The Role of Pitx Proteins in Early <I>Xenopus</I> Development

City University of New York (CUNY) CUNY Academic Works

All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects

9-2017

The Role of Pitx Proteins in Early Xenopus Development

Ye Jin The Graduate Center, City University of New York

How does access to this work benefit ou?y Let us know!

More information about this work at: https://academicworks.cuny.edu/gc_etds/2263 Discover additional works at: https://academicworks.cuny.edu

This work is made publicly available by the City University of New York (CUNY). Contact: [email protected]

THE ROLE OF PITX PROTEINS IN EARLY

XENOPUS DEVELOPMENT

YE JIN

A dissertation submitted to the Graduate Faculty in Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2017

THE ROLE OF PITX PROTEINS IN EARLY XENOPUS DEVELOPMENT

YE JIN

This manuscript has been read and accepted for the Graduate Faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy.

______Date Chair of Examining Committee Dr. Daniel Weinstein, Queens College

______Date Executive Officer Dr. Laurel A. Eckhardt

______Dr. Cathy Savage-Dunn, Queens College

______Dr. Nathalia Holtzman, Queens College

______Dr. Mitchell Goldfarb, Hunter College

______Dr. Xiajun Li, ShanghaiTech University

Supervising Committee

THE CITY UNIVERSITY OF NEW YORK

iii

ABSTRACT

THE ROLE OF PITX PROTEINS IN EARLY XENOPUS DEVELOPMENT

YE JIN

Advisor: Dr. Daniel Weinstein

Embryos of the African clawed frog Xenopus laevis are widely used for the study of early vertebrate development. The cement gland, which secrets mucus to help tadpoles attach to solid supports and live in relative safety, has long been used as a model to study the interplay between cell signaling pathways and transcription factors. It has been proposed that an intermediate level of Bone Morphogenetic

Protein (BMP) signaling is essential for cement gland formation. In addition, several transcription factors have been linked to cement gland development. Among them, the homeodomain proteins Pitx1 and the closely related Pitx2c can generate ectopic cement gland formation; however, the mechanisms underlying this process remain obscure. We report here, for the first time, a requirement for Pitx proteins in cement gland formation, in vivo: knockdown of both Pitx1 and Pitx2c inhibits endogenous cement gland formation. Pitx1 transcriptionally activates downstream target genes through both direct and indirect mechanisms, and functions as a transcriptional activator to inhibit BMP signaling. This inhibition, required for the expression of pitx genes, is partially mediated by Pitx1-dependent follistatin expression. Depletion of

BMP proteins inhibits induction of cement gland markers by Pitx1; furthermore, we

iv find that Pitx1 physically interacts with Smad1, an intracellular transducer of BMP signaling. We propose a model of cement gland formation in which Pitx1 limits local

BMP signaling within the cement gland primordium, and recruits Smad1 to activate cement gland genes.

We also identify the homeobox gene goosecoid, expressed in the pharyngeal endoderm adjacent to the cement gland, as a target of Pitx1. We find that Pitx1 inhibits Activin activity and binds to Smad2/3, an intracellular transducer of

Activin/TGFβ signaling. Our studies suggest that a transactivating complex including

Pitx1 and Smad2/3 stimulates goosecoid transcription during the development of anterior endoderm.

Acknowledgements

The completion my dissertation would be impossible without the support from many people. I would like to express my deepest gratitude to my mentor Dr. Daniel

Weinstein. He has been constantly training me to become an independent scientific researcher with inspiration, motivation and critical thinking. I am grateful for his advice on many aspects during my PhD career. In addition, I am also grateful for his outstanding editing on my proposal, manuscript, dissertation and many other materials. I have absolutely cherished my time studying developmental biology in the Weinstein lab and have enjoyed our many discussions over the years.

I would also like to extend my sincere thanks to all of my fellow lab members, especially to Dr. Tomomi Haremaki for teaching me many experimental techniques and helping me on the initial stages of the project, and to Sushma Teegala for sharing so much time with me in our lab. Moreover, I would like to express my special appreciation to Dr. Cathy Savage-Dunn and Dr. Nathalia Holtzman, who kept monitoring my progress and offered me their precious scientific insights and experience, and to Dr. Mitchell Goldfarb and Dr. Xiajun Li, who have been members of my committee for more than 5 years.

My family and friends provide me with infinite mental support. This dissertation is dedicated to my father Zhenye Xu and mother Changjuan Jin. Actually, my interest in studying biology originates from their contribution to the oncology field. Their understanding and encouragement is the main driving source for me. The strongest

vi gratitude is to my beloved Huijuan Yuan for her long-term company in the aspects of both academia and daily life; to my close friends Xin Zhao and Wenchao Lu for their constant helpfulness. Last, I would like to express my deepest respect for the memory of my grandparents.

vii

Table of Contents

Chapter 1: General introduction ...... 1 1.1 Xenopus ...... 1 1.2 Germ layer formation ...... 2 1.3 The formation of mesoderm and endoderm ...... 4 1.4 The formation of ectoderm...... 9 1.5 Dorsal-ventral patterning in the Xenopus ectoderm ...... 10 1.6 Pituitary homeobox (Pitx) genes ...... 11 Chapter 2: Materials and methods ...... 15 2.1 Synthetic RNA preparation, embryo culture, and explant dissection ...... 15 2.2 Constructs and morpholinos ...... 15 2.3 Dexamethasone and cycloheximide treatment ...... 17 2.4 Reverse transcription-polymerase chain reaction (RT-PCR) ...... 17 2.5 Whole-mount in situ hybridization ...... 18 2.6 Western blot analysis ...... 19 2.7 Co-immunoprecipitation ...... 20 2.8 RNA-sequencing ...... 20 Chapter 3: Pitx1 transcriptionally activates cement gland differentiation genes through both direct and indirect mechanisms ...... 22 3.1 Introduction ...... 22 3.2 Results ...... 25 3.2.1 Misexpression of pitx1 results in extra cement gland formation in Xenopus embryos ...... 25 3.2.2 Knockdown of Pitx1 and Pitx2c inhibits endogenous cement gland formation ...... 26 3.2.3 Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly ...... 28 3.2.4 follistatin is a transcriptional target of Pitx1 in the developing cement gland ...... 31 3.3 Discussion ...... 33

viii

Chapter 4: Intermediate levels of BMP signaling are required for cement gland formation ...... 37 4.1 Introduction ...... 37 4.2 Results ...... 38 4.2.1 Pitx1 functions as a transcriptional activator to inhibit BMP/Smad1 signaling ...... 38 4.2.2 Rescue of Pitx1-mediated BMP signal inhibition represses expression of pitx genes ...... 40 4.2.3 BMP/Smad1 signaling is required for cement gland development ...... 43 4.3 Discussion ...... 46 Chapter 5: Pitx1 regulates goosecoid transcription ...... 48 5.1 Introduction ...... 48 5.2 Results ...... 50 5.2.1 Pitx1 induces goosecoid expression but inhibits Activin activity ...... 50 5.2.2 Pitx1 physically interacts with Smad2/3 ...... 52 5.3 Discussion ...... 53 Chapter 6: Summary ...... 55 References...... 58

Table of Tables

Table 1. List of primers used for RT-PCR...... 18

Table of Figures

Figure 1. Early development of the Xenopus embryo...... 4

Figure 2. Temporal expression of pitx1...... 13

Figure 3. Expression of Xenopus pitx1 and pitx2c at mid-neurula stages...... 13

Figure 4. Drawing of a tailbud stage Xenopus embryo showing anterior structures...... 23

Figure 5. pitx1 misexpression results in extra cement gland formation...... 25

Figure 6. Knockdown of Pitx1 and Pitx2c inhibits endogenous cement gland formation...... 27

Figure 7. Fusion constructs of pitx1...... 30

Figure 8. Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly...... 30

Figure 9. follistatin expression in the cement gland is dependent on Pitx1...... 32

Figure 10. Pitx1 functions as a transcriptional activator to inhibit BMP signaling.... 39

Figure 11. Rescue of Pitx1-mediated inhibition of BMP signaling represses expression of pitx genes...... 41

Figure 12. CA-BMPRIa rescues the C-terminal phosphorylation of Smad1/5 inhibited by either Pitx1 or Noggin...... 42

Figure 13. BMP/Smad1 signaling is required for cement gland development...... 44

Figure 14. Pitx1-Myc, like Pitx1, inhibits BMP activity and promotes cement gland differentiation...... 45

Figure 15. Pitx1 functions as a transcriptional activator to stimulate goosecoid expression...... 51

Figure 16. Pitx1 inhibits Activin activity...... 51

Figure 17. Pitx1 binds to exogenous Smad2/3...... 52

Figure 18. Proposed model of Pitx1-mediated induction of goosecoid transcription...... 55

Figure 19. Proposed model of Pitx1-mediated regulation of cement gland development...... 57

Chapter 1: General introduction

1.1 Xenopus

Xenopus is a genus of highly aquatic frogs belonging to the Pipidae family. In the wild,

Xenopus tend to live in rivers, lakes, and swamps. They are scavengers and have a varied diet in the natural environment. The male and female mate in the water. After external fertilization, fertilized eggs take 3-4 days to develop into swimming tadpoles. Like most amphibians, young Xenopus tadpoles undergo metamorphosis, becoming adult frogs within 12 months (Bernardini, 1999).

Currently, 20 species have been identified as belonging to the Xenopus genus.

Among them, Xenopus laevis (also known as the African clawed frog) has been long used as a model organism to study vertebrate embryology and development (Evans et al., 2015). Several advantages make Xenopus embryos an ideal tool for studies of developmental biology. They are easily maintained in laboratory aquatic systems and can survive more than 10 years. Female frogs are induced to lay eggs by injecting commercially available Human Chorionic Gonadotropin subcutaneously at any time of the year. The minimum interval between priming of each female frog is 3 months. Xenopus embryos are easy to manipulate under the dissecting microscope, due to their relatively large size (~1 mm diameter). Routine manipulations include germ layer dissection, tissue transplantation, and injection of various materials into specific blastomeres. Overexpression or misexpression of a specific gene product is achieved by injecting synthetic RNA encoding the gene into developing embryos at

1 early cleavage stages. Knockdown of target gene expression is accomplished by injecting antisense morpholino oligonucleotides which inhibit either translation or splicing of target RNA. Xenopus embryos, due to their availability in large quantities, can also provide abundant material for high-throughput analysis (Wheeler and

Brandli, 2009). The genome of Xenopus laevis is currently being sequenced and updated annotations are continuously released on the Xenopus database website

Xenbase.

1.2 Germ layer formation

Embryonic development of Xenopus laevis consists of several major steps. A fertilized zygote undergoes a series of cleavages to eventually form a sphere of cells, called the blastula surrounding a cavity called the blastocoel. The blastula embryo is divided into an animal pole, and a vegetal pole, separated by an equatorial marginal zone (Fig. 1A). Cells which undergo more rapid division in the animal pole form a dome-like structure covering the blastocoel; the vegetal pole, which contains heavy yolk-filled cells, is at the base of this cavity (reviewed in Heasman, 2006).

Gastrulation, during which the primary germ layers are determined and spatially rearranged, begins with the formation of the dorsal lip of the blastopore. During this process, cells of the animal pole spread out, in a process called epiboly, to form the outer layer of the embryo, which is called ectoderm; cells of marginal zone and vegetal pole involute through the blastopore into the interior of the embryo, where they form the mid-layer and the inner layer, which are called mesoderm and

2 endoderm respectively. Initial steps in the elongation of the dorsal mesoderm, and thus the embryo, are driven by a process known as convergent extension (reviewed in Winklbauer and Damm, 2012).

Gastrulation is followed by neurulation during which the neural plate folds in to form the neural tube. During and after neurula stages, the three germ layers undergo differentiation to form the tissues and organs of the developing embryo.

The ectoderm develops into the nervous system, epidermis, neural crest-derived and cranial placode-derived tissues. The mesoderm gives rise to the notochord, skeleton, muscles, cartilage, dermis, circulatory system, lymphatic system, kidneys and gonads. The endoderm forms the epithelial lining and associated organs of the digestive, respiratory and urinary systems as well as various endocrine glands (Fig.

1B) (reviewed in Kiecker et al., 2016).

Figure 1. Early development of the Xenopus embryo. (A) Diagram of a blastula stage Xenopus embryo. The animal pole gives rise to ectoderm; the marginal zone becomes mesoderm; vegetal pole forms endoderm. (B) Fate map of the Xenopus embryo. Indicated tissues arise from corresponding regions of the embryo.

1.3 The formation of mesoderm and endoderm

Nieuwkoop and his colleagues demonstrated that animal "cap" explants form ectoderm, and vegetal explants form endoderm, when cultured in isolation. When these two types of explants were co-cultured as aggregates, however, mesendoderm

(a population of cells that differentiate into both mesoderm and endoderm) was induced at the expense of ectoderm, suggesting that an inductive signal secreted from the vegetal cells induces competent presumptive ectodermal cells to become mesendodermal cells (Nieuwkoop, 1967). It was also found that the dorsal region of the vegetal cell mass induces dorsal mesoderm, whereas the ventral region of the

4 vegetal cell mass induces ventral mesoderm (Nieuwkoop, 1973). Prior to

Nieuwkoop's studies, Spemann and Mangold had characterized the ability of the dorsal marginal zone (DMZ) to specify dorsal identity. Transplantation of tissue from this region (later dubbed the "Spemann Organizer") at the onset of gastrulation to the ventral side of a host embryo leads to the formation of a secondary body axis. Most of the tissues in the ectopically-induced twin axis are derived from the host embryo, indicating that the grafted tissue switches the fate of surrounding tissue from ventral to dorsal (reviewed in Harland and Gerhart, 1997).

Studies from Nieuwkoop and others demonstrated that dorsal mesoderm, including the Spemann Organizer, is induced by dorsovegetal cells; the signaling center in the dorsovegetal blastomere is often referred to as "Nieuwkoop Center" (reviewed in

Gerhart, 1999). In the decades following these studies, multiple factors involved in mesendodermal induction have been identified and characterized.

The first protein shown to induce mesoderm in animal cap explants was basic fibroblast growth factor (Slack et al., 1987); FGF, however, does not induce endoderm (Fletcher and Harland, 2008). Moreover, loss of FGF signaling does not completely eliminate mesoderm formation, suggesting that other factors may be responsible for mesendoderm induction. Several studies have proposed that, rather than being the instructive inducer, FGFs establish the competence for mesoderm induction by another group of factors, the transforming growth factor β (TGFβ) family (LaBonne and Whitman, 1994).

The TGFβ family consists of a large number of structurally related signaling proteins that control many cellular processes, including cell proliferation and differentiation.

The family includes TGFβs, Activins, Nodals, Bone Morphogenetic proteins (BMPs) and Growth and Differentiation factors (GDFs). TGFβ signaling begins with the binding of extracellular TGFβ dimers to their corresponding type II receptors, which recruit and activate type I receptors. The activated type I receptor, which functions as a serine/threonine kinase, phosphorylates two C-terminal serine residues of receptor-regulated Smad (R-Smad) proteins. The C-terminally phosphorylated R-

Smads form a complex with the cofactor Smad4 through direct physical association.

The complex enters the nucleus and regulates the transcription of target genes through binding to their promoter regions. There are two branches of the R-Smad family. One group, Smads1/5/8, are intracellular transducers for BMP and GDF signaling, while the other branch, Smads2/3, are responsible for the signal transduction triggered by other ligands in the TGFβ family (reviewed in Moustakas and Heldin, 2009).

Activin A was the first TGFβ factor associated with mesendoderm induction in

Xenopus laevis. Activin A induces a range of dorsal-ventral mesodermal cell fates in a dose-dependent fashion (Green and Smith, 1990). At even higher doses, Activin A treatment also results in the induction of endodermal markers (Ninomiya et al.,

1999). However, claims that Activin A is the primary endogenous mesendodermal- inducing factor remain controversial. Addition of the Activin inhibitor Follistatin fails to strongly affect mesoderm formation in Xenopus embryos (Schulte-Merker et

6 al., 1994). Furthermore, disruption of Activin signaling downstream of the ligand result in much stronger defects of mesendoderm induction than elimination of the ligand itself does, suggesting that other TGFβ factors involved in this process signal via the same pathway (Hemmati-Brivanlou and Melton, 1992).

Loss of function studies revealed that Nodal in the mouse and the Nodal-like proteins Cyclops and Squint in Zebrafish are required for mesoderm and endoderm formation (Conlon et al., 1994; Feldman et al., 1998). Six Xenopus nodal-related (Xnr) genes have been identified. Among them, Xnr5 and Xnr6 are the two earliest zygotically expressed genes, with transcription even detected prior to the midblastula transition (the onset of zygotic transcription of most genes) (reviewed in

Shen, 2007). Simultaneous antisense morpholino-mediated knockdown of these two genes leads to severe defects in mesendoderm formation (Luxardi et al., 2010).

Moreover, similar to Activin, Xnrs induce a variety of dorsoventral mesodermal cell fates in a dose-dependent manner (Jones et al., 1995). It has been shown that Xnrs are expressed more strongly in the dorsal than the ventral vegetal hemisphere. Thus, a model is proposed in which Xnrs, released from the vegetal blastomeres in a dorsal-ventral gradient, induce endoderm at highest levels, dorsal mesoderm at intermediate levels, ventral mesoderm at low levels; ectoderm formation occurs in the absence of the inducer (Agius et al., 2000).

Which factors contribute to the zygotic expression of Xnrs in a DV gradient? Possible factors include maternal transcripts deposited in the egg before fertilization. β-

7 catenin, an intracellular transducer of Wnt signaling initially found throughout the vegetal pole, becomes localized to the dorsal half of the embryo possibly through translocation along microtubules during an event known as cortical rotation triggered by fertilization (Rowning et al., 1997). Additional components of Wnt signaling, such as the adaptor protein Dishevelled, are also transported to the dorsal side of the early embryo (Miller et al., 1999). In addition, similar to results seen in the Spemann Organizer grafting experiments, misexpression of Wnt in ventrovegetal blastomeres results in a complete duplication of the embryonic axis

(McMahon and Moon, 1989). Direct evidence linking Nodal and Wnt/β-catenin signaling came from the demonstration that early Wnt signaling induces Xnr5 and

Xnr6, which are essential for mesendoderm formation (Yang et al., 2002).

Two other maternal factors, Vg1 and VegT, both localized vegetally, are also involved in mesendoderm induction. As a member of the TGFβ family, Vg1 is synthesized as a precursor peptide that requires cleavage and dimerization for activity. Although ectopic expression of Vg1 fails to produce axial duplication, this is possibly due to inefficient processing of the exogenous RNA (Thomsen and Melton,

1993); depletion of Vg1 by either antisense morpholinos or dominant-negative forms results in severe defects in mesendoderm formation (Birsoy et al., 2006;

Joseph and Melton, 1998). Misexpression of the T-domain transcription factor VegT results in the ectopic expression of mesendodermal markers (Horb and Thomsen,

1997). Loss of maternal VegT mRNA causes embryos to display a reduction of mesendoderm and an expansion of ectoderm into the equatorial region;

8 furthermore, vegetal blastomeres depleted in VegT lose the ability to induce mesendoderm in co-cultured animal cap explants (Zhang et al., 1998). VegT is also required for the expression of FGFs and of multiple Xnrs (Kofron et al., 1999). Taken together, vegetally localized maternal Vg1 and VegT together with dorsally localized

Wnt/β-catenin signaling appear to establish a dorsoventral gradient of Nodal- related signaling emanating from the vegetal pole. Distinct levels of Nodal-related signaling induce formation of the endoderm, dorsal mesoderm (including the

Spemann Organizer) and ventral mesoderm. Further germ layer specification requires the involvement of germ layer-specific transcription factors, such as

Brachyury and Eomesodermin for mesoderm, and Sox17 and GATA family proteins for endoderm (reviewed in Kiecker et al., 2016).

1.4 The formation of ectoderm

The ectoderm consists of two layers, an outer epithelial layer and an inner sensorial layer. As described above, addition of mesendoderm-inducing growth factors induce mesoderm or endoderm in ectodermal explants; factors that protect ectodermal cells from excessive inducers are needed for ectoderm formation, both in explants and in vivo. Coco, an antagonist of both BMP and Nodal ligands, is expressed maternally throughout the animal hemisphere of Xenopus embryos (Bell et al., 2003).

Knockdown of Coco results in embryos that display an expansion of endoderm; this effect can be rescued by elimination of either Activin or Xnr5/6 (Bates et al., 2013).

Another maternal factor, with higher transcript distribution on the dorsal side of the animal hemisphere, is Ectodermin, a RING-type E3 ubiquitin ligase that targets

Smad4 for proteasome-mediated degradation. Since Smad4 is a co-Smad for both

Smad2/3 and Smad1/5/8, Ectodermin inhibits both Nodal and BMP signaling.

Knockdown of maternal Ectodermin mRNA results in the expansion of both endoderm and non-neural ectoderm at the expense of mesoderm and neuroectoderm, respectively (Dupont et al., 2005).

Several transcription factors are also involved in the suppression of mesendoderm.

One of these is Xema (Xenopus ectodermally-expressed mesendoderm antagonist), a member of the forkhead box gene family that is expressed zygotically in the animal pole before gastrulation. Ectopic expression of Xema suppresses mesoderm induction, in both animal caps treated with growth factors and in the marginal zone of intact embryos. Conversely, antisense morpholino-mediated knockdown of Xema stimulates the expression of mesendodermal genes in the animal pole (Suri et al.,

2005). Sox3 is another transcription factor that is reported to inhibit mesendoderm induction, by directly repressing Xnr5 expression as a transcriptional repressor

(Zhang et al., 2003). In summary, both extracellular and intracellular factors that inhibit Activin/Nodal-related signaling are required to restrict ectopic mesendoderm induction in the prospective ectoderm.

1.5 Dorsal-ventral patterning in the Xenopus ectoderm

Dorsal-ventral patterning in the Xenopus ectoderm is regulated by signaling through the Bone Morphogenetic Protein receptors (BMPRs), expressed throughout the gastrula stage ectoderm (Hawley et al., 1995; Wilson and Hemmati-Brivanlou, 1995;

Xu et al., 1995). Ventrally, high levels of Bone Morphogenetic Protein (BMP) ligands bind to the BMPRs, initiating a signaling cascade that results in the activation and nuclear retention of the intracellular Smad1/5-Smad4 complex, the transactivation of BMP target genes, and the initiation of the program for epidermal differentiation

(Suzuki et al., 1997; Wilson et al., 1997). Dorsally, extracellular BMP antagonists secreted by the Spemann Organizer bind BMP proteins and prevent them from activating their cognate receptors—Smad1/5 signaling remains inactive, BMP target genes are not activated and the dorsal ectoderm subsequently differentiates as neural tissue, the "default" fate of the ectoderm (reviewed in Weinstein and

Hemmati-Brivanlou, 1999).

1.6 Pituitary homeobox (Pitx) genes

It has been proposed that there is a direct relationship between levels of BMP activity and dorsoventral ectodermal fate (Knecht and Harland, 1997; Wilson et al.,

1997). As an ectodermal organ located in the anterior border of the neural plate, the cement gland can be induced following expression of BMP antagonists (e.g., Chordin,

Noggin and Follistatin) (reviewed in Sive and Bradley, 1996). Neural tissue

(positioned more dorsally) is also induced in concert with the cement gland upon inhibition of BMP signaling, suggesting that other factors are required to discriminate between the cement gland and neural fates. Two Pituitary homeodomain transcription factors, Pitx1 and Pitx2c, have been implicated in cement gland induction (Schweickert et al., 2001a). The zygotic expression of

Xenopus pitx1 begins at late gastrula stages (Fig. 2). At mid-neurula stages, expression of pitx1 is localized in the cement gland primordium and overlying stomodeal/hypophyseal (pituitary) anlage (Fig. 3D) (Schweickert et al., 2001b). The stomodeal/pituitary anlage develops into two separate tissues, the ectodermal mouth plate and Rathke's pouch. The pouch eventually forms the glandular portion of the pituitary gland, which produces and secretes diverse hormones including growth hormone, adrenocorticotropin, thyroid stimulating hormone, gonadotropins and prolactin (reviewed in Kawamura et al., 2002). Pitx1 functions as an early pan- pituitary transcriptional activator during the differentiation of various cell types in the anterior pituitary gland. After the maturation of the pituitary gland, Pitx1 is reported to activate the transcription of the β subunits of Luteinizing Hormone and

Follicle Stimulating Hormone through direct binding to their promoters (reviewed in Bernard et al., 2010). Later, at tailbud stages, Xenopus pitx1 expression is observed in the foregut including the pharyngeal endoderm (Schweickert et al.,

2001b). It has been shown that Pitx1 directly activates Shroom3 transcription to control gut morphogenesis (Chung et al., 2010). In addition, pitx1 mRNA is detected in the lens, midbrain and posterior lateral plate mesoderm. It is reported that Pitx1 is required for the development of hindlimbs.

Figure 2. Temporal expression of pitx1. The zygotic expression of pitx1 begins at late gastrula stages (stage 12). Zygotic expression of the organizer gene chordin begins at early gastrula stages (stage 10) (Sasai et al., 1994). Ornithine decarboxylase (ODC) is used as a loading control (Bassez et al., 1990). RT-PCR analysis was performed on embryos collected at the indicated stages. The -RT lane contains all reagents except reverse transcriptase, and is used as a negative control. UF, unfertilized.

Figure 3. Expression of Xenopus pitx1 and pitx2c at mid-neurula stages. pitx1 and pitx2c are co-expressed in the cement gland primordium (black arrowhead) and the stomodeal/pituitary anlage (yellow arrowhead). (D') and (F') show the sections of the embryos in (D) and (F), respectively. The picture is adapted from Schweickert A, Steinbeisser H, Blum M, 2001, Differential gene expression of Xenopus Pitx1, Pitx2b and Pitx2c during cement gland, stomodeum and pituitary development. Mech Dev 107, 191- 194.

Xenopus pitx2 has several isoforms, among which pitx2c shares the highest overlap in expression, during early embryonic development, with pitx1. Expression of pitx2c at neurula stages, like that of pitx1, is localized in the cement gland primordium and stomodeal/pituitary anlage (Fig. 3F) (Schweickert et al., 2001b). Later, pitx2c is also expressed in the left lateral plate mesoderm and heart anlage (Schweickert et al.,

2000). Pitx2c has been shown to regulate left-right asymmetry of both heart and gut looping in response to Nodal and sonic hedgehog signaling in mouse embryos (Ryan et al., 1998).

Chapter 2: Materials and methods

2.1 Synthetic RNA preparation, embryo culture, and explant dissection

RNA for microinjection was synthesized in vitro in the presence of cap analog using the mMessage mMachine kit (Ambion, AM1340). After in vitro fertilization, synthetic RNA was injected into the animal pole of 2- or 4-cell stage Xenopus embryos cultured in Ficoll solution (Sigma, F4375). 3-4 hours post-injection,

Xenopus embryos were transferred to and cultured in 0.1× MMR. At late blastula stages (stage 8.5-9), animal cap (ectodermal) explants were dissected and cultured in 0.5× MMR with 0.05% BSA until mid-neurula stages (stage 16-18) or late neurula stages (stage 19-21) for collection (Hemmati-Brivanlou and Melton, 1994; Wilson and Hemmati-Brivanlou, 1995).

2.2 Constructs and morpholinos

The coding sequence of pitx1 was isolated by PCR from cDNA derived from mid- neurula stage Xenopus laevis embryos using the following primers: 5'-

GGAATTCACCATGGATTCCTTTAAAG; 5'-CCTCGAGTCAACTGTTATATTGGC, and cloned into the EcoRI and XhoI sites of the pCS2++ vector (Hollemann and Pieler,

1999). Pitx1 fusion constructs were generated by PCR. For VP16-Pitx1, residues

410-490 of the VP16 transcriptional activator were fused upstream of the complete coding sequence of Xenopus pitx1 (Kessler, 1997). For EnR-Pitx1, residues 1-298 of the Drosophila Engrailed transcriptional repressor were fused upstream of the complete coding sequence of Xenopus pitx1 (Kessler, 1997). For Pitx1-Myc, six Myc

15 epitopes were fused downstream of the complete coding sequence of Xenopus pitx1.

A fusion construct of the ligand-binding domain of the human glucocorticoid receptor and Xenopus pitx1 (Pitx1-GR) was a gift from John Wallingford (Chung et al.,

2010). pCS2-hSmad1-WT was a gift from Edward De Robertis (Pera et al., 2003). pBS-XFS-319 was a gift from Ali Hemmati-Brivanlou (Hemmati-Brivanlou et al.,

1994). The constitutively active BMP receptor type Ia was subcloned from pCMV- hBMPR-1A-CA (a gift from Lee Niswander & Peter ten Dijke) into the pCS2++ vector

(Varley et al., 1998). pSP64T-noggin was a gift from Richard Harland (Smith and

Harland, 1992). pCS2-Flag-Smad2 was a gift from Joan Massague (Hata et al., 1997). pCS2-Flag-Smad3 was also a gift from Joan Massague (Kretzschmar et al., 1999).

Morpholinos were heated for 10 minutes at 65°C, then quenched on ice prior to injection at two- or four-cell stages. Antisense morpholino oligonucleotides (Gene

Tools) used in this study are as follows:

Pitx1-MO: 5'-CATGGTCAATCACTTCTGCTCATGA (Chung et al., 2010)

Pitx2-MO: 5'-GGTACAGTACAGTAGGCTCACAGAC

Follistatin-MO: 5'-CCTTTCATTTAACATCCTCAGTGCT

2.3 Dexamethasone and cycloheximide treatment

For studies with Pitx1-GR fusion constructs, 400 pg pitx1-GR RNA was injected into the animal pole of early cleavage stages embryos. pitx1-GR-injected animal cap explants, dissected at late blastula stages, were treated with 10 µM dexamethasone

(Sigma, D1756) at late gastrula stages (stage 12). In the co-treatment condition, dexamethasone was added 10 minutes after addition of 10 µg/ml cycloheximide

(Sigma, C7698) at stage 12 (Chung et al., 2010).

2.4 Reverse transcription-polymerase chain reaction (RT-PCR)

Harvested animal cap explants or embryos were homogenized in Solution D lysis buffer. Cell lysates were subject to RNA Bee (Tel-Test, CS105B) for total RNA isolation and chloroform for phase separation. Isopropanol was used for RNA precipitation. Subsequently, RNA pellets washed by 75% ethanol were dissolved in nuclease-free water. cDNA was synthesized in vitro using M-MLV reverse transcriptase (Promega, M170A). PCR was performed using Taq DNA polymerase

(Denville Scientific, CB4050-1).

Primers used in this study are as follows:

Gene name Forward primer Reverse primer

EF1α 5'-CAGATTGGTGCTGGATATGC 5'-ACTGCCTTGATGACTCCTAG

ODC 5'-AATGGATTTCAGAGACCA 5'-CCAAGGCTAAAGTTGCAG

Xag1 5'-CTGACTGTCCGATCAGAC 5'-GAGTTGCTTCTCTGGCAT

Xcg 5'-ACCAAAAGCACTCCGTCAAC 5'-TTGGCGCAGTGGAACTAAAG

agr2 5'-CCAGCAAAAGTCTCAAAGCC 5'-TGGATACCTTGGTGTTCAGC

nkx3.1 5'-ACATGTCCCATCCAGTCAAG 5'-CGTTTCTGTTGCTGCTTTGC

TGFβ1 5'-AGCTGCGCATGTACAAGAAG 5'-TTTCCTCTGCACGTTTCAGC

bmp4 5'-TGACACGGGCAAGAAGAAAG 5'-TCCAAATGCTCCTCGTGATG

sizzled 5'-CACTAACATGGCAGAAGTCG 5'-GGAACCTGTCACAGTCTAAG

cv2 5'-AATGTGCCTCACCTTTCCTG 5'-TTACAGCGTTCACAGCAAGC

follistatin 5'-AAAAGACTTGCAGGGACGTG 5'-ACAGGCATTTCTTTCCAGCG

pitx2b 5'-GGATTCACCAAAGTGGCAGT 5'-TCAGTTTGTTGGTTCCTCTC

pitx2c 5'-TCCAGCCCAGACACTGCA 5'-TGCATCAGTCCATTGAACTG

pitx1 5'-AAATCCAAGCAGCACTCCAC 5'-ACAACCCGCATAATCCAGAG

goosecoid 5'-AGAGTTCATCTCAGAGAG 5'-TCTTATTCCAGAGGAACC

muscle actin 5'-GCTGACAGAATGCAGAAG 5'-TTGCTTGGAGGAGTGTGT

Xwnt8 5'-GTTCAAGCATTACCCCGGAT 5'-CTCCTCAATTCCATTCTGCG

brachyury 5'-GGATCGTTATCACCTCTG 5'-GTGTAGTCTGTAGCAGCA

Table 1. List of primers used for RT-PCR.

2.5 Whole-mount in situ hybridization

Antisense RNA probes were synthesized in vitro using Digoxigenin RNA Labeling

Mix (Roche, 11277073910). Albino embryos fixed with MEMFA were gradually rehydrated, followed by Proteinase K (10 µg/ml) treatment. Subsequently, embryos refixed with 4% paraformaldehyde were subject to prehybridization for 6 hours and

18 hybridization with RNA probe at 60°C overnight. After multiple washes to remove excess probe, embryos were incubated with blocking reagent (Roche, 11096176001) for 1 hour at room temperature, followed by incubation with anti-digoxygenin antibody at 4°C overnight. Anti-digoxygenin Fab fragments (Roche, 11093274910) coupled to alkaline phosphatase were used at 1:1000 dilution. Chromogenic reactions were performed using NBT (Sigma, N6639) and BCIP (Sigma, B8503) (Suri et al., 2005).

2.6 Western blot analysis

Collected animal cap explants or embryos were homogenized in Mitch's lysis buffer

(Hama et al., 2002). Cell lysates were subject to high-speed centrifuge and the supernatant was transferred to a clean tube. The supernatant with added sample buffer (Novex, NP0007) was boiled for 10 minutes. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4-12% Bis-Tris gel (Novex,

NP0321). Proteins on the gel were transferred to the PVDF membrane (Millipore,

IPVH00010), which was then incubated with blocking buffer for 1 hour at room temperature. Subsequently, the PVDF membrane was incubated with primary antibody at 4°C overnight, followed by secondary antibody incubation for 1 hour at room temperature. Pierce Western blotting Substrate (Thermo Fisher Scientific,

32106) was added to the PVDF membrane for chemiluminescence. Primary antibodies against phospho-Smad1/5 (Cell Signaling Technology, 9516), Smad1

(Cell Signaling Technology, 6944), and β-tubulin (Sigma) were used at 1:500,

1:1,000, and 1:1,000 dilutions, respectively. Secondary antibodies (donkey anti-

19 rabbit IgG, or donkey anti-mouse IgG) coupled to horseradish peroxidase (Jackson

ImmunoResearch, 711-036-152 or 715-036-151) were used at 1:10,000 dilution.

2.7 Co-immunoprecipitation pitx1-myc RNA was injected into early cleavage stages embryos. Injected embryos were harvested at mid-neurula stages. Embryo lysates were incubated with either anti-Smad1 antibodies (333 µg/ml) (Cell Signaling Technology, 6944) at 1:100 dilution or normal Rabbit IgG (1 mg/ml) (Cell Signaling Technology, 2729) at 1:300 dilution, followed by incubation with Dynabeads Protein G (Novex, 10003D). The elution was subject to protein gel electrophoresis (see above). Antibodies against c-

Myc (Sigma, M5546) were used to probe the blot at 1:500 dilution. Secondary antibodies (donkey anti-mouse IgG) coupled to horseradish peroxidase (Jackson

ImmunoResearch, 715-036-151) were used at 1:10,000 dilution.

2.8 RNA-sequencing

400 pg pitx1 RNA was injected into the animal pole of early cleavage stages embryos.

Animal cap explants were dissected from injected embryos and from untreated control embryos at late blastula stages, and harvested at mid-neurula stages. RNA extracted from these two explant pools was sequenced (Genewiz). The sequences of each transcript were aligned to Xenopus laevis Genome version 7.1 and searched for matchable existing sequence linked to a unique identification number (ID) in the database. These IDs, with information including their calculated relative abundance,

20 were arranged in Excel files. Since each ID corresponds to a specific gene in Xenbase, the files created by Genewiz provide us with candidate genes that are positively or negatively regulated by Pitx1.

Chapter 3: Pitx1 transcriptionally activates cement gland differentiation genes through both direct and indirect mechanisms

3.1 Introduction

The cement gland, as its name implies, secretes a sticky mucus that allows amphibian larvae to adhere to hard surfaces before young tadpoles are able to swim freely in the aquatic environment (Sive and Bradley, 1996). The cement gland is an outer layer ectodermal structure positioned at the anterior end of the Xenopus embryo, at the border between dorsal (neural) and ventral (epidermal) ectoderm

(Fig. 4) (Hausen and Riebesell, 1991). The cement gland is a highly visible organ because the apical region of its glandular cells contains oocyte pigment granules

(Lyerla and Pelizzari, 1973). The gland begins differentiation at mid-neurula stages

(stage 16-18), becoming functional before hatching (stage 26-28) and losing its mucus-secreting capability around stage 48 (Bradley et al., 1996). As one of the earliest fully differentiated organs in Xenopus laevis, the cement gland has long been used as a model to study organogenesis from induction through differentiation.

Figure 4. Drawing of a tailbud stage Xenopus embryo showing anterior structures. The cement gland is ventral to the stomodeal/hypophyseal (pituitary) anlage, and anterior to the foregut. The picture is adapted from Hausen P, Riebesell M, 1991, The early development of Xenopus laevis: An atlas of the histology. Springer-Verlag.

In addition to Pitx1 and Pitx2c, another homeodomain transcription factor, Otx2, has also been implicated in cement gland induction. Otx2 is more broadly expressed in the anterodorsal ectodermal domain compared to Pitx proteins (Gammill and Sive,

2000). Otx2 has been shown to promote anterior fates and control cement gland differentiation in the presence of BMP activity (Gammill and Sive, 1997, 2000, 2001).

Otx2-mediated cement gland induction is abrogated by knockdown of Pitx1 or

Pitx2c (Schweickert et al., 2001a). Misexpression of pitx1 or pitx2c induces cement gland formation, through targets that remain elusive (Chang et al., 2001;

Schweickert et al., 2001a). Here, we first confirm that Pitx1 is sufficient to induce cement gland formation in Xenopus embryos. We demonstrate for the first time that the Pitx proteins are required for cement gland formation, in vivo. We also characterize Pitx1 as a transcriptional activator in the context of cement gland induction, and identify direct and indirect target genes of Pitx1, including the BMP antagonist follistatin.

3.2 Results

3.2.1 Misexpression of pitx1 results in extra cement gland formation in Xenopus embryos

It was reported that misexpression of pitx1 induces ectopic cement glands in

Xenopus embryos (Chang et al., 2001; Schweickert et al., 2001a). To confirm this result, we injected pitx1 RNA into the animal pole of early cleavage stages Xenopus embryos, resulting in either enlarged native cement glands or ectopic cement glands

(96% of embryos with extra cement gland formation, n=50) (compare Figs. 5A, B).

Figure 5. pitx1 misexpression results in extra cement gland formation. (A) 96% of tadpole stage embryos (n=50) exhibit either enlarged native cement glands or ectopic cement glands following injection of 100 pg pitx1 RNA. Black arrowheads point to extra cement glands, which contain black pigment and show stickiness as native cement gland. (B) Uninjected control embryos. Black arrow indicates intact native cement gland.

3.2.2 Knockdown of Pitx1 and Pitx2c inhibits endogenous cement gland formation

While Pitx proteins are necessary for ectopic cement gland induction by Otx2

(Schweickert et al., 2001a), a requirement for Pitx proteins in the formation of the native cement gland has not previously been demonstrated. To address this possibility, we designed antisense morpholino oligonucleotides to inhibit translation of Pitx1 and the related Pitx2c, the ectopic expression of which is also sufficient to induce ectopic cement gland formation (Pitx1-MO and Pitx2-MO, respectively) (Chung et al., 2010). No significant effects on cement gland development were seen after injection of either Pitx1-MO or Pitx2-MO, alone (data not shown); co-injection of Pitx1-MO and Pitx2-MO, however, effectively inhibit endogenous cement gland formation in Xenopus embryos (40% of embryos with no observable cement gland, n=42) (compare Figs. 6A, B). Importantly, co-injection of pitx1 RNA with Pitx1-MO/Pitx2-MO rescues cement gland development (8% of embryos with no observable cement gland, n=26) (Fig. 6C) (It would be worth to examine whether co-injection of pitx2c RNA with Pitx1-MO/Pitx2c-MO could rescue cement gland development). These studies demonstrate that Pitx proteins are essential for cement gland differentiation in Xenopus. Our results also suggest redundancy between Pitx1 and Pitx2c during cement gland development.

Figure 6. Knockdown of Pitx1 and Pitx2c inhibits endogenous cement gland formation. (A) 0% of uninjected embryos (n=56) lack an observable cement gland. (B) 40% of tadpole stage embryos (n=42) lack an observable cement gland following injection of 40 ng Pitx1-MO and 80 ng Pitx2-MO. (C) Cement gland in Pitx1-MO and Pitx2-MO-injected embryo is rescued by injection of 100 pg pitx1 RNA: 8% of embryos (n=26) lack an observable cement gland. Black arrows point to actual or expected sites of cement gland primordia.

3.2.3 Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly

It has been shown that misexpression of Pitx1 induces the expression of cement gland markers in animal cap explants (Chang et al., 2001; Schweickert et al., 2001a).

In order to determine whether Pitx1 functions as a transcriptional activator or repressor, we engineered constructs of full-length Pitx1 fused to either the VP16 activation domain (VP16-Pitx1) or the Engrailed repressor domain (EnR-Pitx1) (Fig.

7). Injection of RNA encoding wild-type pitx1 causes the induction of cement gland markers Xag1 and agr2 (Fig. 8A) (Novoselov et al., 2003; Sive et al., 1989). Injection of RNA encoding VP16-pitx1 also leads to an increased expression of cement gland markers, while injection of RNA encoding EnR-pitx1 does not (Fig. 8A). These results suggest that Pitx1 is a transcriptional activator in the context of cement gland formation.

Next, we extracted RNA from pitx1-injected or uninjected explant pools, and sent these samples to Genewiz for RNA-seq analysis to identify potential transcriptional targets of Pitx1. Among the candidates positively regulated by Pitx1 expression, we selected those which we or others have found to be expressed in the cement gland primordium and utilized cycloheximide assays to distinguish between direct and indirect targets of Pitx1. We would expect to see stimulation of genes that are directly activated by Pitx1 in the presence of the protein synthesis inhibitor cycloheximide; indirect targets of Pitx1 that require an intermediate round of protein synthesis for their activation would not be expected to show an increase in

28 expression in the presence of cycloheximide. Thus, only those genes induced by

Pitx1 in the presence of cycloheximide are putative direct targets (Rosa, 1989). In order to prevent Pitx1 from entering the nucleus prior to cycloheximide treatment, we utilized a fusion protein of Pitx1 and the ligand-binding domain of the

Glucocorticoid Receptor (Pitx1-GR) (Chung et al., 2010). We injected RNA encoding pitx1-GR at early cleavage stages, and isolated animal caps from these and control embryos at late blastula stages. At stage 12, the stage at which endogenous zygotic expression of pitx1 is first observed in the presumptive cement gland region, dexamethasone was added to the excised animal caps to stimulate nuclear translocation of Pitx1-GR (Hollemann and Pieler, 1999). Our results show that nkx3.1 and TGFβ1 are strongly induced by Pitx1-GR in both the presence and absence of cycloheximide, suggesting that they are directly activated by Pitx1 (Fig.

8B) (Kondaiah et al., 1990; Newman and Krieg, 2002). Xag1, Xcg and agr2, however, are only weakly induced when protein synthesis is inhibited, indicating that they are likely to be primarily indirect targets of Pitx1 (Fig. 8B) (Jamrich and Sato, 1989; Sive et al., 1989). These studies demonstrate that Pitx1 functions as a transcriptional activator to drive both direct and indirect upregulation of cement gland differentiation genes.

Figure 7. Fusion constructs of pitx1. Full length pitx1 (top) was fused to the 3' end of either the Engrailed repressor domain (EnR) (middle) or the VP16 activation domain (bottom). The number of amino acids in the repressor and activation domains are indicated.

Figure 8. Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly. (A) Injection of VP16-pitx1 RNA, or wild-type pitx1 RNA, leads to induction of cement gland markers. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. RT-PCR analysis was performed on animal caps collected at mid-neurula stages after dissection at late blastula stages. (B) nkx3.1 and TGFβ1, but not Xag1, Xcg, or agr2, are strongly induced by Pitx1-GR in both the presence and absence of cycloheximide. 400 pg pitx1-GR RNA was injected into early cleavage stages embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone and/or cycloheximide until mid-neurula stages. Dexamethasone (10 µM) was added 10 minutes after cycloheximide (10 µg/ml) addition at stage 12, as listed. EF1α is used as a loading control (Krieg et al., 1989). The -RT lane contains all reagents except reverse transcriptase, and is used as a negative control.

3.2.4 follistatin is a transcriptional target of Pitx1 in the developing cement gland

Interestingly, RNA-seq analysis identified the BMP antagonist follistatin as a putative target of Pitx1, a result that we subsequently confirmed by reverse transcription PCR: injection of RNA encoding pitx1 leads to the induction of follistatin expression in mid-neurula stage ectodermal explants (Fig. 9A) (Fainsod et al., 1997). We were not able to determine whether or not follistatin is a direct target of Pitx1, because cycloheximide alone causes an increase in expression of follistatin

(data not shown). To our knowledge, localization of follistatin transcripts has not previously been reported in the cement gland. Whole mount in situ hybridization analysis reveals that follistatin transcripts are indeed present in the presumptive cement gland region in late neurula stage embryos (66% of embryos with detectable follistatin staining, n=29) (Fig. 9B, left panel); we did not observe follistatin transcripts in the cement gland region at earlier neurula stages, presumably because its expression is below the level of detection by in situ hybridization before stage 20.

In order to examine whether follistatin expression in the cement gland is dependent on Pitx1, we injected either pitx1 RNA or Pitx1-MO/Pitx2-MO into the animal pole of early cleavage stages embryos. Knockdown of Pitx1 and Pitx2c results in decreased follistatin staining in the presumptive cement gland region (36% of embryos with detectable follistatin staining, n=33) (Fig. 9B, middle panel), while Pitx1 overexpression results in both expanded and ectopic follistatin expression (77%, n=31) (Fig. 9B, right panel). Taken together, these results demonstrate that Pitx1 is sufficient for ectopic follistatin expression, necessary for follistatin expression

31 within the cement gland primordium, and suggest that Follistatin mediates the induction of cement gland formation by Pitx1.

Figure 9. follistatin expression in the cement gland is dependent on Pitx1. (A) Injection of pitx1 RNA results in the induction of follistatin. 400 pg pitx1 RNA was injected at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) Pitx1 is both sufficient and necessary for follistatin expression. left panel, 66% of uninjected late neurula stage embryos (n=29) have detectable follistatin staining in the presumptive cement gland region; middle panel, 36% of embryos (n=33) have detectable follistatin staining in the cement gland following injection of 40 ng Pitx1-MO and 80 ng Pitx2-MO; right panel, 77% of embryos (n=31) have both expanded and ectopic follistatin staining following injection of 100 pg pitx1 RNA. All embryos are ventral views; anterior is to left.

3.3 Discussion

In this study, we confirm the sufficiency of Pitx1 in cement gland induction, and report that Pitx1 and the related protein Pitx2c are required for endogenous cement gland formation. Pitx1 functions as a transcriptional activator to stimulate cement gland differentiation genes, both directly and indirectly. In addition, follistatin expression in the cement gland primordium is dependent on Pitx activity.

Injection of RNA encoding EnR-pitx1 fails to induce cement gland markers in animal cap explants at a dose 10 times higher than that shown to be effective for VP16-pitx1.

However, we could not rule out the possibility that the EnR-Pitx1 protein is non- functional due to improper folding. To demonstrate EnR-Pitx1 activity, we would need to co-inject wild-type pitx1 and EnR-pitx1 RNA, and determine whether EnR-

Pitx1 can interfere with the activity of wild-type Pitx1.

Pitx1 may activate downstream indirect transcriptional targets through its putative direct target, TGFβ1. It has been shown that misexpression of TGFβ1 alone induces limited expression of Xag1 (Deglincerti et al., 2015). In addition, analysis of the Xag1 promoter revealed that the ATF/CREB-like family members regulate Xag1 expression through an Otx2-dependent pathway (Wardle et al., 2002). It has been reported that ATF-2 plays a crucial role in transducing TGFβ signaling (Kim et al.,

2007; Lim et al., 2005), which raises the possibility that Xag1 expression is under the control of TGFβ1. However, limited induction of cement gland markers by

TGFβ1 alone suggests that other factors are needed to enhance its activity. Coco, a member of the Dan family, is reported to elevate TGFβ1 signaling (including the induction of Xag1 expression) significantly through enhanced binding of TGFβ1 with its cognate receptor complex (Deglincerti et al., 2015). Another extracellular protein, connective tissue growth factor (CTGF), is also involved in the upregulation of

TGFβ1 activity through similar mechanism (Abreu et al., 2002). Since neither factor is expressed in regions close to the cement gland (Mercurio et al., 2004; Vonica and

Brivanlou, 2007), we speculate that other factors that enhance TGFβ1 signaling are present at the anterior end of the Xenopus embryo. Follistatin expressed in the developing cement gland antagonizes BMP activity through similar inhibitory- binding mechanism as Coco and CTGF; it is possible that Follistatin promotes TGFβ1 activity through physical association with the latter. To test this possibility, we have performed TGFβ1 and Follistatin co-expression studies in Xenopus ectoderm.

Follistatin alone induces the expression of cement gland markers, presumably as a

BMP antagonist (Fainsod et al., 1997). In some trials, co-expression of TGFβ1 and

Follistatin indeed leads to an increased expression of cement gland markers compared to misexpression of Follistatin alone (data not shown). However, such results are not repeated in all trials, and we have thus far failed to find highly repeatable, active doses for these two factors.

In addition to the BMP antagonists, Cephalic Hedgehog and Banded Hedgehog, two members of the hedgehog family, have been shown to induce cement gland in both animal cap explants and whole embryos (Ekker et al., 1995). Unlike BMP

34 antagonists, Hedgehog induces expression of both cement gland and anterior pituitary markers, but not the neural marker NCAM, whose expression, based on our

RNA-seq analysis, is also unaffected by Pitx1 (Lai et al., 1995). Since it has been reported that activation of hedgehog signaling by addition of purmorphamine results in significant increases in the expression of both Pitx1 and Pitx2 (Leung et al.,

2013), we postulate that hedgehog signaling may be involved in the activation of pitx genes at the initial stages of cement gland induction.

The expression pattern of pitx1 is highly conserved between Xenopus laevis and Mus musculus. Mouse pitx1 is expressed in the stomodeal epithelium, adenohypophysis, foregut endoderm, first pharyngeal arch and posterior lateral plate (Lanctot et al.,

1997). Although the cement gland does not exist in mouse, expression of pitx1 is localized in other mucus-secreting tissues, such as the submandibular glands. The oral epithelium-derived submandibular glands are a pair of major salivary glands located beneath the mandible, which secretes mucus-containing saliva into the oral cavity. More than 60% of saliva is produced by the submandibular glands (reviewed in Proctor, 2016). It was reported that pitx1-/- mice exhibit loss of the submandibular glands, indicating that Pitx1 is necessary for gland development

(Szeto et al., 1999). The requirement for Pitx1 in both the Xenopus cement gland and mouse submandibular glands suggest that Pitx1 plays an important role in the development of mucus-secreting glands and production of mucus across species.

Pitx1 also serves as a master regulator during development of the anterior pituitary gland, and has been implicated in Follicle Stimulating Hormone (FSH) beta subunit transcription in gonadotrope cells (Bernard et al., 2010). Follistatin is known to suppress Activin-stimulated FSH release from gonadotrope cells by preventing binding of Activin to its receptors (Thompson et al., 2005; Ueno et al., 1987; Ying et al., 1987). follistatin was identified in this study as a target of Pitx1 in the differentiating cement gland, close to the presumptive pituitary gland; dissecting this relationship between Pitx1 and Follistatin may help to both elucidate the regulation of gonadotropins and provide novel insights into early pituitary gland development.

Chapter 4: Intermediate levels of BMP signaling are required for cement gland formation

4.1 Introduction

According to one prominent model of dorsoventral ectodermal patterning, high levels of BMP activity induce ventral epidermis; low levels of BMP signaling result in dorsal neural fate, while intermediate levels of BMP signaling promote the development of tissues located at the neural plate border, such as neural crest, cranial placodes and the cement gland (Knecht and Harland, 1997; Wilson et al.,

1997). Modest BMP inhibition in the ectoderm thus leads to cement gland induction; however, Otx2-induced cement gland markers are abrogated by depletion of BMP ligands (Gammill and Sive, 2000). These results further suggest the role of intermediate BMP signaling in the cement gland formation. The mechanisms underlying the requirement for intermediate level of BMP activity in the cement gland formation is not clear. Here, we find that Pitx1 functions as a transcriptional activator to indirectly inhibit BMP signaling in the presumptive cement gland tissue; this inhibition is partially mediated by Follistatin, whose expression within the cement gland primordium depends on Pitx1. Restoration of BMP signaling inhibited by Pitx1 represses the expression of endogenous pitx2 and pitx1, whereas suppression of BMP signaling attenuates Pitx1-induced cement gland markers.

Finally, we find physical interaction between Pitx1 and Smad1. Considering diverse requirements for levels of BMP activity in the expression of various genes, we find that intermediate level of BMP signaling is optimal for cement gland formation.

4.2 Results

4.2.1 Pitx1 functions as a transcriptional activator to inhibit BMP/Smad1 signaling

Since Follistatin has been shown to antagonize BMP activity through direct binding of extracellular BMP ligands (Fainsod et al., 1997; Iemura et al., 1998), we were interested to see whether Pitx1 regulates BMP signaling through its target

Follistatin. Pitx1 misexpression in animal caps inhibits the BMP-responsive target genes bmp4 and sizzled in a dose dependent manner (Fig. 10A) (Lee et al., 2006;

Reversade and De Robertis, 2005). Moreover, Western blot analysis shows that injection of RNA encoding pitx1 decreases C-terminal phosphorylation of the BMP pathway transducer Smad1/5, without affecting the overall level of Smad1 protein

(Fig. 10B). In order to determine whether Pitx1 works as an activator or repressor in the context of BMP signal inhibition, we examined expression of BMP target genes in animal caps derived from embryos injected with RNA encoding VP16-pitx1 or

EnR-pitx1. VP16-Pitx1 inhibits the BMP-responsive genes bmp4, sizzled and cv2, similar to that seen following injection of wild-type pitx1 RNA, while EnR-Pitx1 does not affect BMP marker gene expression (Fig. 10C) (Ambrosio et al., 2008). These results substantiate the idea that Pitx1-mediated inhibition of BMP signaling is achieved via transcriptional targets of Pitx1. Next, we designed and utilized antisense morpholino oligonucleotides to inhibit translation of Follistatin

(Follistatin-MO). Co-injection of Follistatin-MO and RNA encoding pitx1 partially rescues expression of some but not all of the BMP-responsive genes inhibited by

Pitx1 (Fig. 10D), suggesting that Pitx1 suppresses BMP signaling through activation of both follistatin and additional target genes.

Figure 10. Pitx1 functions as a transcriptional activator to inhibit BMP signaling. (A) Injection of pitx1 RNA inhibits BMP targets in a dose dependent manner. 10 pg to 400 pg pitx1 RNA was injected at early cleavage stages, as listed. (B) Injection of pitx1 RNA results in decreased C-terminal phosphorylation of Smad1/5; total levels of Smad1 are not significantly affected. Embryos were injected with 20 pg, 100 pg and 400 pg pitx1 RNA at early cleavage stages, as listed. β-tubulin is used as a loading control. (C) Injection of VP16- pitx1 RNA leads to down-regulation of BMP target genes, similar to that seen with wild-type pitx1; injection of EnR-pitx1 RNA does not alter expression of BMP-responsive genes. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. (D) Pitx1-mediated inhibition of BMP target genes are partially rescued by co- injection of Follistatin-MO. 400 pg pitx1 RNA and/or 80 ng Follistatin-MO were injected at early cleavage stages. RT-PCR analysis (A, C and D) and Western blot analysis (B) were performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages.

4.2.2 Rescue of Pitx1-mediated BMP signal inhibition represses expression of pitx genes

The cement gland has been proposed to arise from the ectoderm exposed to moderate inhibition of BMP signaling (Knecht and Harland, 1997; Wilson et al.,

1997). Since our studies demonstrate that Pitx1 inhibits BMP signaling, we were interested to see whether enhancement of BMP signaling could interfere with Pitx1- induced cement gland formation. We found that exogenous Smad1 or constitutively active BMP receptor Ia (CA-BMPRIa) rescues expression of the BMP-responsive gene sizzled, but has no effect on Pitx1-induced expression of the cement gland markers Xag1 and Xcg (Fig. 11). CA-BMPRIa also successfully rescues decreased C- terminal phosphorylation of Smad1/5 caused by misexpression of Pitx1 or noggin, suggesting the regulation of BMP signaling by Pitx1 is upstream of the receptor (Fig.

12). Although cement gland differentiation markers appear unaffected, expression of two transcript variants of pitx2 (pitx2b and pitx2c) and endogenous pitx1 were consistently suppressed by elevated BMP signaling (Fig. 11). In conclusion, elevated

BMP signaling has no effect on cement gland-specific marker expression by exogenous Pitx1, but does inhibit expression of pitx2 and endogenous pitx1 in the developing cement gland, suggesting the presence of a Pitx positive feedback loop during cement gland differentiation.

Figure 11. Rescue of Pitx1-mediated inhibition of BMP signaling represses expression of pitx genes. Pitx1-mediated inhibition of the BMP target gene sizzled is rescued by co- expression of either Smad1 or CA-BMPRIa. Pitx1-mediated induction of two pitx2 transcript variants, pitx2b and pitx2c, as well as native pitx1, are down-regulated following enhancement of BMP signaling. 50 pg pitx1 RNA was either injected alone or co-injected with 1.6 ng smad1 or CA-BMPRIa RNA at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages.

Figure 12. CA-BMPRIa rescues the C-terminal phosphorylation of Smad1/5 inhibited by either Pitx1 or Noggin. Decreased C-terminal phosphorylation of Smad1/5 by Pitx1 or Noggin is rescued by injection of 100 pg or 400 pg CA-BMPRIa RNA. Total Smad1 is not significantly affected. GAPDH is used as a loading control (Morgenegg et al., 1986). Western blot analysis was performed on animal caps harvested at mid-neurula stages after isolation at late blastula stages.

4.2.3 BMP/Smad1 signaling is required for cement gland development

Gammill and Sive have demonstrated that coincident BMP4 activity and Otx2 expression correlate with cement gland formation (Gammill and Sive, 2000). We examined the ability of Pitx1 to induce cement gland under conditions of low

BMP/Smad1 signaling. Co-injection of RNA encoding pitx1 and the extracellular BMP antagonist noggin inhibits expression of Pitx1-induced cement gland differentiation genes, including direct target genes of Pitx1 (Fig. 13A); this result indicates that

BMP signaling, or the embryonic environment generated as a consequence of BMP signaling, is required for the activation of cement gland markers by Pitx1. It has been reported that Smad1 physically interacts with Pitx1 in mouse corticotroph cells; this interaction results in the suppression of the Pitx1 target gene proopiomelanocortin (Nudi et al., 2005). We constructed a Pitx1-Myc epitope-tagged vector (Pitx1-Myc), and examined the activity of this construct by injecting synthetic pitx1-Myc RNA into early cleavage stages embryos (Fig. 14). Immunoprecipitation and Western blot analysis demonstrated that native Smad1 binds to exogenous

Pitx1 in Xenopus embryos (Fig. 13B). These studies suggest that the dependence of

Pitx1-mediated cement gland gene transactivation on BMP signaling may involve physical association of Pitx1 and Smad1; this interaction would be expected to stimulate, rather than inhibit, transactivation by Pitx1 in the context of cement gland formation.

Figure 13. BMP/Smad1 signaling is required for cement gland development. (A) Injection of noggin RNA inhibits Pitx1-mediated induction of cement gland differentiation genes. 400 pg pitx1-GR RNA and/or 20 pg noggin RNA were injected into Xenopus embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone until mid-neurula stages. Dexamethasone (10 µM) was added to animal caps at stage 12. (B) Pitx1 binds to Smad1. 2 ng pitx1-myc RNA was injected at early cleavage stages. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Pitx1. Normal rabbit IgG antibody was used in parallel studies as a negative control.

Figure 14. Pitx1-Myc, like Pitx1, inhibits BMP activity and promotes cement gland differentiation. Injection of 2 ng pitx1-Myc RNA induces the expression of cement gland marker Xag1, and inhibits the expression of BMP target genes BMP4 and sizzled, similar to that seen following injection of 400 pg wild-type pitx1 RNA. Injection of 400 pg pitx1-Myc RNA has a much lower activity.

4.3 Discussion

In this study, we report that Pitx1 functions as a transcriptional activator to inhibit

BMP/Smad1 signaling; this inhibition is required for pitx1 and pitx2 expression in the cement gland primordium, and is mediated in part by Pitx1-dependent expression of follistatin. Conversely, suppression of BMP signaling abrogates Pitx1- induced cement gland marker expression; this requirement for BMP signaling may involve the observed physical interaction between Pitx1 and Smad1.

Our studies provide insight into why intermediate levels of BMP signaling are optimal for cement gland development. 1) High BMP activity is not favorable for cement gland development—expression of pitx1 and pitx2 are suppressed by high

BMP pathway activity. The suppression of pitx1 and pitx2 expression by BMP4 has also been reported in mouse dental epithelium (Mitsiadis and Drouin, 2008; St

Amand et al., 2000). 2) Low BMP activity is not favorable for cement gland development—attenuation of BMP signaling causes a strong reduction of cement gland markers induced by either Otx2 or Pitx1 (Gammill and Sive, 2000) (this study).

This may explain why Pitx1- or Otx2-dependent ectopic cement glands are only seen in ventral-lateral regions of the embryo, and never in the dorsal neural plate where

BMP activity is lowest (Chang et al., 2001; Gammill and Sive, 1997). Taken together, these studies suggest that moderate inhibition of BMP signaling in the ectoderm first establishes a zone suitable for expression of pitx1 and pitx2. Once expression of cement gland-inducing factors is initiated, Pitx1 and other transactivators begin to

46 activate cement gland differentiation genes directly, regardless of levels of local

BMP activity. Under native conditions, moderate BMP inhibition may be required throughout the period of cement gland differentiation, in order to maintain expression of pitx genes.

The finding that Pitx1 physically interacts with Smad1 raises an intriguing question: does Pitx1 bind preferentially to either phospho-Smad1 (activated) or non- phospho-Smad1 (non-activated)? We could perform several experiments to address this question. 1) Examine whether pull-down of exogenous Pitx1, using anti-Myc antibody, co-immunoprecipitates phospho-Smad1. 2) Examine whether injection of noggin RNA attenuates the physical interaction between Pitx1 and Smad1; if Pitx1 binds to phospho-Smad1, reduction of phospho-Smad1 by the BMP antagonist noggin may decrease the level of Smad1-bound Pitx1. 3) Examine whether the fusion protein Pitx1-GR physically interacts with Smad1 in the absence of dexamethasone, and whether addition of dexamethasone elevates the interaction between Pitx1 and Smad1. Most Pitx1-GR protein should remain in the cytoplasm in the absence of the ligand dexamethasone, whereas most phospho-Smad1 remains in the nucleus (Massague et al., 2005; Vandevyver et al., 2012). If Pitx1 binds to phospho-Smad1, nuclear translocation of ligand-bound Pitx1-GR, in the presence of dexamethasone, may dramatically increase the level of Smad1-bound Pitx1. 4)

Examine whether Pitx1 binds to Smad4. It is known that phospho-Smad1 and

Smad4 form a complex to regulate the transcription of target genes. If Pitx1 binds to phospho-Smad1, it may also interact with Smad4 in the same complex.

Chapter 5: Pitx1 regulates goosecoid transcription

5.1 Introduction

The homeodomain protein Goosecoid has been implicated as a central regulator of dorsoventral patterning in the gastrula stage Xenopus embryo. Early expression of goosecoid is localized to the Spemann Organizer (De Roberts et al., 1992).

Misexpression of Goosecoid in the ventral marginal zone results in the formation of a secondary body axis, in a non-cell-autonomous manner, partially mimicking the effects caused by transplantation of the Organizer (Cho et al., 1991). The dorsalizing activity of Goosecoid is partially mediated by inhibition of ventralizing BMP signals, through activation of chordin expression and repression of the BMP downstream targets Vent1/2 (Sander et al., 2007; Sasai et al., 1994). The initiation of goosecoid transcription is regulated by multiple factors. Intracellular transduction of Nodal- related signal by a DNA binding complex including Mixer and Smad2/4 requires a distal element in the goosecoid promoter (Germain et al., 2000). Furthermore, Pitx2c is reported to be both sufficient and necessary for early goosecoid expression in response to Nodal signaling (Faucourt et al., 2001).

By the end of neurulation, goosecoid expression is localized to the prechordal plate

(head mesoderm) and pharyngeal endoderm (De Roberts et al., 1992). Mochizuki et al. have proposed that two homeodomain transcription factors, Xlim1 and Otx2, are involved in the maintenance of goosecoid expression in the prechordal plate through direct binding to elements in the goosecoid promoter (Mochizuki et al., 2000). We

48 were interested in identifying other factors that maintain late goosecoid expression during the development of anterior endoderm. Here, we report that Pitx1, functioning as a transcriptional activator, induces goosecoid expression. We also find that Pitx1 binds to exogenous Smad2/3. Taken together, these preliminary studies provide clues to elucidate the mechanisms underlying Pitx1-mediated activation of goosecoid expression.

5.2 Results

5.2.1 Pitx1 induces goosecoid expression but inhibits Activin activity

Our RNA-seq analysis identified homeobox transcription factor goosecoid as a putative target of Pitx1, a result we subsequently confirmed by RT-PCR: injection of wild-type Pitx1 RNA results in the induction of goosecoid expression in animal cap explants; VP16-Pitx1 also induces goosecoid expression, whereas EnR-Pitx1 does not (Fig. 15), suggesting that Pitx1 functions as transcriptional activator to stimulate goosecoid expression. We were not able to determine whether or not goosecoid is a direct target of Pitx1, because cycloheximide alone causes increased expression of goosecoid (data not shown). Other mesodermal markers, such as muscle actin

(dorsal mesoderm), Xwnt8 (ventral mesoderm) and brachyury (pan-mesoderm), cannot be induced by Pitx1 (Christian et al., 1991; Smith et al., 1991; Wilson et al.,

1986). In fact, Pitx1 represses the expression of these mesodermal markers in the presence of the mesoderm-inducing factor Activin (Fig. 16A). Consistently, Activin- induced elongation of animal cap explants (via induction of axial mesoderm) is also completely abrogated by Pitx1 (Compare Figs. 16D, E).

Figure 15. Pitx1 functions as a transcriptional activator to stimulate goosecoid expression. Injection of VP16-pitx1 RNA, or wild-type pitx1 RNA, but not EnR-pitx1 RNA, leads to the induction of goosecoid expression. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. RT-PCR analysis was performed on animal caps collected at late neurula stages after dissection at late blastula stages.

Figure 16. Pitx1 inhibits Activin activity. (A) Expression of Activin-induced mesodermal markers is repressed following injection of 400 pg pitx1 RNA. RT-PCR analysis was performed on animal caps collected at late neurula stages after dissection at late blastula stages. (B) Animal caps isolated from embryos injected with 100 pg pitx1 RNA. (C) Animal caps from uninjected embryos. (D) Animal caps elongate following addition of Activin (0.25 ng/ml). (E) Activin-induced elongation of animal cap explants is abrogated by Pitx1. 100 pg pitx1 RNA was injected at early cleavage stages. Animal caps dissected at late blastula stages were cultured in medium with Activin (0.25 ng/ml), as listed, until late neurula stage.

5.2.2 Pitx1 physically interacts with Smad2/3

As mentioned above, a complex composed of Mixer and Smad2/4 was shown to mediate Nodal-related signaling to activate early expression of goosecoid (Germain et al., 2000). In addition, Pitx1 was reported to interact directly with Smad2/3 in vitro in the context of luteinizing hormone β subunit transactivation (Coss et al.,

2005). These studies suggest that Pitx1-mediated activation of goosecoid may involve physical interaction between Pitx1 and Smad2/3. To test this hypothesis, we co-injected Pitx-Myc RNA with Flag-Smad2 or Flag-Smad3 RNA into the animal pole of early cleavage stages Xenopus embryos. Immunoprecipitation and Western blot analysis revealed that exogenous Pitx1 binds to both exogenous Smad2 and Smad3

(Fig. 17).

Figure 17. Pitx1 binds to exogenous Smad2/3. 1 ng pitx1 RNA was co-injected with either 1 ng Flag-Smad2 or 1 ng Flag-Smad3 into the animal pole of early cleavage stages embryos. Pull down of exogenous Smad2 or Smad3 using anti-Flag antibody from injected embryos results in co-immunoprecipitation of exogenous Pitx1.

5.3 Discussion

In this study, we report that Pitx1 function as a transcriptional activator to stimulate goosecoid expression. Other mesodermal markers, and the morphogenetic movement of explants induced by Activin, are blocked by Pitx1. We also demonstrate physical association between exogenous Pitx1 and Smad2/3 in

Xenopus embryos. We propose that Pitx1 forms a complex with Smad proteins to positively regulate goosecoid transcription during organogenesis.

C-terminal phosphorylation of Smad2/3 is required for binding of the cofactor

Smad4 and translocation into the nucleus to regulate the transcription of target genes (reviewed in Moustakas and Heldin, 2009). Further investigations are needed to address whether Pitx1 binds to phosphor-Smad2/3 or non-phosphorylated

Smads. If phosphorylated Smad2/3 is involved in Pitx1-mediated goosecoid activation, TGFβ1, as a putative direct target of Pitx1, may trigger the activation of

Smad2/3 following Pitx1 misexpression.

Our studies show that Pitx1 strongly inhibits Activin activity. We speculate that

Pitx1-dependent Follistatin, as a potent Activin antagonist, may be involved in this process (Asashima et al., 1991). The anterior ectodermal region, where Pitx1 is expressed, abuts the anterior endoderm (Schweickert et al., 2001b). The inhibition of Activin activity may be required to protect the ectoderm from endoderm- inducing signals. Although both TGFβ1 and Activin can lead to the phosphorylation

53 of Smad2/3, the requirement for TGFβ1 but not Activin in cement gland development highlights the divergent outputs of these related signaling pathways.

The binding site of Otx2 on the goosecoid promoter has been identified as 5'-

TAATCT, which is within the distal element regulating Nodal-related signaling

(Mochizuki et al., 2000). In the context of luteinizing hormone β subunit transcription,

Pitx1 has been shown to bind to its promoter on the site of 5'-TAATCT (Quirk et al.,

2001). It is possible that Pitx1 utilizes the same DNA binding site as Otx2 to drive the transcription of goosecoid.

The expression pattern of goosecoid during mouse embryonic development has two distinct phases: early expression during gastrulation in the developing anterior primitive streak; late expression during organogenesis in the head mesoderm and the first pharyngeal arch (also called the mandibular arch) that gives rise to craniofacial structures (Blum et al., 1992; Gaunt et al., 1993). Homozygous goosecoid mutant mice display defects in the mandible and its associated musculature

(Yamada et al., 1995; Yamada et al., 1997). Interestingly, deletion of pitx1, which is also expressed in the mesenchyme of the first pharyngeal arch, leads to severe mandibular defects in mice (Lanctot et al., 1999). These studies suggest a relationship between Pitx1 and Goosecoid during the formation of the mandible and related structures, supporting our model that late expression of goosecoid is regulated by Pitx1 in Xenopus embryos.

Chapter 6: Summary

This dissertation examines the role of Pitx proteins in the anterior ectoderm and endoderm during the early development of Xenopus laevis, and demonstrates a multi-faceted role for Pitx1 in regulating cement gland development and late goosecoid transcription.

We propose that Pitx1 maintains goosecoid expression required for the development of craniofacial structures, possibly through the formation of a transactivating complex with Smads (Fig. 18). In the future, chromatin immunoprecipitation (ChIP) could be performed to examine whether Pitx1 binds to the distal element in the goosecoid promoter.

Figure 18. Proposed model of Pitx1-mediated induction of goosecoid transcription. A complex composed of Pitx1 and Smads binds to the distal element of goosecoid promoter to activate its transcription. Xtwn, a downstream target of β-catenin signal, can also enhance goosecoid transcription through binding to a proximal element (Laurent et al., 1997).

Much work remains to be done to determine the requirement for Pitx1-mediated goosecoid activation in anterior endoderm development; our understanding of Pitx function during differentiation of the anterior ectoderm is significantly more advanced, and is consistent with a proposed model, in which the cement gland develops at the intersection of three overlapping regions of the ectoderm: Otx2- expressing dorsoanterior cells, ventrolateral cells with adequate BMP signaling, and outer layer ectodermal cells (Wardle and Sive, 2003). Viewing our results in the context of these earlier studies, we propose an updated model for cement gland formation: Pitx proteins within the Otx2-expressing domain stimulate expression of downstream targets within the cement gland primordium through the formation of a transactivating complex that includes Smad1 (Fig. 19A). Meanwhile, Pitx1 restricts local BMP signaling, via targets that include follistatin, to maintain the expression of pitx genes and potentially other factors required for cement gland development (Fig.

19B). In the future, we and others in the lab plan to examine 1) whether Pitx1 physically interacts with phospho-Smad1 (see Discussion in Chapter 4); 2) the effects of enhanced BMP signaling on the spatial expression patterns of pitx genes by whole mount in situ hybridization.

Figure 19. Proposed model of Pitx1-mediated regulation of cement gland development. (A) Pitx1 activates transcription of direct target genes, possibly through the formation of a complex that includes Smad1. (B) Pitx1 limits local BMP signaling within the cement gland primordium, via transcriptional activation of follistatin and additional targets, to maintain the expression of pitx genes and other factors required for cement gland development.

References

Abreu, J.G., Ketpura, N.I., Reversade, B., De Robertis, E.M., 2002. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4, 599-604.

Agius, E., Oelgeschlager, M., Wessely, O., Kemp, C., De Robertis, E.M., 2000. Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development 127, 1173-1183.

Ambrosio, A.L., Taelman, V.F., Lee, H.X., Metzinger, C.A., Coffinier, C., De Robertis, E.M., 2008. Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. Dev Cell 15, 248-260.

Asashima, M., Nakano, H., Uchiyama, H., Sugino, H., Nakamura, T., Eto, Y., Ejima, D., Davids, M., Plessow, S., Cichocka, I., Kinoshita, K., 1991. Follistatin inhibits the mesoderm-inducing activity of activin A and the vegetalizing factor from chicken embryo. Roux Arch Dev Biol 200, 4-7.

Bassez, T., Paris, J., Omilli, F., Dorel, C., Osborne, H.B., 1990. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110, 955-962.

Bates, T.J., Vonica, A., Heasman, J., Brivanlou, A.H., Bell, E., 2013. Coco regulates dorsoventral specification of germ layers via inhibition of TGFbeta signalling. Development 140, 4177-4181.

Bell, E., Munoz-Sanjuan, I., Altmann, C.R., Vonica, A., Brivanlou, A.H., 2003. Cell fate specification and competence by Coco, a maternal BMP, TGFbeta and Wnt inhibitor. Development 130, 1381-1389.

Bernard, D.J., Fortin, J., Wang, Y., Lamba, P., 2010. Mechanisms of FSH synthesis: what we know, what we don't, and why you should care. Fertil Steril 93, 2465-2485.

Bernardini, G., 1999. Atlas of xenopus development. Springer, Milan ; New York.

Birsoy, B., Kofron, M., Schaible, K., Wylie, C., Heasman, J., 2006. Vg 1 is an essential signaling molecule in Xenopus development. Development 133, 15-20.

Blum, M., Gaunt, S.J., Cho, K.W., Steinbeisser, H., Blumberg, B., Bittner, D., De Robertis, E.M., 1992. Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69, 1097-1106.

Bradley, L., Wainstock, D., Sive, H., 1996. Positive and negative signals modulate formation of the Xenopus cement gland. Development 122, 2739-2750.

Chang, W., KhosrowShahian, F., Chang, R., Crawford, M.J., 2001. xPitx1 plays a role in specifying cement gland and head during early Xenopus development. Genesis 29, 78-90.

Cho, K.W., Blumberg, B., Steinbeisser, H., De Robertis, E.M., 1991. Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67, 1111-1120.

Christian, J.L., McMahon, J.A., McMahon, A.P., Moon, R.T., 1991. Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development 111, 1045-1055.

Chung, M.I., Nascone-Yoder, N.M., Grover, S.A., Drysdale, T.A., Wallingford, J.B., 2010. Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut. Development 137, 1339-1349.

Conlon, F.L., Lyons, K.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., Robertson, E.J., 1994. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120, 1919-1928.

Coss, D., Thackray, V.G., Deng, C.X., Mellon, P.L., 2005. Activin regulates luteinizing hormone beta-subunit gene expression through Smad-binding and homeobox elements. Mol Endocrinol 19, 2610-2623.

De Roberts, E.M., Blum, M., Niehrs, C., Steinbeisser, H., 1992. Goosecoid and the organizer. Dev Suppl, 167-171.

Deglincerti, A., Haremaki, T., Warmflash, A., Sorre, B., Brivanlou, A.H., 2015. Coco is a dual activity modulator of TGFbeta signaling. Development 142, 2678-2685.

Dupont, S., Zacchigna, L., Cordenonsi, M., Soligo, S., Adorno, M., Rugge, M., Piccolo, S., 2005. Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase. Cell 121, 87-99.

Ekker, S.C., McGrew, L.L., Lai, C.J., Lee, J.J., von Kessler, D.P., Moon, R.T., Beachy, P.A., 1995. Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis. Development 121, 2337-2347.

Evans, B.J., Carter, T.F., Greenbaum, E., Gvozdik, V., Kelley, D.B., McLaughlin, P.J., Pauwels, O.S., Portik, D.M., Stanley, E.L., Tinsley, R.C., Tobias, M.L., Blackburn, D.C., 2015. Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus, Pipidae) from West and Central Africa. PLoS One 10, e0142823.

Fainsod, A., Deissler, K., Yelin, R., Marom, K., Epstein, M., Pillemer, G., Steinbeisser, H., Blum, M., 1997. The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4. Mech Dev 63, 39-50.

Faucourt, M., Houliston, E., Besnardeau, L., Kimelman, D., Lepage, T., 2001. The pitx2 homeobox protein is required early for endoderm formation and nodal signaling. Dev Biol 229, 287-306.

Feldman, B., Gates, M.A., Egan, E.S., Dougan, S.T., Rennebeck, G., Sirotkin, H.I., Schier, A.F., Talbot, W.S., 1998. Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature 395, 181-185.

Fletcher, R.B., Harland, R.M., 2008. The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus. Dev Dyn 237, 1243- 1254.

Gammill, L.S., Sive, H., 1997. Identification of otx2 target genes and restrictions in ectodermal competence during Xenopus cement gland formation. Development 124, 471-481.

Gammill, L.S., Sive, H., 2000. Coincidence of otx2 and BMP4 signaling correlates with Xenopus cement gland formation. Mech Dev 92, 217-226.

Gammill, L.S., Sive, H., 2001. otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation. Dev Biol 240, 223-236.

Gaunt, S.J., Blum, M., De Robertis, E.M., 1993. Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall. Development 117, 769-778.

Gerhart, J., 1999. Pieter Nieuwkoop's contributions to the understanding of meso- endoderm induction and neural induction in chordate development. Int J Dev Biol 43, 605-613.

Germain, S., Howell, M., Esslemont, G.M., Hill, C.S., 2000. Homeodomain and winged- helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif. Genes Dev 14, 435-451.

Green, J.B., Smith, J.C., 1990. Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347, 391-394.

Hama, J., Suri, C., Haremaki, T., Weinstein, D.C., 2002. The molecular basis of Src kinase specificity during vertebrate mesoderm formation. J Biol Chem 277, 19806- 19810.

Harland, R., Gerhart, J., 1997. Formation and function of Spemann's organizer. Annu Rev Cell Dev Biol 13, 611-667.

Hata, A., Lo, R.S., Wotton, D., Lagna, G., Massague, J., 1997. Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature 388, 82-87.

Hausen, P., Riebesell, M., 1991. The early development of Xenopus laevis : an atlas of the histology. Verlag der Zeitschrift für Naturforschung ; Springer-Verlag, Tübingen Berlin ; New York.

Hawley, S.H., Wunnenberg-Stapleton, K., Hashimoto, C., Laurent, M.N., Watabe, T., Blumberg, B.W., Cho, K.W., 1995. Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction. Genes Dev 9, 2923-2935.

Heasman, J., 2006. Patterning the early Xenopus embryo. Development 133, 1205- 1217.

Hemmati-Brivanlou, A., Kelly, O.G., Melton, D.A., 1994. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77, 283-295.

Hemmati-Brivanlou, A., Melton, D.A., 1992. A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature 359, 609-614.

Hemmati-Brivanlou, A., Melton, D.A., 1994. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell 77, 273-281.

Hollemann, T., Pieler, T., 1999. Xpitx-1: a homeobox gene expressed during pituitary and cement gland formation of Xenopus embryos. Mech Dev 88, 249-252.

Horb, M.E., Thomsen, G.H., 1997. A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation. Development 124, 1689-1698.

Iemura, S., Yamamoto, T.S., Takagi, C., Uchiyama, H., Natsume, T., Shimasaki, S., Sugino, H., Ueno, N., 1998. Direct binding of follistatin to a complex of bone- morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. Proc Natl Acad Sci U S A 95, 9337-9342.

Jamrich, M., Sato, S., 1989. Differential gene expression in the anterior neural plate during gastrulation of Xenopus laevis. Development 105, 779-786.

Jones, C.M., Kuehn, M.R., Hogan, B.L., Smith, J.C., Wright, C.V., 1995. Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development 121, 3651-3662.

Joseph, E.M., Melton, D.A., 1998. Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos. Development 125, 2677-2685.

Kawamura, K., Kouki, T., Kawahara, G., Kikuyama, S., 2002. Hypophyseal development in vertebrates from amphibians to mammals. Gen Comp Endocrinol 126, 130-135.

Kessler, D.S., 1997. Siamois is required for formation of Spemann's organizer. Proc Natl Acad Sci U S A 94, 13017-13022.

Kiecker, C., Bates, T., Bell, E., 2016. Molecular specification of germ layers in vertebrate embryos. Cell Mol Life Sci 73, 923-947.

Kim, E.S., Sohn, Y.W., Moon, A., 2007. TGF-beta-induced transcriptional activation of MMP-2 is mediated by activating transcription factor (ATF)2 in human breast epithelial cells. Cancer Lett 252, 147-156.

Knecht, A.K., Harland, R.M., 1997. Mechanisms of dorsal-ventral patterning in noggin-induced neural tissue. Development 124, 2477-2488.

Kofron, M., Demel, T., Xanthos, J., Lohr, J., Sun, B., Sive, H., Osada, S., Wright, C., Wylie, C., Heasman, J., 1999. Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors. Development 126, 5759-5770.

Kondaiah, P., Sands, M.J., Smith, J.M., Fields, A., Roberts, A.B., Sporn, M.B., Melton, D.A., 1990. Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis. J Biol Chem 265, 1089-1093.

Kretzschmar, M., Doody, J., Timokhina, I., Massague, J., 1999. A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras. Genes Dev 13, 804-816.

Krieg, P.A., Varnum, S.M., Wormington, W.M., Melton, D.A., 1989. The mRNA encoding elongation factor 1-alpha (EF-1 alpha) is a major transcript at the midblastula transition in Xenopus. Dev Biol 133, 93-100.

LaBonne, C., Whitman, M., 1994. Mesoderm induction by activin requires FGF- mediated intracellular signals. Development 120, 463-472.

Lai, C.J., Ekker, S.C., Beachy, P.A., Moon, R.T., 1995. Patterning of the neural ectoderm of Xenopus laevis by the amino-terminal product of hedgehog autoproteolytic cleavage. Development 121, 2349-2360.

Lanctot, C., Lamolet, B., Drouin, J., 1997. The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. Development 124, 2807-2817.

Lanctot, C., Moreau, A., Chamberland, M., Tremblay, M.L., Drouin, J., 1999. Hindlimb patterning and mandible development require the Ptx1 gene. Development 126, 1805-1810.

Laurent, M.N., Blitz, I.L., Hashimoto, C., Rothbacher, U., Cho, K.W., 1997. The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer. Development 124, 4905-4916.

Lee, H.X., Ambrosio, A.L., Reversade, B., De Robertis, E.M., 2006. Embryonic dorsal- ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. Cell 124, 147-159.

Leung, A.W., Kent Morest, D., Li, J.Y., 2013. Differential BMP signaling controls formation and differentiation of multipotent preplacodal ectoderm progenitors from human embryonic stem cells. Dev Biol 379, 208-220.

Lim, J.Y., Park, S.J., Hwang, H.Y., Park, E.J., Nam, J.H., Kim, J., Park, S.I., 2005. TGF- beta1 induces cardiac hypertrophic responses via PKC-dependent ATF-2 activation. J Mol Cell Cardiol 39, 627-636.

Luxardi, G., Marchal, L., Thome, V., Kodjabachian, L., 2010. Distinct Xenopus Nodal ligands sequentially induce mesendoderm and control gastrulation movements in parallel to the Wnt/PCP pathway. Development 137, 417-426.

Lyerla, T.A., Pelizzari, J.J., 1973. Histological development of the cement gland in Xenopus laevis: a light microscopic study. J Morphol 141, 491-501.

Massague, J., Seoane, J., Wotton, D., 2005. Smad transcription factors. Genes Dev 19, 2783-2810.

McMahon, A.P., Moon, R.T., 1989. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis. Cell 58, 1075-1084.

Mercurio, S., Latinkic, B., Itasaki, N., Krumlauf, R., Smith, J.C., 2004. Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex. Development 131, 2137-2147.

Miller, J.R., Rowning, B.A., Larabell, C.A., Yang-Snyder, J.A., Bates, R.L., Moon, R.T., 1999. Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation. J Cell Biol 146, 427-437.

Mitsiadis, T.A., Drouin, J., 2008. Deletion of the Pitx1 genomic locus affects mandibular tooth morphogenesis and expression of the Barx1 and Tbx1 genes. Dev Biol 313, 887-896.

Mochizuki, T., Karavanov, A.A., Curtiss, P.E., Ault, K.T., Sugimoto, N., Watabe, T., Shiokawa, K., Jamrich, M., Cho, K.W., Dawid, I.B., Taira, M., 2000. Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter. Dev Biol 224, 470-485.

Morgenegg, G., Winkler, G.C., Hubscher, U., Heizmann, C.W., Mous, J., Kuenzle, C.C., 1986. Glyceraldehyde-3-phosphate dehydrogenase is a nonhistone protein and a possible activator of transcription in neurons. J Neurochem 47, 54-62.

Moustakas, A., Heldin, C.H., 2009. The regulation of TGFbeta signal transduction. Development 136, 3699-3714.

Newman, C.S., Krieg, P.A., 2002. Xenopus bagpipe-related gene, koza, may play a role in regulation of cell proliferation. Dev Dyn 225, 571-580.

Nieuwkoop, P.D., 1967. The "organization centre". II. Field phenomena, their origin and significance. Acta Biotheor 17, 151-177.

Nieuwkoop, P.D., 1973. The organization center of the amphibian embryo: its origin, spatial organization, and morphogenetic action. Adv Morphog 10, 1-39.

Ninomiya, H., Takahashi, S., Tanegashima, K., Yokota, C., Asashima, M., 1999. Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus. Dev Growth Differ 41, 391-400.

Novoselov, V.V., Alexandrova, E.M., Ermakova, G.V., Zaraisky, A.G., 2003. Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo. Gene Expr Patterns 3, 225-230.

Nudi, M., Ouimette, J.F., Drouin, J., 2005. Bone morphogenic protein (Smad)- mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity. Mol Endocrinol 19, 1329-1342.

Pera, E.M., Ikeda, A., Eivers, E., De Robertis, E.M., 2003. Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. Genes Dev 17, 3023-3028.

Proctor, G.B., 2016. The physiology of salivary secretion. Periodontol 2000 70, 11-25.

Quirk, C.C., Lozada, K.L., Keri, R.A., Nilson, J.H., 2001. A single Pitx1 binding site is essential for activity of the LHbeta promoter in transgenic mice. Mol Endocrinol 15, 734-746.

Reversade, B., De Robertis, E.M., 2005. Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field. Cell 123, 1147-1160.

Rosa, F.M., 1989. Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. Cell 57, 965-974.

Rowning, B.A., Wells, J., Wu, M., Gerhart, J.C., Moon, R.T., Larabell, C.A., 1997. Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs. Proc Natl Acad Sci U S A 94, 1224-1229.

Ryan, A.K., Blumberg, B., Rodriguez-Esteban, C., Yonei-Tamura, S., Tamura, K., Tsukui, T., de la Pena, J., Sabbagh, W., Greenwald, J., Choe, S., Norris, D.P., Robertson, E.J., Evans, R.M., Rosenfeld, M.G., Izpisua Belmonte, J.C., 1998. Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394, 545-551.

Sander, V., Reversade, B., De Robertis, E.M., 2007. The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. EMBO J 26, 2955-2965.

Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L.K., De Robertis, E.M., 1994. Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell 79, 779-790.

Schulte-Merker, S., Smith, J.C., Dale, L., 1994. Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction? EMBO J 13, 3533-3541.

Schweickert, A., Campione, M., Steinbeisser, H., Blum, M., 2000. Pitx2 isoforms: involvement of Pitx2c but not Pitx2a or Pitx2b in vertebrate left-right asymmetry. Mech Dev 90, 41-51.

Schweickert, A., Deissler, K., Blum, M., Steinbeisser, H., 2001a. Pitx1 and Pitx2c are required for ectopic cement gland formation in Xenopus laevis. Genesis 30, 144-148.

Schweickert, A., Steinbeisser, H., Blum, M., 2001b. Differential gene expression of Xenopus Pitx1, Pitx2b and Pitx2c during cement gland, stomodeum and pituitary development. Mech Dev 107, 191-194.

Shen, M.M., 2007. Nodal signaling: developmental roles and regulation. Development 134, 1023-1034.

Sive, H., Bradley, L., 1996. A sticky problem: the Xenopus cement gland as a paradigm for anteroposterior patterning. Dev Dyn 205, 265-280.

Sive, H.L., Hattori, K., Weintraub, H., 1989. Progressive determination during formation of the anteroposterior axis in Xenopus laevis. Cell 58, 171-180.

Slack, J.M., Darlington, B.G., Heath, J.K., Godsave, S.F., 1987. Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature 326, 197-200.

Smith, J.C., Price, B.M., Green, J.B., Weigel, D., Herrmann, B.G., 1991. Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell 67, 79-87.

Smith, W.C., Harland, R.M., 1992. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 829-840.

St Amand, T.R., Zhang, Y., Semina, E.V., Zhao, X., Hu, Y., Nguyen, L., Murray, J.C., Chen, Y., 2000. Antagonistic signals between BMP4 and FGF8 define the expression of Pitx1 and Pitx2 in mouse tooth-forming anlage. Dev Biol 217, 323-332.

Suri, C., Haremaki, T., Weinstein, D.C., 2005. Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm. Development 132, 2733-2742.

Suzuki, A., Chang, C., Yingling, J.M., Wang, X.F., Hemmati-Brivanlou, A., 1997. Smad5 induces ventral fates in Xenopus embryo. Dev Biol 184, 402-405.

Szeto, D.P., Rodriguez-Esteban, C., Ryan, A.K., O'Connell, S.M., Liu, F., Kioussi, C., Gleiberman, A.S., Izpisua-Belmonte, J.C., Rosenfeld, M.G., 1999. Role of the Bicoid- related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. Genes Dev 13, 484-494.

Thompson, T.B., Lerch, T.F., Cook, R.W., Woodruff, T.K., Jardetzky, T.S., 2005. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding. Dev Cell 9, 535-543.

Thomsen, G.H., Melton, D.A., 1993. Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell 74, 433-441.

Ueno, N., Ling, N., Ying, S.Y., Esch, F., Shimasaki, S., Guillemin, R., 1987. Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone. Proc Natl Acad Sci U S A 84, 8282-8286.

Vandevyver, S., Dejager, L., Libert, C., 2012. On the trail of the glucocorticoid receptor: into the nucleus and back. Traffic 13, 364-374.

Varley, J.E., McPherson, C.E., Zou, H., Niswander, L., Maxwell, G.D., 1998. Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. Dev Biol 196, 107-118.

Vonica, A., Brivanlou, A.H., 2007. The left-right axis is regulated by the interplay of Coco, Xnr1 and derriere in Xenopus embryos. Dev Biol 303, 281-294.

Wardle, F.C., Sive, H.L., 2003. What's your position? the Xenopus cement gland as a paradigm of regional specification. Bioessays 25, 717-726.

Wardle, F.C., Wainstock, D.H., Sive, H.L., 2002. Cement gland-specific activation of the Xag1 promoter is regulated by co-operation of putative Ets and ATF/CREB transcription factors. Development 129, 4387-4397.

Weinstein, D.C., Hemmati-Brivanlou, A., 1999. Neural induction. Annu Rev Cell Dev Biol 15, 411-433.

Wheeler, G.N., Brandli, A.W., 2009. Simple vertebrate models for chemical genetics and drug discovery screens: lessons from zebrafish and Xenopus. Dev Dyn 238, 1287-1308.

Wilson, C., Cross, G.S., Woodland, H.R., 1986. Tissue-specific expression of actin genes injected into Xenopus embryos. Cell 47, 589-599.

Wilson, P.A., Hemmati-Brivanlou, A., 1995. Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331-333.

Wilson, P.A., Lagna, G., Suzuki, A., Hemmati-Brivanlou, A., 1997. Concentration- dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1. Development 124, 3177-3184.

Winklbauer, R., Damm, E.W., 2012. Internalizing the vegetal cell mass before and during amphibian gastrulation: vegetal rotation and related movements. Wiley Interdiscip Rev Dev Biol 1, 301-306.

Xu, R.H., Kim, J., Taira, M., Zhan, S., Sredni, D., Kung, H.F., 1995. A dominant negative bone morphogenetic protein 4 receptor causes neuralization in Xenopus ectoderm. Biochem Biophys Res Commun 212, 212-219.

Yamada, G., Mansouri, A., Torres, M., Stuart, E.T., Blum, M., Schultz, M., De Robertis, E.M., Gruss, P., 1995. Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death. Development 121, 2917-2922.

Yamada, G., Ueno, K., Nakamura, S., Hanamure, Y., Yasui, K., Uemura, M., Eizuru, Y., Mansouri, A., Blum, M., Sugimura, K., 1997. Nasal and pharyngeal abnormalities

70 caused by the mouse goosecoid gene mutation. Biochem Biophys Res Commun 233, 161-165.

Yang, J., Tan, C., Darken, R.S., Wilson, P.A., Klein, P.S., 2002. Beta-catenin/Tcf- regulated transcription prior to the midblastula transition. Development 129, 5743- 5752.

Ying, S.Y., Becker, A., Swanson, G., Tan, P., Ling, N., Esch, F., Ueno, N., Shimasaki, S., Guillemin, R., 1987. Follistatin specifically inhibits pituitary follicle stimulating hormone release in vitro. Biochem Biophys Res Commun 149, 133-139.

Zhang, C., Basta, T., Jensen, E.D., Klymkowsky, M.W., 2003. The beta-catenin/VegT- regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Development 130, 5609-5624.

Zhang, J., Houston, D.W., King, M.L., Payne, C., Wylie, C., Heasman, J., 1998. The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94, 515-524.

The Role of Pitx Proteins in Early &lt;I&gt;Xenopus&lt;/I&gt; Development

The Role of Pitx Proteins in Early <I>Xenopus</I> Development