexus but showed formation of EC clusters positive for Flk1. Taken together, these results suggest that canonical

Wnt signaling contributes to the vessel forming ability of allantoises ex vivo, and in the absence of this signal the ECs fail to form a primary vascular plexus.

Tbx4 and Wnt2 genetically interact during the formation of the chorio-allantoic placenta

Since canonical Wnt signaling is necessary for formation of allantois vessels ex vivo we tested whether Tbx4 mediated control of canonical Wnt signaling alone was responsible for vessel formation in allantoises in vivo. Wnt2 is the only known canonical Wnt expressed in the allantois and thus we analyzed Tbx4+/-;Wnt2+/- and Tbx4+/-;Wnt2-/- allantoises. Allantoises from both these genotypes fused with the chorion normally at E8.5 and formed normal umbilical vessels at E10.5 indicating no genetic interactions between Tbx4 and Wnt2 during allantois vessel formation in vivo. We hypothesized that lack of a phenotype in the Tbx4+/-;Wnt2-/- allantoises could be due to activation of β-catenin by another pathway. Thus, in order to create a mesenchymal deletion of β-catenin, we used a Tbx4-cre (Naiche et al., 2011) and a β-catenin conditional allele (Brault et al., 2001). Tbx4cre/+; βcatenincond/cond embryos did not show an allantoic vascular phenotype.

Chorio-allantoic placenta development of Tbx4+/-;Wnt2+/- embryos was analyzed later in development. At E11.5, there is a decreased amount of perivascular stroma in the chorio- allantoic plate (CAP) of double heterozygous placentas (Figure 2.4A-D). The extent of vascularization of the CAP was analyzed using H & E sections of controls and double heterozygote placentas (Figure 2.4A,B). Quantification of the vessels shows a significantly lower number of vessels present in double heterozygotes compared to controls (Mann Whitney U test p=0.001). At E13.5, double heterozygotes showed interdigitation of the spongiotrophoblast and labyrinth (Figure 2.4E,F,G,H). Additionally, at E13.5 there was a significant reduction in

55 the labyrinthine layer as seen by the ratio of area of labyrinth to the combined area of labyrinth and spongiotrophoblast in double heterozygotes (Figure 2.4I, Mann Whitney U test p=0.002).

Discussion

Tbx4 affects multiple processes important for allantois development including chorio-allantoic fusion, cavitation, cell proliferation and vasculogenesis (Naiche and Papaioannou, 2003). Here we show evidence that Tbx4 acts as a regulator of all these processes by regulating expression of a variety of genes including ECM genes, signaling molecules and transcription factor genes either directly or indirectly. We show that expression of ECM molecules Versican, Has2, Itgα5, transcription factors Snai1 and Twist and signaling molecules Bmp2, Notch2 and Wnt2, was absent in the Tbx4 mutant allantoises. Individual mutations in some of the genes identified downstream of Tbx4 are enough by themselves to explain the EC phenotype of the mutant allantoises. For example, Snai1 epiblast-specific mutant allantoises are incapable of forming a vascular plexus in the embryo proper and in the allantois explants, although they show the presence of differentiated Pecam-positive ECs (Lomeli et al., 2009) and Snai1 is absent in Tbx4 mutant allantoises. Itgα5 (Francis et al., 2002) and Wnt2 mutant EBs (Wang et al., 2007) that are differentiated down the vascular pathway show Pecam-positive ECs but do not form a vascular plexus and both these genes are not detected in the Tbx4 mutant allantoises.

Four of the genes absent in Tbx4 mutant allantoises, Vcan, Jag1, Snai1 and Twist, are downstream targets of the canonical Wnt signaling pathway in different systems

(http://www.stanford.edu/group/nusselab/cgi-bin/Wnt/target_genes) indicating a role for canonical Wnt signaling in development of allantois vasculature downstream of Tbx4.

Additionally, since Tbx4 is not expressed in ECs of the allantois but still causes a drastic effect on the assembly of ECs into a vascular plexus, a secreted molecule like Wnt2 could explain such

56 a non-cell-autonomous effect. In addition, Wnt2 a canonical Wnt known to be expressed in the allantois, was absent in Tbx4 mutants. There is evidence that Wnt2 plays an important role in EC proliferation and network formation specifically in hepatic sinusoidal ECs (HSEC’s). Wnt inhibitors lead to reduced proliferation and reduced endothelial tube forming ability of HSEC’s on matrigel (Klein et al., 2008). Additionally, Wnt2 null EBs fail to form a Pecam-positive vascular plexus and the vessel forming ability could be rescued by the exogenous addition of

Wnt2 conditioned media (Wang et al., 2007). We were able to rescue the vessel-forming potential of the Tbx4 mutant allantoises by activating the Wnt signaling pathway using LiCl and by adding Wnt2 conditioned media. We were also able to phenocopy the mutant phenotype by addition of a tankyrase inhibitor IWR1. Tankyrase destabilizes axin and prevents β-catenin from degradation, thus in the presence of the inhibitor β-catenin is degraded, blocking the canonical

Wnt signaling pathway (Karner et al., 2010). Taken together, our results suggest that canonical

Wnt signaling contributes to the vessel forming ability of allantoises ex vivo, and in the absence of this signal the ECs fail to form a primary vascular plexus.

Tbx4 and Wnt2 did not show interactions during allantoic vessel formation in vivo. One possible explanation for the lack of genetic interaction is that other, as yet unknown, canonical

Wnts are expressed in the allantois. Alternatively, β-catenin could be activated downstream of

Tbx4 by other pathways that are still active in Tbx4+/-;Wnt2+/- and Tbx4+/-;Wnt2-/- allantoises. It has already been shown that conditional excision of β-catenin in ECs does not affect allantois vascular development (Cattelino et al., 2003). Thus, neither an endothelial, nor a mesenchymal deletion of β-catenin (our study) reproduces the Tbx4 mutant phenotype, indicating that possibly pathways other than canonical Wnt signaling that are regulated by Tbx4 play a role in development of the allantois vasculature in vivo. An alternative explanation is that the lack of a

57 phenotype in the allantoises of Tbx4cre/+; βcatenincond/cond embryos is due to delayed or incomplete excision of β-catenin in the allantois mesenchyme with the Tbx4cre. There is a lag in reporter gene expression when using the Tbx4-cre as compared to Tbx4 expression (Naiche et al.,

2011). Since the process of allantois development from the appearance of bud to chorio- allantoic fusion occurs in a short span of about 12 hours, the deletion of β-catenin may not occur soon enough to see a phenotype prior to chorio-allantoic fusion. Brachyurycre is another mesodermal cre that can excise β-catenin early in development, but excision of β-catenin in the primitive streak, where Brachyury is also expressed, can produce streak related phenotypes that will overshadow an allantoic phenotype.

We show that although Tbx4 and Wnt2 do not interact in allantois vascular network formation in vivo, they interact during the formation of the fetal labyrinthine layer. Wnt2 by itself is important for the proper formation of the different placental layers and Wnt2 null mutants show disruptions in the placental labyrinthine layer (Monkley et al., 1996). In addition, canonical Wnt signaling has been shown to be very important for placenta development; deficiency in a number of genes involved in Wnt/β-catenin signaling show mid-gestation lethality due to placental insufficiency. For example, conditional inactivation of β-catenin in ECs shows a phenotype similar to Wnt2;Tbx4 double heterozygotes, where the labyrinthine layer is smaller and less vascularized (Cattelino et al., 2003). Rspondin3, a secretory molecule whose proposed function is to promote the canonical Wnt signaling pathway, is expressed in the allantois. Fetal vessels of Rspo3 null embryos fail to penetrate the chorion leading to lethality during mid-gestation (Aoki et al., 2007). Similarly Wnt receptor Fzd5 is expressed in the labyrinthine layer and null mutants for Fzd5 die due to lack of penetration of fetal vessels into the chorion (Ishikawa et al., 2001). We show that the Wnt2;Tbx4 double heterozgygous placentas

58 show decreased vascular coverage in the chorioallantoic plate, again suggesting the importance of Wnt2 in the vessel forming potential of ECs derived from the allantois. Furthermore, double heterozygous placentas show a mixing of placental layers reduced fetal placental layer. Thus, although Wnt2 does not genetically interact with Tbx4 in the process of vessel formation in the allantois in vivo, these genes do interact in formation of the vasculature of the CAP and the labyrinthine layer of the placenta.

Figure 2.1 Tbx4 mutant allantoises fail to form a vascular plexus ex vivo. (A-C) Tbx4+/+ (D-F), Tbx4+/- and (G-I) Tbx4-/- allantoises isolated from LHF-6 somite stage embryos were cultured for 24 hours, stained with anti-Flk1 antibody and scored for formation of isolated EC clusters, ETs or a vascular plexus. Cultures in B, E, H were counterstained with methylene blue. (J) Stacked bar graphs show the relative proportions of allantoises that formed EC clusters, ETs, or a vascular plexus amongst allantoises from different genotypes.

Figure 2.2 Identification of genes downstream of Tbx4 in the allantois using a candidate gene approach and ISH or IHC. (A-E) Expression of ECM genes Vcan, Has2 and Itgα5 was undetectable whereas Cola1(I) and Fn were unaffected in Tbx4 mutants. Inset in E shows secondary antibody control in the absence of primary antibody against Fn. (F-L) Expression of signaling molecules Bmp2, Bmp7, Wnt2, Jag1, and Notch2 was undetectable; Wnt5a was unaffected; Dll4 was expressed in an EC-like pattern in the Tbx4 mutants. (M-N) Expression of transcription factors (TF) Snai1 and Twist expression was undetectable in Tbx4 mutant allantoises. In each panel the image on the left is a control and on the right is a Tbx4 mutant allantois.

Figure 2.3 Wnt signaling is essential for vessel formation in allantoises ex vivo. (A-B) Vascular plexus formation is rescued in Tbx4 mutant allantoises in the presence of 5 mM LiCl. (C) Quantification of the number of ET interconnections in the absence and presence of LiCl using notched box plots (bars represent standard deviations). When the notches do not overlap, the medians are significantly different at ~95% confidence level. (D-E) Wnt2 conditioned media but not control conditioned media rescues plexus formation in mutant allantoises. (F-K) IWR1 inhibits plexus formation in control allantoises. (H) Quantification of number of ET interconnections formed in allantoises cultured starting EHF-LHF with or without IWR1. (K) Quantification of number of ET interconnections formed in allantoises cultured starting LHF- 1som with or without IWR1. Notched box plots (H,K) show significantly lower number of interconnections in the presence of IWR1 (bars represent ranges). Cultures were stained with anti-Flk1 antibody.

Figure 2.4 Genetic interactions between Tbx4 and Wnt2 in formation of the chorio-allantoic placenta. (A-B’) Placenta morphology of controls and Tbx4+/-;Wnt2+/- at 11.5. A’ and B’ are higher magnification views of the chorio-allantoic plate. Brackets delineate the extent of the

CAP. (C,D) Pecam staining of chorio-allantoic plate at E11.5. Arrowheads point to vessels. (E- F’) At E13.5, interdigitation of spongiotrophoblast, demarcated by dashed lines, and labyrinthine layers is observed in double heterozygote placentas, unlike controls. E’ and F’ are higher magnification views of boxed regions in E and F. (G,H) Pecam staining of E13.5 placentas. (I) Notched box plots show a significantly lower ratio of area of labyrinth/(area of spongiotrophoblast + labyrinth) in the double heterozygotes compared to controls at E13.5 (bars represent standard deviations). sp, spongiotrophoblast, lab, labyrinthine layer. Scale bars: 500µm.

Chapter 3. Multiple Roles and interactions of Tbx4 and Tbx5 in development of the respiratory system

Introduction

The development of the respiratory system represents an evolutionary hallmark that allowed vertebrates to survive on land utilizing air as a source of oxygen. Because the respiratory system is dispensable for embryonic survival in mammals, defects in development of the respiratory system manifest at or after birth. Indeed, abnormal development of the respiratory system in humans is associated with multiple disorders including but not limited to tracheal/bronchial atresia, TEF, bronchogenic cysts, pulmonary/lobar atresia and pulmonary hypoplasia (Whitsett et al., 2004). Thus, understanding the genetic basis of development of the respiratory system is important and this has been largely investigated using the mouse as a model.

Tbx4 and Tbx5 are the most closely related T-box genes with 94% homology in their T- box binding domain (Epstein et al., 2008). Although a role for these genes has not been analyzed in tissues that coexpress them – lung, trachea and genital tubercle – these genes can substitute for each other during limb formation suggesting similar roles in initiation and maintenance of limb outgrowth (Minguillon et al., 2005). Both Tbx4 and Tbx5 are expressed in the mesenchyme of the developing lung and trachea and although multiple genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. Thus, we investigated the roles of Tbx4 and Tbx5 in lung and trachea development. We studied three distinct processes important for development of the respiratory system, namely 1) lung bud and trachea specification, 2) lung branching morphogenesis, and 3) tracheal cartilage formation.

Materials and methods

Mouse strains, crosses and embryo collection

Mice carrying the following alleles were genotyped as previously described: a Tbx4 conditional

‘floxed’ allele, Tbx4tm1.2Pa (Naiche and Papaioannou, 2007b), hereafter referred to as Tbx4fl; a

Tbx5 conditional floxed allele, Tbx5tm1.2Jse (Mori et al., 2006), hereafter referred to as Tbx5fl; an

Fgf10 null allele (Min et al., 1998); ROSA26CRE-ERT2, a ubiquitous tamoxifen-inducible cre transgene (de Luca et al., 2005), hereafter referred to as CreER; Tbx4-cre, an insertion into the endogenous Tbx4 allele resulting in a bicistronic allele that expresses both cre and Tbx4 in all areas of Tbx4 expression including lung and trachea (Luria et al., 2008; Naiche et al., 2011), hereafter referred to as Tbx4cre; and a R26RlacZ reporter (Soriano, 1999). All lines of mice were kept on mixed genetic backgrounds. Embryos were dissected from timed matings and yolk sacs were removed for PCR genotyping. The dark period was 19.00 to 05.00 h and noon on the day a mating plug was identified as E0.5.

Tamoxifen injections

Tamoxifen (Sigma) at a concentration of 20 mg/ml in sunflower oil (Sigma) was administered to pregnant females by intraperitoneal injection between 15.30 and 19.30 hours on E8.5 or between

23.00 and 24.00 hours on E9.0.

In situ hybridization, immunohistochemistry and histology

Whole-mount ISH, immunohistochemistry (IHC) and ISH on cryosections was performed as described previously (Davis, 1993; Wilkinson and Nieto, 1993; Grieshammer et al., 2004).

Primary antibodies used were anti-PECAM (Pharmingen, catalog number 01951D) and anti-E- cadherin (Takara clone ECCD-2). All secondary antibodies were peroxidase-conjugated donkey

IgG from Jackson Immunochemicals.

For histology embryos were removed from the uterine horns, dissected out of the decidua and the embryos were fixed in Bouin’s fixative (Sigma). After dehydration in ethanol, embryos were embedded in paraffin wax, sectioned at 8 µm thickness and stained with hematoxylin and eosin

(H & E).

Alcian blue staining

Alcian blue staining was performed according to standard protocols (Nagy et al., 2009). Briefly, lungs and trachea were dissected out at different stages, fixed in Bouin’s fixative and washed with 70% ethanol. The tissue was then equilibrated in 5% acetic acid and stained with 0.05% alcian blue in 5% acetic acid for 2 hours. The tissue was washed in 5% acetic acid to remove excess stain and dehydrated in 100% methanol, cleared in BABB (benzyl alcohol, benzyl benzoate) and photographed. For alcian blue staining on sections, 8 µm paraffin sections were rehydrated, treated with 0.05% alcian blue in 5% acetic acid and then counterstained with nuclear fast red.

Foregut and lung bud culture

Foregut culture was carried out as described previously (Chen et al., 2010). Briefly, foreguts were isolated from 8-16 somite stage embryos using tungsten needles and cultured at 37oC in the presence of 95% air and 5% CO2 on Transwell-Col filters (Fisher Scientific), containing 1.5 ml

BGJb media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml vitamin

C (Sigma) and 2 µM 4-OH tamoxifen (Sigma). For lung bud culture, lung buds were dissected at E11.5 in phosphate buffered saline (PBS) with 0.1% bovine serum albumin (Sigma) and cultured in media containing DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen) and 1 µM 4-OH tamoxifen on 3.0 µm filters (Millipore) or

0.4 µm Transwell filters (Fisher Scientific). Similar results were obtained using both types of

67 filter; results reported are for experiments with Millipore filters. Where specified, Fgf10 (R&D) was added after 1 day of culture at a concentration of 500 ng/ml. In some experiments heparin beads coated with Fgf10 (100µg/ml) or BSA (100µg/ml) were placed near the branching tips of the explants after 1 day of culture. Transwell filters were used for the bead experiments.

Lung branching analysis

Lungs were stained with either E-cadherin antibody using IHC or E-cadherin RNA probe using

ISH. In case of whole mount lungs, different lobes were separated and photographed to count the number of branching tips. The cultured lungs were photographed and the branching tips were counted. For some experiments, after E-cadherin ISH, lungs were post fixed in 4% PFA, washed with PBT, dehydrated in 100% methanol, cleared in BABB and then photographed.

Results

Expression of Tbx4 and Tbx5 in the developing lung and trachea

Tbx5 expression is first detected using in situ hybridization (ISH) at E9.0 (24 somites) in the mesenchyme of the lung and trachea primordia, concurrent with Nkx2.1 expression in the ventral foregut epithelium (Figure 3.1A,B). Tbx4 expression is detected in the lung buds when they first appear a few hours later at E9.25 (28 somites) in a pattern similar to Tbx5 (Figure 3.1C, D).

Tbx4 and Tbx5 are expressed at E11.5, E13.5 and E15.5 throughout the lung mesenchyme but not the epithelium (Figure 3.1E-J) (Chapman et al., 1996).

Both Tbx4 and Tbx5 show a dynamic expression pattern in the developing trachea. At

E11.5 and E13.5 both genes are expressed throughout the tracheal mesenchyme surrounding the epithelium but are excluded from the epithelium and the outermost layer, the mesothelium

(Figure 3.1E,F,K,K’,L,L’). At these stages, Tbx4 and Tbx5 are expressed in ventral tracheal cells that also express Sox9 (Figure 3.1L,L’) (Elluru and Whitsett, 2004) and in dorsal tracheal cells

68 that also express SM22α (Figure 3.1M,M’) (Badri et al., 2008). Within the ventral mesenchyme at E15.5, Tbx4 and Tbx5 expression is restricted to mesenchyme between and surrounding cartilage condensations (Figure 3.1O,P) in a pattern complementary to that of Sox9, which is restricted to condensing mesenchyme of the cartilage rings at this stage (Figure 3.1Q).

Efficiency of recombination of conditional alleles

The genotypes of embryos used in this study and the corresponding descriptive shorthand nomenclature are shown in Table 3.1. In order to determine the efficiency of recombination of the conditional alleles by the CreER transgene, we used PCR genotyping. For embryos carrying the CreER transgene, a dose of 8 mg tamoxifen was injected into pregnant females at E9.0 and embryos were dissected at E12.5. Yolk sacs of these embryos were analyzed as an estimate of recombination in the whole embryo. Both alleles of Tbx4fl/fl embryos were completely recombined at E12.5 to produce the mutant allele at this dose of tamoxifen (Figure 3.2A), but the single floxed allele of Tbx5fl/+ embryos was only partially recombined to the mutant form (Figure

3.2E). Doses of tamoxifen higher than 8 mg at E9.0 lead to a loss of pregnancy. When 7 mg tamoxifen was injected at E8.5, complete recombination of the Tbx4fl alleles (Figure 3.2B) and the single Tbx5fl allele (Figure 3.2F) was obtained at E13.5.

In lung bud cultures a concentration of 1µM 4-OH tamoxifen produced near-complete recombination of the Tbx4fl allele after 24 hours (Figure 3.2C) whereas the Tbx5fl allele was only partially recombined (Figure 3.2G). Virtually complete recombination for both the Tbx4fl and the Tbx5fl alleles was achieved after 4 days of culture (Figure 3.2D,H). These data suggest that the Tbx5fl allele has a lower efficiency of Cre-mediated recombination than the Tbx4fl allele.

Thus we assume that there may be some residual Tbx5 activity from the Tbx5fl allele in the in vivo experiments, even in the presence of the CreER or Tbx4cre allele.

Early loss of Tbx5, but not Tbx4, leads to a unilateral loss of lung bud specification and absence of tracheal specification

To explore the role of Tbx4 and Tbx5 in the earliest stages of lung and trachea specification, foregut culture was used (Chen et al., 2010) with Nkx2.1 as a marker of specification. This ex vivo technique allows for analysis of mutants in culture, circumventing early embryonic lethality of the Tbx4 homozygous mutants due to allantois defects and Tbx5 homozygous mutants due to heart defects. When foreguts and surrounding tissue are isolated at E8.75 (8-16 somites), the foregut tube is devoid of Nkx2.1 expression and the lung buds are not present (Harris-Johnson et al., 2009). At the end of 3 or 4 days of culture the lung buds and trachea have formed as seen by

Nkx2.1 expression (Figure 3.3A,D,G). Expression of Tbx4 and Tbx5 was confirmed in control foreguts that were cultured for 4 days (Figure 3.3B,C). Foreguts from E8.5 embryos of various

Tbx4 and Tbx5 genotypes, with and without the CreER transgene were cultured in the presence of 4-hydroxyl (OH) tamoxifen and analyzed for Nkx2.1 expression. Reduction of Tbx5 alone lead to a lack of Nkx2.1 expression in one of the lung buds after 3 days of culture (Figure 3.3E) and 4 days of culture (Figure 3.3H) suggesting a unilateral loss of lung bud specification. Removal of

Tbx4 alone did not affect Nkx2.1 expression after 3 days of culture and removal of Tbx4 alleles in addition to Tbx5 alleles did not exacerbate the Tbx5 phenotype (Figure 3.3F,I). Therefore, Tbx5 but not Tbx4 is important for the bilateral specification of lung buds ex vivo.

With respect to tracheal specification, Nkx2.1 expression was not observed in the foregut tube after 3 or 4 days of culture in embryos lacking Tbx5 (arrowheads in Figure 3.3E,H) suggesting a lack of tracheal specification in the foregut tube in the absence of Tbx5. Additional loss of Tbx4 alleles did not alter the phenotype (Figure 3.3F,I), supporting a role for Tbx5 in the specification of the trachea, independent of Tbx4.

Lung branching is severely affected by the loss of Tbx4 and Tbx5

To analyze the effect of loss of Tbx4 and Tbx5 on lung branching in vivo we made use of the tamoxifen inducible CreER transgene. Tamoxifen was injected at E8.75 (8-16 somites), late enough to bypass lethality but well before lung branching begins. Embryos with CreER and different combinations of the Tbx4fl and Tbx5fl alleles were examined at E12.5 and E13.5.

Conditional Tbx4 null lungs were similar to controls at E12.5 but had fewer branching tips at

E13.5 (Figure 3.4A,B,E,F). Conditional Tbx4;Tbx5 double heterozygous lungs (Figure 3.4C) were smaller in size and had fewer branching tips than the conditional Tbx4 null lungs at E13.5

(Figure 3.4B) but were more advanced developmentally (Figure 3.4E,F) than the conditional

Tbx4 null;Tbx5 heterozygous lungs (Figure 3.4D), which were severely retarded in their development. Lobation in the right lung was disrupted in the conditional Tbx4 null;Tbx5 heterozygous lungs: the accessory lobe was missing and only rudimentary cranial and medial lobes were present (Figure 3.4H and Figure 3.7A,B). The lobes had a fused appearance (Figure

3.4H’) suggesting a failure of separation. When these mutant lungs were sectioned and compared with controls, there were no obvious structural defects observed other than an overall reduction in size (Figure 3.4G,G’,H,H’).

The conditional Tbx4 null;Tbx5 heterozygous mutants could not be analyzed later than

E13.5, due to hematopoietic defects throughout the embryo because of Cre-induced apoptosis

(Naiche and Papaioannou, 2007a) and it was not possible to analyze the conditional Tbx5 null embryos using the inducible CreER in vivo due to lethal heart defects. To circumvent these limitations, we used the Tbx4cre allele, which is expressed in the lung and trachea but not in the heart (Luria et al., 2008). From this allele, Cre is expressed in the majority of cells of the developing lung and trachea mesenchyme, as seen with a lacZ reporter expression at E13.5

(Figure 3.4I,J) (Naiche et al., 2011).

Lung-specific Tbx5 null mutants, carrying a single copy of Tbx4cre, showed a range of phenotypes from an apparently normal lung to a severe decrease in lung size. We hypothesize that this variability in phenotype is due to a variable extent of recombination of the Tbx5fl allele

(Figure 3.2). All lung-specific Tbx4 heterozygous;Tbx5 null pups became cyanotic at birth and died shortly thereafter due to respiratory distress. Unlike the variable lung size in the lung- specific Tbx5 null mutants, the lungs of these mutants were consistently smaller than the controls at E13.5 (Figure 3.4K,L) and at birth (Figure 3.4N,O) and had significantly fewer branching tips compared to controls at E13.5 (Figure 3.4M). At P0 the lung histology of the lung-specific Tbx4 heterozygous;Tbx5 null lungs is comparable to controls although the mutant lungs and trachea show accumulation of a mucus like substance (Figure 3.4P,Q). These lungs show lobation defects very similar to the conditional Tbx4 null;Tbx5 heterozygous mutant lungs. The accessory lobe was absent in most embryos but when present showed less branching; the cranial and caudal lobes also showed decreased branching. The cranial, medial and caudal lobes had a fused appearance (Figure 3.5A,B). In addition to having fewer branches these lungs also showed reduction in lateral growth of the branching lung lobes. Although tertiary dorsal branches were present, they were crowded together and the secondary lateral branches had outgrown a shorter distance compared to controls (Figure 3.5C,D)

Using Tbx4cre, it is not possible to study the conditional double nulls as Tbx4 is also expressed from this allele. Thus, to further explore branching morphogenesis in conditions where both alleles of Tbx4 and Tbx5 could be deleted, we made use of a lung bud culture system in which lung buds from embryos with or without the CreER transgene were cultured in the presence of 4-OH tamoxifen (Figure 3.6). Conditional Tbx4 null;Tbx5 heterozygous, or conditional Tbx4 heterozygous;Tbx5 null lung buds showed reduced branching (Figure

3.6A,B,C) consistent with the reduced number of branching tips observed in vivo at E13.5. The conditional double null lungs showed a complete branching arrest by 3 days of culture (Figure

3.6D,E,F). The existing branches continued to elongate as seen at 4 days of culture (arrow in

Figure 3.6E). Therefore, Tbx4 and Tbx5 are essential for continuing branching morphogenesis ex vivo.

Loss of Tbx4 and Tbx5 affects the Fgf10 signaling pathway and Wnt2 expression

Expression of the lung mesenchymal marker Fgf10 as well as epithelial targets of the Fgf10 signaling pathway, Bmp4, Spry2 and Etv5 (Weaver et al., 2000; Firnberg and Neubuser, 2002;

Horowitz and Simons, 2008), was analyzed in lungs with reduced Tbx4 and Tbx5 expression.

Fgf10 is expressed in mesenchyme surrounding the distal epithelial tips that mark the site of future bud formation (Figure 3.7A) (Bellusci et al., 1997). Consistent with the smaller overall lung size, there were fewer foci of Fgf10 expression in the conditional Tbx4 null;Tbx5 heterozygous lungs (Figure 3.7B) and in the lung-specific Tbx4 heterozygous;Tbx5 nulls (Figure

3.7C). The primary receptor for this pathway, Fgfr2 (Arman et al., 1999), is expressed normally in the epithelium of lung-specific Tbx4 heterozygous;Tbx5 null lungs (Figure 3.7D,E). Bmp4

(Figure 3.7F,G,H) and Spry2 (Figure 3.7I,J) were downregulated in the Tbx4 and Tbx5-deficient lungs but Etv5 (Figure 3.7N,O) expression was not affected, although it was drastically reduced in the conditional double nulls cultured ex vivo (see Figure 3.8J,K).

Wnt2, which is normally expressed in the developing lung mesenchyme (Figure 3.7K)

(Goss et al., 2009), was greatly reduced at E13.5 in conditional Tbx4 null;Tbx5 heterozygous

(Figure 3.7L) and lung-specific Tbx4 heterozygous;Tbx5 null lungs (Figure 3.7M). In addition to

Fgf10 and canonical Wnt2 signaling pathways, the Shh signaling pathway has also been implicated in the process of branching morphogenesis in the lung (Pepicelli et al., 1998).

Epithelial Shh (Figure 3.7S,T) and its mesenchymal receptor Ptc (Figure 3.7U,V) showed normal expression in the lung-specific Tbx4 heterozygous;Tbx5 null lungs. The epithelial marker Nkx2.1 which is necessary for lung branching (Kimura et al., 1996) and showed unilateral expression in the conditional Tbx5 null foreguts at the time of specification (Figure 2), showed expression similar to controls at E13.5 in the lung-specific Tbx4 heterozygous;Tbx5 null lungs (Figure

3.7W,X). At E13.5, expression of the vascular marker Pecam indicated normal development of vessels around the individual bronchioles of the Tbx4 and Tbx5-deficient mutants (Figure

3.7P,Q,R).

Tbx4 and Tbx5 interact with Fgf10 during lung development

Since mesenchymal Fgf10 expression and expression of epithelial targets was affected in mutants with reduced Tbx4 and Tbx5, we investigated the interactions between the Tbx4, Tbx5 and the Fgf10 signaling pathway during branching morhphogenesis using double and triple heterozygotus mutants. Tbx4;Tbx5 double heterozygous lungs were smaller than control lungs at

E18.5 (Figure 3.8A,B). The Tbx4;Tbx5;Fgf10 triple heterozygous lungs were smaller than the

Tbx4;Tbx5 double heterozygous lungs (Figure 3.8B,C), suggesting that Tbx4 and Tbx5 genetically interact with the Fgf10 signaling pathway during lung development. Removing one copy of Fgf10 from the lung-specific Tbx4 heterozygous;Tbx5 nulls did not reduce lung size further (Figure 3.8D,E).

Fgf10 genetically interacts with Tbx4 and Tbx5; loss of Tbx4 and Tbx5 leads to downregulation of Fgf10 and of genes activated downstream of the Fgf10 signaling pathway in the epithelium (Figure 3.7). However, the addition of exogenous Fgf10 to lung bud cultures of conditional double nulls did not rescue branching (Figure 3.8F-I). The lack of change in Etv5 expression, an Fgf10 target, indicated that the Fgf10 signaling pathway was not activated in the

74 presence of exogenous Fgf10 in the conditional double null lungs (Figure 3.8J-M). To ensure that the Fgf10 used for the rescue experiments was active, Fgf10 coated heparin beads were placed near the branching tips of lung explants. The tips of both control and conditional double null lungs swelled up in response to Fgf10 beads but not BSA coated heparin beads (Figure

3.8N,O) (Izvolsky et al., 2003b). Since Fgfr2 is expressed normally in the Tbx4 and Tbx5- deficient lungs (Figure 3.8E) and the conditional double null lungs, it is not surprising that the conditional double null lung tips can respond to Fgf10. However the conditional double null lungs fail to undergo branching in the presence of Fgf10. Thus, in addition to Fgf10 there must be other factors under the control of Tbx4 and Tbx5 important for activation of the Fgf10 signaling pathway leading to branching morphogenesis.

Loss of Tbx4 and Tbx5 causes tracheal-bronchial ring defects

To analyze the development of the tracheal-bronchial cartilage in embryos with reduced Tbx4 and Tbx5, cartilage rings were visualized at birth using alcian blue staining. The lung-specific

Tbx5 nulls and lung-specific Tbx4 heterozygous;Tbx5 null embryos had defective cartilage ring development (Figure 3.9A-C). There were some normal rings (arrows in Figure 3.9B,C) and isolated foci of cartilage (black arrowheads in Figure 3.9B,C). The tracheal and bronchial lumen of newborn pups was expanded in the controls (Figure 3.9D,E) but collapsed in lung-specific

Tbx4 heterozygous;Tbx5 nulls (Figure 3.9F,G). To assess the development of cartilage rings at earlier stages, Sox9 expression was analyzed at E12.5 and E13.5 in lung-specific Tbx4 heterozygous;Tbx5 nulls. At E12.5, these mutant tracheas have Sox9 expression on the ventral aspect of the trachea similar to the controls (Figure 3.9H,I) but fail to undergo mesenchymal condensations at E13.5 (Figure 3.9J,K). Expression of the genes downstream of Sox9, Sox6

(Figure 3.9L,M) and Sox5 (Figure 3.9N,O), was downregulated at E13.5 whereas Col2α1,

75 another Sox9 target, was expressed at apparently normal levels in those rings that were present

(Figure 3.9P,Q). The smooth muscle marker SM22α was analyzed to assess the development of the dorsal trachealis muscle. SM22α was expressed in an expanded domain and there was a loss of the characteristic banding pattern in the mutant tracheas (Figure 3.9R,S) indicating a disruption in smooth muscle formation due to loss of Tbx4 and Tbx5.

Tbx4 and Tbx5 do not interact with Fgf10 during trachea development

Tracheas of controls (Figure 3.10A), Tbx4;Fgf10 double heterozygotes (Figure 3.10B) and

Tbx5;Fgf10 double heterozygotes (Figure 3.10C) showed a normal pattern of 10-11 cartilage rings, suggesting a lack of genetic interactions between Tbx4 or Tbx5 and Fgf10 in trachea formation. Fgf10 null mutants do not form lungs but have a truncated trachea with 6-8 cartilage rings, some of which are aberrantly formed (Sala et al., 2011) (Figure 3.10D). Tbx4;Tbx5 double heterozygous mice also have tracheas with 6-8 cartilage rings but also have main stem bronchial cartilage rings (Figure 3.10E). Removing a copy of Fgf10 in these double heterozygotes did not alter the phenotype (Figure 3.10F) showing an Fgf10-independent role for Tbx4 and Tbx5 in the formation of tracheal-bronchial cartilage rings. Also, removing a copy of Fgf10 in the lung- specific Tbx4 heterozygous;Tbx5 nulls did not alter the phenotype of the tracheal-bronchial cartilage rings (Figure 3.10G,H). Thus, Tbx4 and Tbx5 affect lung development via control of

Fgf10 expression but affect tracheal-bronchial cartilage development independently of the Fgf10 signaling pathway.

Discussion

Loss of Tbx5 affects specification of the lung buds and trachea

Tbx5 is expressed around the lung/trachea primordia at the same time that Nkx2.1, a marker of specification, is first expressed in the primordia of the foregut endoderm. Tbx4 is expressed

76 slightly later at the time of lung bud formation. In our study, loss of Tbx5, but not Tbx4, leads to unilateral loss of lung bud formation, indicating that Tbx5 is important for lung bud specification.

In contrast, in the chick, although ectopic expression of Tbx4 in the esophagus can specify lung fate, expression of a dominant negative form of Tbx4 leads to a lack of primary budding in only a third of the mutants analyzed (Sakiyama et al., 2003). However, the dominant negative Tbx4 could also be affecting the expression of Tbx5 targets as the repressor construct utilized the complete T-box domain and Tbx4 and Tbx5 have 94% amino acid identity in their T-box domains (Epstein et al., 2008). This interpretation is compatible with our hypothesis that Tbx5 plays a more important role in vertebrate lung primordia specification than Tbx4 (Figure 3.11A).

Loss of Tbx5 alone leads to a lack of tracheal specification ex vivo, a phenotype similar to that of mice mutant for Bmpr1 and Bmpr2. In these mutants, although lung bud specification occurs, the ventral foregut fails to acquire tracheal identity (Domyan et al., 2011). Thus, Tbx5 either acts upstream of or in parallel with the BMP signaling pathway in specification of the foregut into a trachea (Figure 3.11A).

Tbx4 and Tbx5 interactions in lung development

At the next stage of lung development, in lung branching morphogenesis, Tbx4 and Tbx5 genetically interact with one another. Although, neither Tbx5 nor Tbx4 single heterozygous lungs show a branching defect, double heterozygous lungs are smaller at E13.5 and E18.5 with a reduced number of branching tips at E13.5. Because these genes are closely related (Epstein et al., 2008), they could potentially activate the same targets by binding to a similar T-box binding element, independent of one another. Alternatively, Tbx4 and Tbx5 could physically interact with each other as heterodimers to activate and/or repress transcription of downstream targets, as has been suggested for other T-box genes (Goering et al., 2003).

Further reduction of Tbx4 or Tbx5 in conditional Tbx4 nulls;Tbx5 heterozygous and lung- specific Tbx4 heterozygous;Tbx5 null lungs leads to a severe reduction in branching, failure of lobe separation and loss of lung lobe specification, while absence of Tbx4 and Tbx5 leads to a branching arrest. These results are in line with the published observations that antisense RNAs used against both Tbx4 and Tbx5 inhibit lung branching in culture and that this effect is explained by loss of Fgf10 expression (Cebra-Thomas et al., 2003). The drastic effect on lung branching in our study is explained by loss of expression of the important regulatory genes Fgf10 and Wnt2 in the lung mesenchyme. The Fgf10 targets Bmp4 and Spry2 show very low levels of expression in the epithelium of Tbx4 and Tbx5-deficient mutants and Etv5, another Fgf10 target, shows greatly reduced expression in the conditional double null lungs, leading to the hypothesis that the Fgf10 signaling pathway is activated downstream of Tbx4 and Tbx5 in the developing lung and that Fgf10 genetically interacts with Tbx4 and Tbx5. Lungs that are triple heterozygous for Tbx4, Tbx5 and Fgf10 are reduced in size compared to Tbx4;Tbx5 double heterozygous lungs, which supports this hypothesis. Lung-specific Tbx4 heterozygote;Tbx5 null lungs are severely retarded but lung size is not affected by further loss of Fgf10, demonstrating epistasis and supporting the hypothesis that Fgf10 lies downstream of Tbx4 and Tbx5. Lack of rescue of branch formation and lack of activation of the Fgf10 signaling pathway by exogenously supplied

Fgf10 in culture suggests that there are other factors downstream of Tbx4 and Tbx5 that affect

Fgf10 signaling. Although, lungs deficient in Tbx4 and Tbx5 retain the ability to respond to

Fgf10 coated beads (our study and (Cebra-Thomas et al., 2003)), they fail to activate additional factors necessary for branching morphogenesis (Figure 3.11B).

One possibility for such a factor is a mesenchymal signaling molecule that communicates with the epithelium and activates downstream target(s) required for the activation of the Fgf10

78 pathway. We examined expression of Ptc and Shh to determine whether the Shh pathway was involved, but Shh signaling is not affected in the lung-specific Tbx4 heterozygous;Tbx5 nulls or in the conditional double null cultures. The extracellular matrix (ECM) molecules, heparan sulfate (HS) proteoglycans aid in Fgf10-Fgfr2 interactions during lung development (Izvolsky et al., 2003b) and hence are good candidates for factors missing in the Tbx4 and Tbx5-deficient mutants. Inhibition of heparanase decreases submandibular gland morphogenesis in culture due to deficiency of Fgf signaling (Patel et al., 2007). Additionaly, HS-deficient null mouse embryos fail to respond to Fgf signaling and the spatiotemporal expression of cell surface-tethered HS chains regulates the local reception of Fgf-signaling activity during embryonic development

(Shimokawa et al., 2011). Tbx4 regulates ECM molecules in the developing allantois, specifically the chondroitin sulfate proteoglycan versican (Fig. 2.2), suggesting that ECM might be one of the targets for T-box genes in regulating development of other organs as well.

Tbx4 and Tbx5 interactions in tracheal-bronchial cartilage development

Appropriate dorsal smooth muscle development and ventral tracheal-bronchial cartilage development is important for the normal functioning of the trachea. Smooth muscle provides tracheal flexibility and the cartilaginous rings prevent tracheal collapse. Defects in trachea formation may result in tracheomalacia or tracheal stenosis. Reduction of Tbx4 and Tbx5 causes defects in both formation of the tracheal cartilage rings and development of the trachealis smooth muscle indicating interactions between Tbx4 and Tbx5. Tbx4 heterozygous and Tbx5 heterozygous tracheas have 10-11 cartilage rings but Tbx4;Tbx5 double heterozygous tracheas have 6-8 cartilage rings, some of which are incomplete. Additional reduction of Tbx4 and Tbx5 in the lung-specific Tbx4 heterozygous;Tbx5 null lungs leads to a complete disruption of

79 cartilage ring formation further supporting genetic interactions between Tbx4 and Tbx5 in trachea formation.

Sox9, a master regulator of the process of chondrogenesis is expressed normally at E12.5 throughout the ventral mesenchyme in lung-specific Tbx4 heterozygous;Tbx5 null tracheas but at

E13.5 these tracheas show a lack of characteristic mesenchymal condensations and reduced Sox9 expression. Either Tbx4 and Tbx5 control of Sox9 expression becomes more sensitive to dosage at E13.5 or Tbx4 and Tbx5 control another factor, possibly an ECM molecule, which is important for formation of mesenchymal condensations and, in the absence of these condensations, there is a down regulation of Sox9 expression (Figure 3.11C). Expression of Sox5 and Sox6, genes genetically downstream of Sox9 and important for condensation and cartilage formation, is downregulated at E13.5, concordant with an aberration in the process of chondrogenesis.

While Tbx4 and Tbx5 regulate lung branching by controlling Fgf10 signaling, their control of tracheal-bronchial cartilage formation is independent of the Fgf10 signaling pathway.

Fgf10 homozygous mutants have 6-8 tracheal cartilage rings, although the spacing between them is reduced and the rings do not always form the characteristic C shape (Min et al., 1998; Sekine et al., 1999; Sala et al., 2011). Neither Tbx4;Fgf10 double heterozygous tracheas nor

Tbx5;Fgf10 double heterozygous tracheas show cartilage condensation defects or a reduction in the number of cartilage rings present. In contrast, Tbx4;Tbx5 double heterozygotes show a shorter trachea with 6-8 tracheal cartilage rings suggesting Tbx4 and Tbx5 interact with each other during trachea formation but do not interact with Fgf10. The triple heterozygous tracheas do not show an exacerbation of the Tbx4;Tbx5 double heterozygous trachea phenotype.

Additionally, aberrant Fgf10 signaling has been shown to affect tracheal cartilage formation and loss of Fgf10 affects Shh expression but not Sox9 expression (Sala et al., 2011). In contrast, in

80 the Tbx4 and Tbx5-deficient mutants Shh expression appears to be unaffected whereas Sox9 expression is reduced at E13.5. Hence, Tbx4 and Tbx5 control tracheal-bronchial cartilage formation via Sox9, independent of the Fgf10 signaling pathway.

Figure 3.1 Expression of Tbx4 and Tbx5 in the developing lung and trachea. (A-J) Tbx4 and Tbx5 expression analyzed using ISH on lungs. Tbx5 is first expressed at E9.0 (B) when the specification of lung primordia occurs, as seen by Nkx2.1 expression (A). Tbx4 is first expressed at E9.5 along with Tbx5 in the newly formed lung buds (C,D). Expression is seen in lung whole mounts at E11.5 (E,F), E13.5 (G,H) and in lung mesenchyme in cryosections at E15.5 (I,J). (K- Q) Tbx4 and Tbx5 expression analyzed using ISH on tracheas. Tbx4 and Tbx5 are expressed throughout tracheal mesenchyme (m) at E13.5 (K,K’,L,L’) but not in the epithelium (e) or the mesothelium (arrowheads). Caudal view of cut tracheas after whole mount ISH (K-N). ISH on cryosections (K’-N’). At E13.5, Sox9 is expressed in the mesenchyme on the ventral side

(M,M’) and SM22α is expressed on the dorsal side (N,N’) of the trachea. D-dorsal; V-ventral; R- right; L-left. At E15.5, Tbx4 and Tbx5 are expressed around the condensing cartilage mesenchyme and in the intercartilage mesenchyme (O,P). Sox9 is expressed in the condensing cartilage rings (Q). Asterisk indicates areas of cartilage condensation. Inset in (O) and (Q) shows ISH on E15.5 sagittal cryosections with Tbx4 and Sox9 probe respectively. Scale bars represent 50 µm.

Figure 3.2 Analysis of excision efficiency of Tbx4fl and Tbx5fl. Females carrying embryos with Tbx4fl, Tbx5fl and CreER alleles were injected with either 8mg tamoxifen at E9.0 and dissected at E12.5 (A,E) or 7mg tamoxifen at E8.5 and dissected at E13.5 (B,F). Tbx4fl/fl was completely excised to mutant Tbx4-/- at both doses and times (A,B) whereas the single conditional allele of Tbx5fl/+ was incompletely excised by injection at E9.0 (E) but completely excised by injection at E8.5 (F). In addition when lungs with the genotype Tbx4fl/fl; Tbx5fl/fl; CreER were treated with 1µm tamoxifen in culture, the Tbx4 locus was nearly completely excised at the end of 1 day of culture (C) but the Tbx5 locus was only partially excised (G). At the end of a 4 day culture both Tbx4 (D) and Tbx5 (H) loci achieved virtually complete excision. fl, floxed conditional PCR band; WT, wild type PCR band; Mut, excised PCR band.

Figure 3.3 Early loss of Tbx5 leads to aberrant lung bud and trachea specification. (A-I) Foreguts were isolated between 8-16 somite stages and analyzed by ISH following culture. Nkx2.1 (A), Tbx4 (B) and Tbx5 (C) expression in lung buds (arrows) and tracheal primordia (yellow arrowhead) was confirmed in control cultures. Recombination of Tbx5 using 4-OH tamoxifen in conditional null foreguts with CreER leads to the loss of Nkx2.1 expression in one of the lung buds at 3 (E) or 4 days (H). Conditional double nulls (F,I) carrying CreER show a phenotype similar to the conditional Tbx5 nulls, suggesting additional removal of Tbx4 does not exacerbate the phenotype. Nkx2.1 expression was absent in the foregut tube of conditional Tbx5 null and conditional double null foreguts after 3 or 4 days of culture (yellow arrowheads in E,F,H,I) compared to controls (D,G).

Figure 3.4 Loss of Tbx4 and Tbx5 causes reduced lung branching and lethality at birth. (A-H’) Tbx4fl and Tbx5fl alleles were excised using CreER by injecting tamoxifen at E8.75 and lungs were dissected at different time points. Loss of Tbx4 alone or both Tbx4 and Tbx5 leads to lung hypoplasia at E13.5 (A-D). The number of branching tips was quantitated following E- cadherin staining for different genotypes at E12.5 (E) and E13.5 (F). H & E on histological sections shows a decrease in lung size at E12.5 in the conditional Tbx4 null;Tbx5 heterozygous

86 lungs (G,H,). (G’) and (H’) are magnified views of the boxed regions in (G) and (H) respectively. Black arrowheads indicate separation between the lobes of the right lung in the control (G’) and lack of separation in the mutants (H’). (I-Q) Tbx4cre was utilized for lung and trachea-specific excision of Tbx4fl and Tbx5fl alleles. Tbx4cre was expressed in most of the trachea mesenchyme (I) and lung mesenchymal cells (J) as seen by a R26RlacZ reporter expression at E13.5. Lung-specific Tbx4 heterozygous;Tbx5 null lungs show hypoplasia at E13.5 (K,L) and at birth (N,O). The number of branching tips in control lungs and the lung- specific Tbx4 heterozygous;Tbx5 null lungs, labeled as experimental in the box plot (M), are significantly different at E13.5. H & E staining on sections shows lung morphology for control (P) and mutants (Q) at birth. Scale bars represent 100 µm.

Figure 3.5 Loss of Tbx4 and Tbx5 has multiple affects on branching morphogenesis. (A,B) H & E sections of lung specific Tbx4 heterozygous;Tbx5 null lungs (B) show lack of separation of the cranial (cr), medial (m) and caudal (cd) lobes in the right lung as compared to control lungs (A). Black arrowheads point to the space between the lobes in the control lungs (A) and to the corresponding regions of the mutant lungs (B). (C,D) Control lungs stained for E-cadherin at E13 show greater outgrowth (yellow brackets) of lateral branches (C) than the lung specific Tbx4 heterozygous;Tbx5 null lungs (D). Scale bars represent 100 µm.

Figure 3.6 Loss of Tbx4 and Tbx5 leads to branching arrest ex vivo. Lung buds were isolated at E11.5 and cultured in the presence of 4-OH tamoxifen. Airways were visualized using E- cadherin ISH. Conditional Tbx4 homozygous;Tbx5 heterozygous (B) and conditional Tbx4 heterozygous;Tbx5 null (C) lungs with CreER showed reduced branching after 4 days of culture compared to controls (A). Control, without CreER, (D) and conditional double null lung buds, with CreER, (E) were cultured for 4 days and photographed at 1 day, 2 days, 3 days and 4 days. Arrow in E shows the progression of an elongating airway. A plot of the number of branching tips as a function of time for controls and the conditional double nulls (F), shows a branching arrest of the conditional double null lungs after 2 days of culture.

Figure 3.7 Loss of Tbx4 and Tbx5 affects the Fgf10 signaling pathway and Wnt2 expression. (A-X) Marker analysis of control and Tbx4 and Tbx5-deficient lungs using whole mount ISH (A- O, S-X) and IHC (P-R): Fewer foci of Fgf10 expression were seen in the Tbx4 and Tbx5- deficient lungs (B,C) compared to control (A). cr, cranial; m, medial; cd, caudal; a, accesory lobes. Fgf10 target genes Bmp4 (F,G,H) and Spry2 (I,J) and canonical Wnt2 (K,L,M) were downregulated in Tbx4 and Tbx5-deficient lungs compared to controls. Fgfr2 (D,E), Etv5 (N,O), Pecam (P,Q,R), Shh (S,T), Ptc (U,V) and Nkx2.1 (W,X) were expressed similarly in controls and Tbx4 and Tbx5-deficient lungs.

Figure 3.8 Tbx4 and Tbx5 interact with Fgf10 but FGF10 fails to rescue Tbx4 and Tbx5- deficient lungs. (A-E) Lungs of different genotypes at E18.5. Tbx4;Tbx5 double heterozygous lungs (B) are smaller than control lungs (A). Tbx4;Tbx5;Fgf10 triple heterozygous lungs (C) are smaller than the double heterozygous lungs (B) but comparable in size to the lung-specific Tbx4 heterozyous;Tbx5 null lungs (D). Removing an additional copy of Fgf10 from the lung-specific Tbx4 heterozyous;Tbx5 null lungs does not further affect lung size (E). ht, heart. (F-I) E-cad expression in epithelium of the control lungs and conditional double null lungs in the absence (F,G) and presence (H,I) of exogeneous Fgf10 showing a lack of rescue of branching morphogenesis. (J-M) Etv5 expression in control lungs and conditional double null lungs in the absence (J,K) and presence (L,M) of exogeneous Fgf10. Etv5 expression remains unchanged after addition of Fgf10. (N,O) Both control and conditional double null lungs respond to FGF10 coated beads (f) but not to BSA coated beads (b) as seen by swelling of tips (arrows) in proximity to the FGF10 bead.

Figure 3.9 Loss of Tbx4 and Tbx5 disrupts tracheal-bronchial cartilage and smooth muscle formation. (A-G) Alcian blue staining was used to visualize tracheal-bronchial cartilage. Ten- eleven cartilage rings were seen in control tracheas (E18.5, A); normal (arrow) and incomplete rings were seen in tracheas and bronchi (black and red arrowheads respectively) of lung-specific Tbx5 null (E18.5, B) and lung-specific Tbx4 heterozygous;Tbx5 nulls (post natal day (P)0, C). Asterisk in (A-C) is the point of bronchial separation from the trachea. Alcian blue on P0 sections showed that the control trachea (D) and main stem bronchial lumen (E) were expanded and the chondrocytes were present as C shaped rings (arrowheads). The lung-specific Tbx4 heterozygous;Tbx5 null tracheal lumen (F) and main stem bronchial lumen (G) were collapsed and filled with a mucus like substance (arrows) and alcian blue positive foci were seen (arrowheads). TL, Tracheal lumen; RBL, Right bronchial lumen; LBL, Left bronchial lumen. (H-S) Genes important for chondrogenesis and smooth muscle development. Sox9 expression at E12.5 showed a comparable ventral expression pattern in control (H) and lung-specific Tbx4 heterozygous;Tbx5 null tracheas (I). At E13.5, Sox9 positive cells begin to condense and appear in a ring like pattern in controls (J) but not in the mutant tracheas (K). Sox6 (L,M) and Sox5 (N,O) expression was downregulated in the lung-specific Tbx4 heterozygous;Tbx5 null tracheas at E13.5. Col21 was expressed in its characteristic ring like pattern (P) similar to Sox9 in the control tracheas but there were fewer rings with apparently normal expression in the lung-

92 specific Tbx4 heterozygous;Tbx5 null tracheas (Q). SM22α expression was analyzed on the dorsal trachea at E13.5 in the control (R) and in the lung-specific Tbx4 heterozygous;Tbx5 null tracheas (S). Arrowheads in (S) point to ectopic expression in an uncharacteristic pattern in the mutants. Scale bars represent 100 µm.

Figure 3.10 Lack of genetic interactions between Tbx4, Tbx5 and Fgf10 in trachea formation. Alcian blue staining was used to analyze tracheal-bronchial cartilage development in tracheas from different genotypes at E18.5. Control tracheas (A), Tbx4;Fgf10 double heterozygous tracheas (B) and Tbx5;Fgf10 double heterozygous tracheas (C) have 10-11 C- shaped, ventral tracheal cartilage rings and the trachea forms the two main stem bronchi which have lateral C-shaped cartilage rings. Fgf10 homozygous mutants (D), Tbx4;Tbx5 double heterozygous mutants (E) and Tbx4;Tbx5;Fgf10 triple heterozygous mutants (F) each form fewer (6-8) tracheal cartilage rings, some irregularly shaped or incomplete (arrowheads). Additionally, Fgf10 mutants lack bronchi and hence any bronchial cartilage rings, but the double and triple heterozygotes form normal lateral bronchial cartilage rings (arrows in E and F). Lung-specific Tbx4 heterozygous;Tbx5 nulls (G) show severe disruptions in formation of tracheal-bronchial cartilage rings, a phenotype that is unchanged with the removal of an Fgf10 allele (H). Asterisk shows the point of bronchial separation from the trachea.

Figure 3.11 Model for the role of Tbx4 and Tbx5 in lung and trachea development. (A) Lung and trachea specification begins at E9.0 in the ventral foregut and at this time Tbx5 expression (light purple) is adjacent to the presumptive endoderm. Later, Tbx4 and Tbx5 expression (dark purple) is in mesenchyme associated with the lung and trachea. Tbx5 but not Tbx4 is important for specification of bilateral lung buds and the trachea. (B) Magnification of box shown in (A) representing the events in the growing tip during branching morphogenesis. Grey denotes epithelium and purple denotes mesenchyme. Tbx4 and Tbx5 interact with each other and act upstream of the Fgf10 signaling pathway. Decrease in Tbx4 and Tbx5 affects mesenchymal Fgf10 expression and expression of its targets in the epithelium – Bmp4, Spry2 and Etv5 – but not the expression of the epithelial Fgf10 receptor Fgfr2. In addition to Fgf10 expression in the mesenchyme, Tbx4 and Tbx5 also control the expression of an unknown factor (X) that is essential for activation of the Fgf10 signaling pathway. Furthermore, Tbx4 and Tbx5 act upstream of Wnt2 in the mesenchyme. (C) In the trachea and the main stem bronchi Tbx4 and Tbx5 either control Sox9 expression, which in turn regulates cartilage condensation, or Tbx4 and Tbx5 regulate another factor (X) essential for chondrogenesis secondarily affecting Sox9 expression.

Table 3.1 Different allelic combinations and the descriptive nomenclature used in this chapter.

Genotype Nomenclature

Tbx4fl/fl;Tbx5+/+; CreER Conditional Tbx4 null

Tbx4+/+;Tbx5fl/fl; CreER Conditional Tbx5 null

Tbx4fl/+;Tbx5fl/+; CreER Conditional Tbx4;Tbx5 double heterozygous

Tbx4fl/fl;Tbx5fl/+; CreER Conditional Tbx4 null;Tbx5 heterozygous

Tbx4fl/fl;Tbx5fl/fl; CreER Conditional double null

Tbx4cre/+;Tbx5fl/fl Lung-specific Tbx5 null

Tbx4cre/fl;Tbx5fl/fl Lung-specific Tbx4 heterozygous;Tbx5 null

1Tbx4cre/-;Tbx5fl/fl

1Tbx4cre/fl;Tbx5fl/-

1Tbx4- and Tbx5- alleles are generated by recombination of the floxed alleles in the germ line due to germ line expression of the Tbx4cre allele (Douglas et al., 2011; Naiche et al., 2011)

Chapter 4. T-box gene function in organogenesis

Our studies on the allantois and the respiratory system suggest common themes for T-box function.

T-box genes lie upstream of Fgf signaling

T-box genes have been implicated upstream of Fgf signaling in different organs. Fgf8 interacts genetically with Tbx1 during pharyngeal endoderm, mesoderm and aortic arch development

(Vitelli et al., 2002b; Mesbah et al., 2012). Fgf8 expression is upregulated in the secondary heart field and the outflow tract of Tbx2-/-;Tbx3-/- double mutants suggesting that these T-box genes repress Fgf8 expression during heart development (Mesbah et al., 2012). In Tbx4 mutant hindlimbs Fgf10 expression is initiated but not maintained (Naiche and Papaioannou, 2003). In

Tbx5 mutant forelimbs expression of Fgf8, Fgf10 and a downstream target of Fgf10 signaling,

Pea3, is not present at initiation (Agarwal et al., 2003) nor during later outgrowth (Rallis et al.,

2003). A similar role for Tbx5 upstream of Fgf10 has been suggested in pectoral fin development in zebrafish (Ng et al., 2002) and in limb development in the chick (Takeuchi et al.,

2003). Further the Fgf10 promoter has a conserved T-box binding site which can be activated in vitro by TBX5 protein (Agarwal et al., 2003). This suggests that Fgf10 is a direct target of Tbx5 and potentially of Tbx4 since both these proteins have a highly homologous T-box amino acid domain. In the chick lung bud ectopic expression of Tbx4 leads to ectopic expression of Fgf10 and expression of a dominant negative construct of Tbx4 leads to reduced Fgf10 expression in the lung bud mesenchyme (Sakiyama et al., 2003). Further, when lung bud cultures are treated with both Tbx4 and Tbx5 antisense RNA, Fgf10 expression is reduced in the branching lung

(Cebra-Thomas et al., 2003). Our in vivo studies in the lung are in line with this role of T-box

97 genes, where upon reduction in both Tbx4 and Tbx5, Fgf10 expression in the mesenchyme and

Fgf10 target expression in the epithelium – Bmp4 and Spry2 is greatly reduced. We also see that

Tbx4, Tbx5 and Fgf10 interact in lung branching morphogenesis by means of triple heterozygous mutants. In contrast, it is important to note that although regulation of Fgf10 expression by T- box genes is a common theme, this does not happen in all developing organs. In the developing trachea Fgf10 does not interact with Tbx4 or Tbx5 although aberrant Fgf10 expression by itself affects tracheal cartilage formation.

T-box genes interact with canonical Wnt signaling

Another signaling pathway that lies downstream of T-box genes or interacts with them is the canonical Wnt signaling pathway. In zebrafish, both brachyury homologs no tail and brachyury are essential and sufficient for expression of canonical wnt3a and wnt8 in the mesoderm progenitors for formation of the posterior somites (Martin and Kimelman, 2008). In the mouse embryo T and Tbx6 interact with canonical wnt signaling to activate notch signaling in the presomitic mesoderm (Hofmann et al., 2004). Tbx16 or spadetail regulates the canonical Wnt signaling pathway in the intermediate mesoderm of the developing zebrafish, which is also the site of primitive red blood cell formation (Mueller et al., 2010). In Tbx5 mutant forelimb limb buds Lef1, Tcf1 and Snai1, all targets of canonical Wnt signaling, are absent suggesting that Tbx5 lies upstream of canonical Wnt signaling during forelimb bud growth (Agarwal et al., 2003). In zebrafish tbx5 and wnt2b interact to initiate and specify forelimb identity (Ng et al., 2002). We show that Tbx4 lies upstream of Wnt2, and four genes suggested to be direct downstream targets of canonical Wnt signaling – Snai1, Twist, Versican and Jagged1– in the developing allantois.

Further, both Tbx4 and Wnt2 interact in allantois-derived vascular network formation in the chorio-allantoic plate and in the placental labyrinth. Additionally, our studies in the developing

98 lung suggest that Tbx4 and Tbx5 regulate Wnt2 expression during lung branching morphogenesis.

T-box genes regulate expression of major components of extracellular matrix

Finally our work suggests a role for Tbx4 and Tbx5 in the regulation of extracellular matrix during organ formation. There have been multiple studies linking T-box gene function to matrix regulation indicating that this might be a common theme through which Tbx genes act in different tissues. Decrease in expression of eight Tbx genes Tbx1, Tbx2, Tbx3, Tbx4, Tbx5,

Tbx15, Tbx18, Tbx21 during hind limb chondrogenesis was observed when comparing expression in E11.5 anlagen, E12.5 condensations and E13.5 cartilage anlagen (Cameron et al.,

2009). This decrease in expression suggests that T-box genes are expressed in mesenchyme that has chondrogenic potential but not in cells that are actively undergoing condensation. These results are similar to the results we obtained in the tracheal cartilage, where Tbx4 and Tbx5 are expressed in the chondrogenic mesenchyme at E11.5 but not expressed in mesenchymal condensations and the cartilage anlagen at E15.5. In primary avian endocardial cells, Tbx20 overexpression leads to repression of chondroitin sulphate proteoglycans Aggrecan and

Versican, and increased expression of Mmp9 and Mmp13. Further, Tbx20 plays a role in repression of extracellular matrix remodeling and activation of mesenchymal proliferation

(Shelton and Yutzey, 2007). In zebrafish the T-box gene spadetail, regulates the transcription of cell adhesion molecule paraxial protocadherin in the trunk mesoderm during morphogenesis

(Yamamoto et al., 1998). Over-expression of Tbx2 in human embryonic kidney cells also upregulates three genes involved in extracellular matrix formation and remodeling – Mmp2, Ctgf and Acta2 (Butz et al., 2004). In another study over-expression of Tbx2 in NIH3T3 cells leads to upregulation of Caveolin, Pleiotrophin (osf-1), Osteoblast-specific factor-2 (osf-2) and collagen

99 type Iα and downregulation of Cadherin 3 and Tenascin C (Chen et al., 2001) further highlighting the transcriptional regulation of extracellular matrix components by the T-box genes. Our work highlights different extracellular matrix genes Has2, Vcan and Itgα5 regulated by Tbx4 in the developing allantois. Amongst these genes both Has2 and Vcan are implicated in the process of cavitation which is important for the maturation of the developing allantois. In the branching lung, inability of Fgf10 to rescue the Tbx4 and Tbx5 mutant phenotype suggests that these T-box genes regulate additional matrix molecules that aid in Fgf10-Fgfr2 interactions.

Finally, Tbx4 and Tbx5 deficient tracheas show defective cartilage condensations, although Sox9 a master regulator for cartilage formation is initially expressed normally in these tracheas. This further suggests a role for Tbx4 and Tbx5 in regulating appropriate ECM to support mesenchymal condensations and cartilage ring formation.

Future perspectives

The research described in this thesis addresses the role of Tbx4 in allantois development and proposes different functions for Tbx4 and Tbx5 in the development of the respiratory system.

However, there are still many unanswered questions and possible hypotheses to explore, some of which are described below: i) Our studies describe Tbx4 as a master regulator of allantois vascular development. This study raises the question, if there is a general role of T-box genes in vasculogenesis? Although Tbx2 and Tbx3 are expressed in the allantois, null mutations in neither of these T-box genes produce an allantois vascular phenotype. On the other hand, Tbx20 null mutant embryos show defects in the development of yolk sac vasculature and Tbx3 has been implicated in development of the yolk sac although the vasculature was not specifically analyzed. Thus, an interesting possibility

100 is that different T-box genes could potentially regulate a similar sub-set of genes important for vascular development and this hypothesis should be tested further. ii) Tbx4 mutant allantoises provide for a good tool for studying the process of endothelial cell tube formation using the adherent allantois cultures. As used to study the canonical Wnt signaling pathway, these mutant allantois cultures could be used to screen small molecule agonists and antagonists of different pathways for a rescue of Tbx4 mutant phenotype to discover additional molecules that affect endothelial cell assembly. iii) We show that Tbx4 and Tbx5 interact during the development of the respiratory system, but whether they have overlapping functions or distinct functions is unclear. Tbx5 is expressed before Tbx4 in the lung/trachea primordia and loss of Tbx5 but not Tbx4 at this early time leads to aberrant specification pointing to a role for Tbx5 during specification independent of Tbx4 function. On the other hand, increasing the number of Tbx4 and Tbx5 null alleles leads to an increase in the severity of the lung branching morphogenesis and tracheal cartilage phenotype, suggesting interactions. Whether these interactions are due to additive, overlapping or distinct functions is unclear. This is also the first study where the interactions and functions of Tbx4 and

Tbx5 are studied in a tissue where these genes are co-expressed. This hints at possible redundant and discreet functions of these genes in other tissues where they are co-expressed, for example the genital tubercle. iv) Sox9 is considered to be the master transcriptional regulator of cartilage formation. In our

Tbx4 and Tbx5-deficient mutants, Sox9 is expressed normally to begin with, but cartilage fails to form. These data indicate that there are unknown regulators of chondrogenesis downstream of

Tbx4 and Tbx5, in the absence of which, Sox9 is insufficient to induce mesenchymal condensations.

101 v) The smooth muscle expansion defect in the Tbx4 and Tbx5-deficient mutants could be secondary to cartilage defect, as has been seen for other mutants. Alternatively, since the two ectopic stripes of smooth muscle expression in the mutants are very close in position to the two vagus nerves that run across the length of the trachea, the ectopic smooth muscle expression could be in these nerves. This can be addressed by staining for markers of nerve differentiation and SM22α and analyzing colocalization. This analysis would shed light on the normal differentiation pathway of the airway smooth muscle and the nerve components of the trachea and their mutual interactions, if any, during development.

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References

Ackerman, K. G., Wang, J., Luo, L., Fujiwara, Y., Orkin, S. H. and Beier, D. R. (2007). Gata4 Is Necessary for Normal Pulmonary Lobar Development. Am. J. Respir. Cell Mol. Biol. 36, 391-397.

Ackerman, K. G., Herron, B. J., Vargas, S. O., Huang, H., Tevosian, S. G., Kochilas, L., Rao, C., Pober, B. R., Babiuk, R. P., Epstein, J. A. et al. (2005). Fog2 Is Required for Normal Diaphragm and Lung Development in Mice and Humans. PLoS Genet 1, e10.

Agarwal, P., Wylie, J. N., Galceran, J., Arkhitko, O., Li, C., Deng, C., Grosschedl, R. and Bruneau, B. G. (2003). Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development 130, 623-633.

Aisha, A., M. Abu-Salah, K., Ismail, Z. and Abdul Majid, A. (2010). Screening For Antiangiogenesis Activity In Natural Products: A Review. WebmedCentral PHARMACOLOGY 1.

Akhurst, R. J., Lehnert, S. A., Faissner, A. and Duffie, E. (1990). TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis. Development 108, 645-656.

Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A. and de Crombrugghe, B. (2002). The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16, 2813- 2828.

Aoki, M., Mieda, M., Ikeda, T., Hamada, Y., Nakamura, H. and Okamoto, H. (2007). R- spondin3 is required for mouse placental development. Dev Biol 301, 218-226.

Argraves, K. M., Wilkerson, B. A., Argraves, W. S., Fleming, P. A., Obeid, L. M. and Drake, C. J. (2004). Sphingosine-1-phosphate signaling promotes critical migratory events in vasculogenesis. J Biol Chem 279, 50580-50590.

Argraves, W. S., Larue, A. C., Fleming, P. A. and Drake, C. J. (2002). VEGF signaling is required for the assembly but not the maintenance of embryonic blood vessels. Dev Dyn 225, 298-304.

103

Arman, E., Haffner-Krausz, R., Gorivodsky, M. and Lonai, P. (1999). Fgfr2 is required for limb outgrowth and lung-branching morphogenesis. Proc Natl Acad Sci U S A 96, 11895-11899.

Astorga, J. and Carlsson, P. (2007). Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4. Development 134, 3753-3761.

Badri, K. R., Zhou, Y. and Schuger, L. (2008). Embryological origin of airway smooth muscle. Proc Am Thorac Soc 5, 4-10.

Baumer, S., Keller, L., Holtmann, A., Funke, R., August, B., Gamp, A., Wolburg, H., Wolburg-Buchholz, K., Deutsch, U. and Vestweber, D. (2006). Vascular endothelial cell- specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development. Blood 107, 4754-4762.

Begemann, G. and Ingham, P. W. (2000). Developmental regulation of Tbx5 in zebrafish embryogenesis. Mech Dev 90, 299-304.

Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. (1996). Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122, 1693-1702.

Bellusci, S., Grindley, J., Emoto, H., Itoh, N. and Hogan, B. L. (1997). Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung. Development 124, 4867-4878.

Betsholtz, C., Karlsson, L. and Lindahl, P. (2001). Developmental roles of platelet-derived growth factors. Bioessays 23, 494-507.

Bimber, B., Dettman, R. W. and Simon, H. G. (2007). Differential regulation of Tbx5 protein expression and sub-cellular localization during heart development. Dev Biol 302, 230-242.

Bongers, E. M., Duijf, P. H., van Beersum, S. E., Schoots, J., Van Kampen, A., Burckhardt, A., Hamel, B. C., Losan, F., Hoefsloot, L. H., Yntema, H. G. et al. (2004). Mutations in the human TBX4 gene cause small patella syndrome. Am J Hum Genet 74, 1239-1248.

Brault, V., Moore, R., Kutsch, S., Ishibashi, M., Rowitch, D. H., McMahon, A. P., Sommer, L., Boussadia, O. and Kemler, R. (2001). Inactivation of the beta-catenin gene by Wnt1-Cre- mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128, 1253-1264.

104

Brown, J. J. and Papaioannou, V. E. (1993). Ontogeny of hyaluronan secretion during early mouse development. Development 117, 483-492.

Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E. et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106, 709-721.

Butz, N. V., Campbell, C. E. and Gronostajski, R. M. (2004). Differential target gene activation by TBX2 and TBX2VP16: evidence for activation domain-dependent modulation of gene target specificity. Gene 342, 67-76.

Byrd, N. and Grabel, L. (2004). Hedgehog signaling in murine vasculogenesis and angiogenesis. Trends Cardiovasc Med 14, 308-313.

Byrd, N., Becker, S., Maye, P., Narasimhaiah, R., St-Jacques, B., Zhang, X., McMahon, J., McMahon, A. and Grabel, L. (2002). Hedgehog is required for murine yolk sac angiogenesis. Development 129, 361-372.

Camenisch, T. D., Spicer, A. P., Brehm-Gibson, T., Biesterfeldt, J., Augustine, M. L., Calabro, A., Jr., Kubalak, S., Klewer, S. E. and McDonald, J. A. (2000). Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest 106, 349-360.

Cameron, T. L., Belluoccio, D., Farlie, P. G., Brachvogel, B. and Bateman, J. F. (2009). Global comparative transcriptome analysis of cartilage formation in vivo. BMC Dev Biol 9, 20.

Cardoso, W. V. and Lu, J. (2006). Regulation of early lung morphogenesis: questions, facts and controversies. Development 133, 1611-1624.

Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C. et al. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439.

Carmeliet, P., Lampugnani, M. G., Moons, L., Breviario, F., Compernolle, V., Bono, F., Balconi, G., Spagnuolo, R., Oosthuyse, B., Dewerchin, M. et al. (1999). Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98, 147-157.

105

Cattelino, A., Liebner, S., Gallini, R., Zanetti, A., Balconi, G., Corsi, A., Bianco, P., Wolburg, H., Moore, R., Oreda, B. et al. (2003). The conditional inactivation of the beta- catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol 162, 1111-1122.

Cebra-Thomas, J. A., Bromer, J., Gardner, R., Lam, G. K., Sheipe, H. and Gilbert, S. F. (2003). T-box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lung. Dev Dyn 226, 82-90.

Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev Dyn 206, 379-390.

Charite, J., McFadden, D. G. and Olson, E. N. (2000). The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development. Development 127, 2461-2470.

Chen, F., Cao, Y., Qian, J., Shao, F., Niederreither, K. and Cardoso, W. V. (2010). A retinoic acid-dependent network in the foregut controls formation of the mouse lung primordium. J Clin Invest 120, 2040-2048.

Chen, J., Zhong, Q., Wang, J., Cameron, R. S., Borke, J. L., Isales, C. M. and Bollag, R. J. (2001). Microarray analysis of Tbx2-directed gene expression: a possible role in osteogenesis. Mol Cell Endocrinol 177, 43-54.

Chen, J. C. and Holinger, L. D. (1994). Congenital tracheal anomalies: pathology study using serial macrosections and review of the literature. Pediatr Pathol 14, 513-537.

Cheng, C. W., Yeh, J. C., Fan, T. P., Smith, S. K. and Charnock-Jones, D. S. (2008). Wnt5a- mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration. Biochem Biophys Res Commun 365, 285-290.

Christoffels, V. M., Hoogaars, W. M., Tessari, A., Clout, D. E., Moorman, A. F. and Campione, M. (2004). T-box transcription factor Tbx2 represses differentiation and formation of the cardiac chambers. Dev Dyn 229, 763-770.

Chuang, P. T., Kawcak, T. and McMahon, A. P. (2003). Feedback control of mammalian Hedgehog signaling by the Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching morphogenesis of the lung. Genes Dev 17, 342-347.

106

Corbel, C., Salaun, J., Belo-Diabangouaya, P. and Dieterlen-Lievre, F. (2007). Hematopoietic potential of the pre-fusion allantois. Dev Biol 301, 478-488.

Crosby, C. V., Fleming, P. A., Argraves, W. S., Corada, M., Zanetta, L., Dejana, E. and Drake, C. J. (2005). VE-cadherin is not required for the formation of nascent blood vessels but acts to prevent their disassembly. Blood 105, 2771-2776.

Davenport, T. G., Jerome-Majewska, L. A. and Papaioannou, V. E. (2003). Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome. Development 130, 2263-2273.

Davis, C. A. (1993). Whole-mount immunohistochemistry. Methods Enzymol 225, 502-516. de Crombrugghe, B., Lefebvre, V. and Nakashima, K. (2001). Regulatory mechanisms in the pathways of cartilage and bone formation. Curr Opin Cell Biol 13, 721-727.

De Langhe, S. P., Sala, F. G., Del Moral, P. M., Fairbanks, T. J., Yamada, K. M., Warburton, D., Burns, R. C. and Bellusci, S. (2005). Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung. Dev Biol 277, 316-331.

De Langhe, S. P., Carraro, G., Tefft, D., Li, C., Xu, X., Chai, Y., Minoo, P., Hajihosseini, M. K., Drouin, J., Kaartinen, V. et al. (2008). Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by beta-catenin signaling. PLoS One 3, e1516. de Luca, C., Kowalski, T. J., Zhang, Y., Elmquist, J. K., Lee, C., Kilimann, M. W., Ludwig, T., Liu, S. M. and Chua, S. C., Jr. (2005). Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. J Clin Invest 115, 3484-3493.

De Val, S. (2011). Key transcriptional regulators of early vascular development. Arterioscler Thromb Vasc Biol 31, 1469-1475.

DeLise, A. M., Fischer, L. and Tuan, R. S. (2000). Cellular interactions and signaling in cartilage development. Osteoarthritis Cartilage 8, 309-334.

Desai, T. J., Malpel, S., Flentke, G. R., Smith, S. M. and Cardoso, W. V. (2004). Retinoic acid selectively regulates Fgf10 expression and maintains cell identity in the prospective lung field of the developing foregut. Dev Biol 273, 402-415.

107

Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995). Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 121, 1845-1854.

Dieterlen-Lievre, F. and Jaffredo, T. (2009). Decoding the hemogenic endothelium in mammals. Cell Stem Cell 4, 189-190.

Domyan, E. T. and Sun, X. (2011). Patterning and plasticity in development of the respiratory lineage. Dev Dyn 240, 477-485.

Domyan, E. T., Ferretti, E., Throckmorton, K., Mishina, Y., Nicolis, S. K. and Sun, X. (2011). Signaling through BMP receptors promotes respiratory identity in the foregut via repression of Sox2. Development 138, 971-981.

Douglas, N. C., Heng, K., Sauer, M. V. and Papaioannou, V. E. (2011). Dynamic expression of Tbx2 subfamily genes in development of the mouse reproductive system. Dev Dyn.

Downs, K. M. (2006). In vitro methods for studying vascularization of the murine allantois and allantoic union with the chorion. Methods Mol Med 121, 241-272.

Downs, K. M., Gifford, S., Blahnik, M. and Gardner, R. L. (1998). Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis. Development 125, 4507-4520.

Downs, K. M., Temkin, R., Gifford, S. and McHugh, J. (2001). Study of the murine allantois by allantoic explants. Dev Biol 233, 347-364.

Drake, C. J. and Fleming, P. A. (2000). Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95, 1671-1679.

Dumont, D. J., Fong, G. H., Puri, M. C., Gradwohl, G., Alitalo, K. and Breitman, M. L. (1995). Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev Dyn 203, 80-92.

Dyer, M. A., Farrington, S. M., Mohn, D., Munday, J. R. and Baron, M. H. (2001). Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development 128, 1717-1730.

108

Elluru, R. G. and Whitsett, J. A. (2004). Potential role of Sox9 in patterning tracheal cartilage ring formation in an embryonic mouse model. Arch Otolaryngol Head Neck Surg 130, 732-736.

Epstein, C., Erickson, R. and Wynshaw-Boris, A. (2008). Inborn Errors of Development: Oxford University Press.

Fenderson, B. A., Stamenkovic, I. and Aruffo, A. (1993). Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis. Differentiation 54, 85-98.

Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O'Shea, K. S., Powell-Braxton, L., Hillan, K. J. and Moore, M. W. (1996). Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439-442.

Firnberg, N. and Neubuser, A. (2002). FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region. Dev Biol 247, 237-250.

Fischer, A., Schumacher, N., Maier, M., Sendtner, M. and Gessler, M. (2004). The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev 18, 901-911.

Fleming, P. A., Argraves, W. S., Gentile, C., Neagu, A., Forgacs, G. and Drake, C. J. (2010). Fusion of uniluminal vascular spheroids: a model for assembly of blood vessels. Dev Dyn 239, 398-406.

Fong, G. H., Zhang, L., Bryce, D. M. and Peng, J. (1999). Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 126, 3015-3025.

Francis, S. E., Goh, K. L., Hodivala-Dilke, K., Bader, B. L., Stark, M., Davidson, D. and Hynes, R. O. (2002). Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies. Arterioscler Thromb Vasc Biol 22, 927- 933.

French, W. J., Creemers, E. E. and Tallquist, M. D. (2008). Platelet-derived growth factor receptors direct vascular development independent of vascular smooth muscle cell function. Mol Cell Biol 28, 5646-5657.

Gale, N. W., Dominguez, M. G., Noguera, I., Pan, L., Hughes, V., Valenzuela, D. M., Murphy, A. J., Adams, N. C., Lin, H. C., Holash, J. et al. (2004). Haploinsufficiency of delta-

109 like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A 101, 15949-15954.

Gambardella, L., Hemberger, M., Hughes, B., Zudaire, E., Andrews, S. and Vermeren, S. (2010). PI3K signaling through the dual GTPase-activating protein ARAP3 is essential for developmental angiogenesis. Sci Signal 3, ra76.

Gentile, C., Fleming, P. A., Mironov, V., Argraves, K. M., Argraves, W. S. and Drake, C. J. (2008). VEGF-mediated fusion in the generation of uniluminal vascular spheroids. Dev Dyn 237, 2918-2925.

George, E. L., Georges-Labouesse, E. N., Patel-King, R. S., Rayburn, H. and Hynes, R. O. (1993). Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119, 1079-1091.

Gerety, S. S., Wang, H. U., Chen, Z. F. and Anderson, D. J. (1999). Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell 4, 403-414.

Gibson-Brown, J. J., S, I. A., Silver, L. M. and Papaioannou, V. E. (1998). Expression of T- box genes Tbx2-Tbx5 during chick organogenesis. Mech Dev 74, 165-169.

Goering, L. M., Hoshijima, K., Hug, B., Bisgrove, B., Kispert, A. and Grunwald, D. J. (2003). An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm. Proc Natl Acad Sci U S A 100, 9410-9415.

Gory-Faure, S., Prandini, M. H., Pointu, H., Roullot, V., Pignot-Paintrand, I., Vernet, M. and Huber, P. (1999). Role of vascular endothelial-cadherin in vascular morphogenesis. Development 126, 2093-2102.

Goss, A. M., Tian, Y., Cheng, L., Yang, J., Zhou, D., Cohen, E. D. and Morrisey, E. E. (2011). Wnt2 signaling is necessary and sufficient to activate the airway smooth muscle program in the lung by regulating myocardin/Mrtf-B and Fgf10 expression. Dev Biol 356, 541-552.

Goss, A. M., Tian, Y., Tsukiyama, T., Cohen, E. D., Zhou, D., Lu, M. M., Yamaguchi, T. P. and Morrisey, E. E. (2009). Wnt2/2b and [beta]-Catenin Signaling Are Necessary and Sufficient to Specify Lung Progenitors in the Foregut. Developmental Cell 17, 290-298.

110

Grieshammer, U., Le, M., Plump, A. S., Wang, F., Tessier-Lavigne, M. and Martin, G. R. (2004). SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev Cell 6, 709-717.

Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J. R. and Tsujimoto, Y. (1999). Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality. Development 126, 3415-3424.

Harding, R., Pinkerton, K. and Plopper, C. (2003). The Lung: Development, Aging and The Environment Academic Press.

Hardingham, T. E., Oldershaw, R. A. and Tew, S. R. (2006). Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat 209, 469-480.

Harrelson, Z. and Papaioannou, V. E. (2006). Segmental expression of the T-box transcription factor, Tbx2, during early somitogenesis. Dev Dyn 235, 3080-3084.

Harris-Johnson, K. S., Domyan, E. T., Vezina, C. M. and Sun, X. (2009). beta-Catenin promotes respiratory progenitor identity in mouse foregut. Proc Natl Acad Sci U S A 106, 16287- 16292.

Hasson, P., DeLaurier, A., Bennett, M., Grigorieva, E., Naiche, L. A., Papaioannou, V. E., Mohun, T. J. and Logan, M. P. (2010). Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning. Dev Cell 18, 148-156.

Hatcher, C. J., Diman, N. Y., Kim, M. S., Pennisi, D., Song, Y., Goldstein, M. M., Mikawa, T. and Basson, C. T. (2004). A role for Tbx5 in proepicardial cell migration during cardiogenesis. Physiol Genomics 18, 129-140.

Hofmann, M., Schuster-Gossler, K., Watabe-Rudolph, M., Aulehla, A., Herrmann, B. G. and Gossler, A. (2004). WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos. Genes Dev 18, 2712- 2717.

Horowitz, A. and Simons, M. (2008). Branching Morphogenesis. Circ Res 103, 784-795.

Hynes, R. O. (2007). Cell-matrix adhesion in vascular development. J Thromb Haemost 5 Suppl 1, 32-40.

111

Hynes, R. O., Lively, J. C., McCarty, J. H., Taverna, D., Francis, S. E., Hodivala-Dilke, K. and Xiao, Q. (2002). The diverse roles of integrins and their ligands in angiogenesis. Cold Spring Harb Symp Quant Biol 67, 143-153.

Inman, K. E. and Downs, K. M. (2006a). Brachyury is required for elongation and vasculogenesis in the murine allantois. Development 133, 2947-2959.

Inman, K. E. and Downs, K. M. (2006b). Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation. Gene Expr Patterns 6, 783-793.

Inman, K. E. and Downs, K. M. (2007). The murine allantois: emerging paradigms in development of the mammalian umbilical cord and its relation to the fetus. Genesis 45, 237-258.

Ishikawa, T., Tamai, Y., Zorn, A. M., Yoshida, H., Seldin, M. F., Nishikawa, S. and Taketo, M. M. (2001). Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis. Development 128, 25-33.

Izvolsky, K. I., Zhong, L., Wei, L., Yu, Q., Nugent, M. A. and Cardoso, W. V. (2003a). Heparan sulfates expressed in the distal lung are required for Fgf10 binding to the epithelium and for airway branching. Am J Physiol Lung Cell Mol Physiol 285, L838-846.

Izvolsky, K. I., Shoykhet, D., Yang, Y., Yu, Q., Nugent, M. A. and Cardoso, W. V. (2003b). Heparan sulfate-FGF10 interactions during lung morphogenesis. Dev Biol 258, 185-200.

Jerome, L. A. and Papaioannou, V. E. (2001). DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat Genet 27, 286-291.

Jungers, K. A., Le Goff, C., Somerville, R. P. and Apte, S. S. (2005). Adamts9 is widely expressed during mouse embryo development. Gene Expr Patterns 5, 609-617.

Kanemura, H., Satoh, T., Bilasy, S. E., Ueda, S., Hirashima, M. and Kataoka, T. (2009). Impaired vascular development in the yolk sac and allantois in mice lacking RA-GEF-1. Biochem Biophys Res Commun 387, 754-759.

Karner, C. M., Merkel, C. E., Dodge, M., Ma, Z., Lu, J., Chen, C., Lum, L. and Carroll, T. J. (2010). Tankyrase is necessary for canonical Wnt signaling during kidney development. Dev Dyn 239, 2014-2023.

112

Kazanskaya, O., Ohkawara, B., Heroult, M., Wu, W., Maltry, N., Augustin, H. G. and Niehrs, C. (2008). The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development 135, 3655-3664.

Kimura, S., Hara, Y., Pineau, T., Fernandez-Salguero, P., Fox, C. H., Ward, J. M. and Gonzalez, F. J. (1996). The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10, 60-69.

Kispert, A. and Herrmann, B. G. (1993). The Brachyury gene encodes a novel DNA binding protein. EMBO J 12, 3211-3220.

Kispert, A., Vainio, S., Shen, L., Rowitch, D. H. and McMahon, A. P. (1996). Proteoglycans are required for maintenance of Wnt-11 expression in the ureter tips. Development 122, 3627- 3637.

Klein, D., Demory, A., Peyre, F., Kroll, J., Augustin, H. G., Helfrich, W., Kzhyshkowska, J., Schledzewski, K., Arnold, B. and Goerdt, S. (2008). Wnt2 acts as a cell type-specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway. Hepatology 47, 1018-1031.

Koo, B. H., Coe, D. M., Dixon, L. J., Somerville, R. P., Nelson, C. M., Wang, L. W., Young, M. E., Lindner, D. J. and Apte, S. S. (2010). ADAMTS9 is a cell-autonomously acting, anti- angiogenic metalloprotease expressed by microvascular endothelial cells. Am J Pathol 176, 1494-1504.

Kowanetz, M. and Ferrara, N. (2006). Vascular endothelial growth factor signaling pathways: therapeutic perspective. Clin Cancer Res 12, 5018-5022.

Krause, A., Zacharias, W., Camarata, T., Linkhart, B., Law, E., Lischke, A., Miljan, E. and Simon, H. G. (2004). Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development. Dev Biol 273, 106-120.

Lanzer, P. and Topol, E. J. (2002). Pan Vascular Medicine: Integrated Clinical Management.: Springer.

Larsson, J., Goumans, M. J., Sjostrand, L. J., van Rooijen, M. A., Ward, D., Leveen, P., Xu, X., ten Dijke, P., Mummery, C. L. and Karlsson, S. (2001). Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice. EMBO J 20, 1663- 1673.

113

Lee, D., Park, C., Lee, H., Lugus, J. J., Kim, S. H., Arentson, E., Chung, Y. S., Gomez, G., Kyba, M., Lin, S. et al. (2008). ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification. Cell Stem Cell 2, 497-507.

Leimeister, C., Externbrink, A., Klamt, B. and Gessler, M. (1999). Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech Dev 85, 173-177.

Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts, R. W., Burns, C. G. and Poss, K. D. (2006). A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127, 607-619.

Leptin, M. (1991). twist and snail as positive and negative regulators during Drosophila mesoderm development. Genes Dev 5, 1568-1576.

Li, C., Xiao, J., Hormi, K., Borok, Z. and Minoo, P. (2002). Wnt5a participates in distal lung morphogenesis. Dev Biol 248, 68-81.

Li, C., Hu, L., Xiao, J., Chen, H., Li, J. T., Bellusci, S., Delanghe, S. and Minoo, P. (2005). Wnt5a regulates Shh and Fgf10 signaling during lung development. Dev Biol 287, 86-97.

Lin, F. J., Tsai, M. J. and Tsai, S. Y. (2007). Artery and vein formation: a tug of war between different forces. EMBO Rep 8, 920-924.

Litingtung, Y., Lei, L., Westphal, H. and Chiang, C. (1998). Sonic hedgehog is essential to foregut development. Nat Genet 20, 58-61.

Liu, Y., Wada, R., Yamashita, T., Mi, Y., Deng, C. X., Hobson, J. P., Rosenfeldt, H. M., Nava, V. E., Chae, S. S., Lee, M. J. et al. (2000). Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest 106, 951-961.

Lomeli, H., Starling, C. and Gridley, T. (2009). Epiblast-specific Snai1 deletion results in embryonic lethality due to multiple vascular defects. BMC Res Notes 2, 22.

Luria, V., Krawchuk, D., Jessell, T. M., Laufer, E. and Kania, A. (2008). Specification of motor axon trajectory by ephrin-B:EphB signaling: symmetrical control of axonal patterning in the developing limb. Neuron 60, 1039-1053.

114

Maeda, Y., Dave, V. and Whitsett, J. A. (2007). Transcriptional control of lung morphogenesis. Physiol Rev 87, 219-244.

Mahlapuu, M., Enerback, S. and Carlsson, P. (2001). Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128, 2397-2406.

Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S., Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H., Papadopoulos, N. et al. (1997). Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60.

Majumdar, A., Vainio, S., Kispert, A., McMahon, J. and McMahon, A. P. (2003). Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development. Development 130, 3175-3185.

Martin, B. L. and Kimelman, D. (2008). Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation. Dev Cell 15, 121-133.

Masckauchan, T. N., Agalliu, D., Vorontchikhina, M., Ahn, A., Parmalee, N. L., Li, C. M., Khoo, A., Tycko, B., Brown, A. M. and Kitajewski, J. (2006). Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2. Mol Biol Cell 17, 5163-5172.

McGill, R., Tukey, J. W. and Larsen, W. A. (1978). Variations of Box Plots. The American Statistician 32, 12-16.

McGowan, S. E. (1992). Extracellular matrix and the regulation of lung development and repair. FASEB J 6, 2895-2904.

Mendelsohn, C., Lohnes, D., Decimo, D., Lufkin, T., LeMeur, M., Chambon, P. and Mark, M. (1994). Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development 120, 2749-2771.

Mesbah, K., Rana, M. S., Francou, A., van Duijvenboden, K., Papaioannou, V. E., Moorman, A. F., Kelly, R. G. and Christoffels, V. M. (2012). Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and arterial pole morphogenesis. Hum Mol Genet 21, 1217-1229.

115

Metzger, R., Wachowiak, R. and Kluth, D. (2011). Embryology of the early foregut. Semin Pediatr Surg 20, 136-144.

Metzger, R. J., Klein, O. D., Martin, G. R. and Krasnow, M. A. (2008). The branching programme of mouse lung development. Nature 453, 745-750.

Miller, L. A., Wert, S. E., Clark, J. C., Xu, Y., Perl, A. K. and Whitsett, J. A. (2004). Role of Sonic hedgehog in patterning of tracheal-bronchial cartilage and the peripheral lung. Dev Dyn 231, 57-71.

Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B. D., Tarpley, J. E., DeRose, M. and Simonet, W. S. (1998). Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 12, 3156-3161.

Minguillon, C., Del Buono, J. and Logan, M. P. (2005). Tbx5 and Tbx4 are not sufficient to determine limb-specific morphologies but have common roles in initiating limb outgrowth. Dev Cell 8, 75-84.

Moloney, D. M., Slaney, S. F., Oldridge, M., Wall, S. A., Sahlin, P., Stenman, G. and Wilkie, A. O. (1996). Exclusive paternal origin of new mutations in Apert syndrome. Nat Genet 13, 48-53.

Monkley, S. J., Delaney, S. J., Pennisi, D. J., Christiansen, J. H. and Wainwright, B. J. (1996). Targeted disruption of the Wnt2 gene results in placentation defects. Development 122, 3343-3353.

Mori, A. D., Zhu, Y., Vahora, I., Nieman, B., Koshiba-Takeuchi, K., Davidson, L., Pizard, A., Seidman, J. G., Seidman, C. E., Chen, X. J. et al. (2006). Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis. Dev Biol 297, 566-586.

Moser, M. and Patterson, C. (2005). Bone morphogenetic proteins and vascular differentiation: BMPing up vasculogenesis. Thromb Haemost 94, 713-718.

Motoyama, J., Liu, J., Mo, R., Ding, Q., Post, M. and Hui, C. C. (1998). Essential function of Gli2 and Gli3 in the formation of lung, trachea and oesophagus. Nat Genet 20, 54-57.

Mueller, R. L., Huang, C. and Ho, R. K. (2010). Spatio-temporal regulation of Wnt and retinoic acid signaling by tbx16/spadetail during zebrafish mesoderm differentiation. BMC Genomics 11, 492.

116

Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R. (2009). Alcian Blue Staining of the Mouse Fetal Cartilaginous Skeleton. Cold Spring Harbor Protocols 2009, pdb.prot5169.

Naiche, L. A. and Papaioannou, V. E. (2003). Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development 130, 2681-2693.

Naiche, L. A. and Papaioannou, V. E. (2007a). Cre activity causes widespread apoptosis and lethal anemia during embryonic development. Genesis 45, 768-775.

Naiche, L. A. and Papaioannou, V. E. (2007b). Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth. Development 134, 93-103.

Naiche, L. A., Harrelson, Z., Kelly, R. G. and Papaioannou, V. E. (2005). T-box genes in vertebrate development. Annu Rev Genet 39, 219-239.

Naiche, L. A., Arora, R., Kania, A., Lewandoski, M. and Papaioannou, V. E. (2011). Identity and fate of Tbx4-expressing cells reveal developmental cell fate decisions in the allantois, limb, and external genitalia. Dev Dyn 240, 2290-2300.

Ng, J. K., Kawakami, Y., Buscher, D., Raya, A., Itoh, T., Koth, C. M., Rodriguez Esteban, C., Rodriguez-Leon, J., Garrity, D. M., Fishman, M. C. et al. (2002). The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129, 5161- 5170.

Ohlsson, R., Falck, P., Hellstrom, M., Lindahl, P., Bostrom, H., Franklin, G., Ahrlund- Richter, L., Pollard, J., Soriano, P. and Betsholtz, C. (1999). PDGFB regulates the development of the labyrinthine layer of the mouse fetal placenta. Dev Biol 212, 124-136.

Palis, J., McGrath, K. E. and Kingsley, P. D. (1995). Initiation of hematopoiesis and vasculogenesis in murine yolk sac explants. Blood 86, 156-163.

Papaioannou, V. E. (2001). T-box genes in development: from hydra to humans. Int Rev Cytol 207, 1-70.

Parera, M. C., van Dooren, M., van Kempen, M., de Krijger, R., Grosveld, F., Tibboel, D. and Rottier, R. (2005). Distal angiogenesis: a new concept for lung vascular morphogenesis. Am J Physiol Lung Cell Mol Physiol 288, L141-149.

117

Park, J., Zhang, J. J., Moro, A., Kushida, M., Wegner, M. and Kim, P. C. (2010). Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage. Dev Dyn 239, 514-526.

Patan, S. (2000). Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J Neurooncol 50, 1-15.

Patan, S. (2004). Vasculogenesis and angiogenesis. Cancer Treat Res 117, 3-32.

Patel-Hett, S. and D'Amore, P. A. (2011). Signal transduction in vasculogenesis and developmental angiogenesis. Int J Dev Biol 55, 353-363.

Patel, V. N., Knox, S. M., Likar, K. M., Lathrop, C. A., Hossain, R., Eftekhari, S., Whitelock, J. M., Elkin, M., Vlodavsky, I. and Hoffman, M. P. (2007). Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis. Development 134, 4177-4186.

Pepicelli, C. V., Lewis, P. M. and McMahon, A. P. (1998). Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 8, 1083-1086.

Perryn, E. D., Czirok, A. and Little, C. D. (2008). Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility. Dev Biol 313, 545-555.

Pozzi, A. and Zent, R. (2011). Extracellular matrix receptors in branched organs. Curr Opin Cell Biol 23, 547-553.

Que, J., Luo, X., Schwartz, R. J. and Hogan, B. L. (2009). Multiple roles for Sox2 in the developing and adult mouse trachea. Development 136, 1899-1907.

Que, J., Wilm, B., Hasegawa, H., Wang, F., Bader, D. and Hogan, B. L. (2008). Mesothelium contributes to vascular smooth muscle and mesenchyme during lung development. Proc Natl Acad Sci U S A 105, 16626-16630.

Que, J., Okubo, T., Goldenring, J. R., Nam, K. T., Kurotani, R., Morrisey, E. E., Taranova, O., Pevny, L. H. and Hogan, B. L. (2007). Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm. Development 134, 2521-2531.

118

Rallis, C., Bruneau, B. G., Del Buono, J., Seidman, C. E., Seidman, J. G., Nissim, S., Tabin, C. J. and Logan, M. P. (2003). Tbx5 is required for forelimb bud formation and continued outgrowth. Development 130, 2741-2751.

Rao, A. S., Kremenevskaja, N., Resch, J. and Brabant, G. (2005). Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signalling. Eur J Endocrinol 153, 929-938.

Ribatti, D., Nico, B. and Crivellato, E. (2009). Morphological and molecular aspects of physiological vascular morphogenesis. Angiogenesis 12, 101-111.

Rossant, J. and Howard, L. (2002). Signaling pathways in vascular development. Annu Rev Cell Dev Biol 18, 541-573.

Rossig, L., Urbich, C., Bruhl, T., Dernbach, E., Heeschen, C., Chavakis, E., Sasaki, K., Aicher, D., Diehl, F., Seeger, F. et al. (2005). Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells. J Exp Med 201, 1825- 1835.

Rutter, M. and Post, M. (2005). Shh/Gli Signaling During Murine Lung Development. In Madame Curie Bioscience Database: Landes Bioscience

Sakiyama, J., Yamagishi, A. and Kuroiwa, A. (2003). Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development. Development 130, 1225-1234.

Sala, F. G., Del Moral, P. M., Tiozzo, C., Alam, D. A., Warburton, D., Grikscheit, T., Veltmaat, J. M. and Bellusci, S. (2011). FGF10 controls the patterning of the tracheal cartilage rings via Shh. Development 138, 273-282.

Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron- Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Qin, Y. (1995). Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70-74.

Schulte-Merker, S., van Eeden, F. J., Halpern, M. E., Kimmel, C. B. and Nusslein-Volhard, C. (1994). no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120, 1009-1015.

Scott, D. A. (2009). Esophageal Atresia/Tracheoesophageal Fistula Overview. In Gene reviews: University of Washington, Seattle.

119

Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat Genet 21, 138-141.

Serls, A. E., Doherty, S., Parvatiyar, P., Wells, J. M. and Deutsch, G. H. (2005). Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development 132, 35-47.

Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L. and Schuh, A. C. (1995). Failure of blood-island formation and vasculogenesis in Flk-1- deficient mice. Nature 376, 62-66.

Shaw-Smith, C. (2010). Genetic factors in esophageal atresia, tracheo-esophageal fistula and the VACTERL association: roles for FOXF1 and the 16q24.1 FOX transcription factor gene cluster, and review of the literature. Eur J Med Genet 53, 6-13.

Shelton, E. L. and Yutzey, K. E. (2007). Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression. Dev Biol 302, 376-388.

Shimokawa, K., Kimura-Yoshida, C., Nagai, N., Mukai, K., Matsubara, K., Watanabe, H., Matsuda, Y., Mochida, K. and Matsuo, I. (2011). Cell surface heparan sulfate chains regulate local reception of FGF signaling in the mouse embryo. Dev Cell 21, 257-272.

Shu, W., Jiang, Y. Q., Lu, M. M. and Morrisey, E. E. (2002). Wnt7b regulates mesenchymal proliferation and vascular development in the lung. Development 129, 4831-4842.

Singer, J. B., Harbecke, R., Kusch, T., Reuter, R. and Lengyel, J. A. (1996). Drosophila brachyenteron regulates gene activity and morphogenesis in the gut. Development 122, 3707- 3718.

Solloway, M. J. and Robertson, E. J. (1999). Early embryonic lethality in Bmp5;Bmp7 double mutant mice suggests functional redundancy within the 60A subgroup. Development 126, 1753- 1768.

Somanath, P. R., Ciocea, A. and Byzova, T. V. (2009). Integrin and growth factor receptor alliance in angiogenesis. Cell Biochem Biophys 53, 53-64.

Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70-71.

120

Spagnuolo, R., Corada, M., Orsenigo, F., Zanetta, L., Deuschle, U., Sandy, P., Schneider, C., Drake, C. J., Breviario, F. and Dejana, E. (2004). Gas1 is induced by VE-cadherin and vascular endothelial growth factor and inhibits endothelial cell apoptosis. Blood 103, 3005-3012.

Sparrow, M. P., Warwick, S. P. and Mitchell, H. W. (1994). Foetal airway motor tone in prenatal lung development of the pig. Eur Respir J 7, 1416-1424.

Spooner, B. S. and Wessells, N. K. (1970). Mammalian lung development: interactions in primordium formation and bronchial morphogenesis. J Exp Zool 175, 445-454.

Stefater, J. A., 3rd, Lewkowich, I., Rao, S., Mariggi, G., Carpenter, A. C., Burr, A. R., Fan, J., Ajima, R., Molkentin, J. D., Williams, B. O. et al. (2011). Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells. Nature 474, 511-515.

Stennard, F. A., Costa, M. W., Elliott, D. A., Rankin, S., Haast, S. J., Lai, D., McDonald, L. P., Niederreither, K., Dolle, P., Bruneau, B. G. et al. (2003). Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev Biol 262, 206-224.

Stupack, D. G. and Cheresh, D. A. (2002). ECM remodeling regulates angiogenesis: endothelial integrins look for new ligands. Sci STKE 2002, pe7.

Stupack, D. G. and Cheresh, D. A. (2004). Integrins and angiogenesis. Curr Top Dev Biol 64, 207-238.

Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N. and Yancopoulos, G. D. (1996). Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-1180.

Takashima, S., Kitakaze, M., Asakura, M., Asanuma, H., Sanada, S., Tashiro, F., Niwa, H., Miyazaki Ji, J., Hirota, S., Kitamura, Y. et al. (2002). Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci U S A 99, 3657-3662.

Takeuchi, J. K., Koshiba-Takeuchi, K., Suzuki, T., Kamimura, M., Ogura, K. and Ogura, T. (2003). Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade. Development 130, 2729-2739.

121

Tamura, K., Yonei-Tamura, S. and Izpisua Belmonte, J. C. (1999). Differential expression of Tbx4 and Tbx5 in Zebrafish fin buds. Mech Dev 87, 181-184.

Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Iseki, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J. and Arai, H. (2006). Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid. J Biol Chem 281, 25822-25830.

Tempel, C., Gilead, A. and Neeman, M. (2000). Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle. Biol Reprod 63, 134-140.

Theveniau-Ruissy, M., Dandonneau, M., Mesbah, K., Ghez, O., Mattei, M. G., Miquerol, L. and Kelly, R. G. (2008). The del22q11.2 candidate gene Tbx1 controls regional outflow tract identity and coronary artery patterning. Circ Res 103, 142-148.

Tien, J. Y. and Spicer, A. P. (2005). Three vertebrate hyaluronan synthases are expressed during mouse development in distinct spatial and temporal patterns. Dev Dyn 233, 130-141.

Tiozzo, C., De Langhe, S., Carraro, G., Alam, D. A., Nagy, A., Wigfall, C., Hajihosseini, M. K., Warburton, D., Minoo, P. and Bellusci, S. (2009). Fibroblast growth factor 10 plays a causative role in the tracheal cartilage defects in a mouse model of Apert syndrome. Pediatr Res 66, 386-390.

Tollet, J., Everett, A. W. and Sparrow, M. P. (2001). Spatial and temporal distribution of nerves, ganglia, and smooth muscle during the early pseudoglandular stage of fetal mouse lung development. Dev Dyn 221, 48-60.

Tremblay, K. D., Dunn, N. R. and Robertson, E. J. (2001). Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development 128, 3609-3621.

Tseng, Y. R., Su, Y. N., Lu, F. L., Jeng, S. F., Hsieh, W. S., Chen, C. Y., Chou, H. C. and Peng, S. S. (2007). Holt-Oram syndrome with right lung agenesis caused by a de novo mutation in the TBX5 gene. Am J Med Genet A 143A, 1012-1014.

Vermot, J., Niederreither, K., Garnier, J. M., Chambon, P. and Dolle, P. (2003). Decreased embryonic retinoic acid synthesis results in a DiGeorge syndrome phenotype in newborn mice. Proc Natl Acad Sci U S A 100, 1763-1768.

122

Villa, N., Walker, L., Lindsell, C. E., Gasson, J., Iruela-Arispe, M. L. and Weinmaster, G. (2001). Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mech Dev 108, 161-164.

Vitelli, F., Morishima, M., Taddei, I., Lindsay, E. A. and Baldini, A. (2002a). Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum Mol Genet 11, 915-922.

Vitelli, F., Taddei, I., Morishima, M., Meyers, E. N., Lindsay, E. A. and Baldini, A. (2002b). A genetic link between Tbx1 and fibroblast growth factor signaling. Development 129, 4605- 4611.

Vokes, S. A., Yatskievych, T. A., Heimark, R. L., McMahon, J., McMahon, A. P., Antin, P. B. and Krieg, P. A. (2004). Hedgehog signaling is essential for endothelial tube formation during vasculogenesis. Development 131, 4371-4380.

Wang, H., Gilner, J. B., Bautch, V. L., Wang, D. Z., Wainwright, B. J., Kirby, S. L. and Patterson, C. (2007). Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies. J Biol Chem 282, 782-791.

Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741-753.

Watson, E. D. and Cross, J. C. (2005). Development of structures and transport functions in the mouse placenta. Physiology (Bethesda) 20, 180-193.

Weaver, M., Dunn, N. R. and Hogan, B. L. (2000). Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis. Development 127, 2695-2704.

Weaver, M., Yingling, J. M., Dunn, N. R., Bellusci, S. and Hogan, B. L. (1999). Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development 126, 4005-4015.

West, D. C., Hampson, I. N., Arnold, F. and Kumar, S. (1985). Angiogenesis induced by degradation products of hyaluronic acid. Science 228, 1324-1326.

White, P. H. and Chapman, D. L. (2005). Dll1 is a downstream target of Tbx6 in the paraxial mesoderm. Genesis 42, 193-202.

123

Whitsett, J. A., Wert, S. E. and Trapnell, B. C. (2004). Genetic disorders influencing lung formation and function at birth. Hum Mol Genet 13 Spec No 2, R207-215.

Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225, 361-373.

Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 9, 2105- 2116.

Xu, B., Chen, C., Chen, H., Zheng, S. G., Bringas, P., Jr., Xu, M., Zhou, X., Chen, D., Umans, L., Zwijsen, A. et al. (2011). Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development. Development 138, 925-935.

Xue, Y., Gao, X., Lindsell, C. E., Norton, C. R., Chang, B., Hicks, C., Gendron-Maguire, M., Rand, E. B., Weinmaster, G. and Gridley, T. (1999). Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 8, 723-730.

Yamagishi, H., Olson, E. N. and Srivastava, D. (2000). The basic helix-loop-helix transcription factor, dHAND, is required for vascular development. J Clin Invest 105, 261-270.

Yamaguchi, T. P., Bradley, A., McMahon, A. P. and Jones, S. (1999). A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126, 1211- 1223.

Yamamoto, A., Amacher, S. L., Kim, S. H., Geissert, D., Kimmel, C. B. and De Robertis, E. M. (1998). Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Development 125, 3389-3397.

Yamamura, H., Zhang, M., Markwald, R. R. and Mjaatvedt, C. H. (1997). A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse. Dev Biol 186, 58- 72.

Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M. and Nakao, K. (2000). Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92-96.

Yanagisawa, K. O., Fujimoto, H. and Urushihara, H. (1981). Effects of the brachyury (T) mutation on morphogenetic movement in the mouse embryo. Dev Biol 87, 242-248.

124

Yin, Y., Wang, F. and Ornitz, D. M. (2011). Mesothelial- and epithelial-derived FGF9 have distinct functions in the regulation of lung development. Development 138, 3169-3177.

Yin, Y., White, A. C., Huh, S. H., Hilton, M. J., Kanazawa, H., Long, F. and Ornitz, D. M. (2008). An FGF-WNT gene regulatory network controls lung mesenchyme development. Dev Biol 319, 426-436.

Yuan, H. T., Suri, C., Landon, D. N., Yancopoulos, G. D. and Woolf, A. S. (2000). Angiopoietin-2 is a site-specific factor in differentiation of mouse renal vasculature. J Am Soc Nephrol 11, 1055-1066.

Zaret, K. S. (2002). Regulatory phases of early liver development: paradigms of organogenesis. Nat Rev Genet 3, 499-512.

Zeigler, B. M., Sugiyama, D., Chen, M., Guo, Y., Downs, K. M. and Speck, N. A. (2006). The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential. Development 133, 4183-4192.

Zhang, H. and Bradley, A. (1996). Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 2977-2986.

Zudaire, E., Gambardella, L., Kurcz, C. and Vermeren, S. (2011). A computational tool for quantitative analysis of vascular networks. PLoS One 6, e27385.