The Evolutionary Transition from Lungs to a Gas Bladder: Evidence from Immunohistochemistry, Rna-Seq, and Morphology

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THE EVOLUTIONARY TRANSITION FROM LUNGS TO A GAS BLADDER: EVIDENCE FROM IMMUNOHISTOCHEMISTRY, RNA-SEQ, AND MORPHOLOGY

A Dissertation Presented to the Faculty of the Graduate School

of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

by Emily Funk December 2020

THE EVOLUTIONARY TRANSITION FROM LUNGS TO A GAS BLADDER: EVIDENCE FROM IMMUNOHISTOCHEMISTRY, RNA-SEQ, AND MORPHOLOGY

Emily Funk, Ph. D. Cornell University 2020

Key to understanding the evolutionary origin and modification of phenotypic traits is revealing the underlying developmental genetic mechanisms. An important morphological trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe- finned fishes. While gas bladders and lungs are similar in many ways, the distinguishing morphological difference between these organs is the general direction of budding from the foregut during development. Lungs bud ventrally and the gas bladder buds dorsally from the foregut endoderm. To compare lung and gasbladder development, the relevant taxa include bichir and bowfin. Bichir are the only living ray-finned fish that develops ventrally budding lungs. Bowfin, an early-diverging lineage, sister to teleosts, develops a gas bladder and exhibits a number of ancestral characteristics. Additionally, we included zebrafish as a representative of teleost fishes. I investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction using immunohistochemistry and RNA sequencing to determine whether gene expression patterns show a dorsoventral inversion paralleling the morphological inversion in budding direction. I also characterize morphological budding direction in bowfin, a purported transitional form, using nano-CT scanning. Taken together, the results of our gene expression and morphological studies of gasbladder development suggest that the inversion and modification of expression patterns of an ancestral lung-gene network underlies the evolution of a dorsal gas bladder from ventral lungs. The bowfin gas bladder does indeed bud dorsally from the

foregut and does not represent an intermediate, laterally-budding morphology between ventral lungs and a dorsal gas bladder. We suggest that a regulatory change producing the dorsoventral inversion of expression of Tbx5, a known lung-regulatory gene, in the foregut during gasbladder development could facilitate the inversion of expression of downstream genes, such as Tbx4, Wnt2, Fgf10, and Bmp-signaling. Furthermore, this gene network may have been modified during the evolution of the gas bladder leading to the expression of Bmp16, rather than the orthologous Bmp4, to regulate gasbladder development in ray-finned fishes. Changes in the timing and spatial expression of lung-regulatory genes appear to induce the dorsal budding of the gas bladder during development.

BIOGRAPHICAL SKETCH

Emily Funk received her a Bachelor of Science in Ecology and Evolutionary Biology from the University of Connecticut in 2013. While at UConn, Emily completed her Honors Thesis research with Dr. Eric Schultz on the genetic basis for osmoregulatory adaptation to freshwater in alewife (Alosa pseudoharengus). Emily’s experience during her undergraduate education sparked her interest in the genetic underpinnings of evolution especially in fishes. After graduating, Emily joined Amy McCune’s lab in the Department of Ecology and Evolutionary Biology at Cornell to study the evolution of morphological novelty.

ACKNOWLEDGMENTS

Many thanks to my adviser, Amy McCune, and committee members, Natasza

Kurpios and Bob Reed, for stimulating discussions, methods-development advice, and invaluable feedback throughout my graduate career. Thank you to Ezra Lencer for being a great lab mate and for sharing his bioinformatics expertise as well as providing thoughtful feedback on this thesis. I am grateful to all members of the Kurpios Lab for welcoming me into their lab group and entertaining stimulating discussions. My sincere thanks goes to my undergraduate research assistants, Catie Breen, who assisted with immunohistochemistry assays and Eda Birol, who helped with the nano-CT image analyses. Thank you to Ken Zeedyk, Dr. Joe Fetcho, Nikki McGuire, for graciously providing fish embryo samples, and thanks to Francis Feng for boat access to bowfin spawning grounds. I would also like to thank Drs. Ingo Braasch and Andrew

Thomson for early access to the bowfin genome. Many thanks to the Cornell

Biotechnology Resource Center Imaging Facility, and in particular, Teresa Porri, for guidance with nano-CT scanning and image analyses.

Thanks to the great friends and colleagues who have supported me throughout grad school including, Andrea Attenasio, Lina Arcila, Annie Scofield, Cora Demler,

Joe Welkin, Jake Berv, Michelle Wong, Bridget Darby, and many more. A special thanks to the SeaPigs, Erin Larson, Lauren Brzozowski, Lizzie Lombardi, Jenny

Uehling, Kelsey Jensen, Karin Vanderburg, Emily Rodekohr, Olivia Graham, and

Kara Andres, for being the most awesome running and adventure friends ever. I cannot thank my family enough for supporting me in anything and everything I choose

to pursue in life. Thank you to my Mom and Dad, and my brothers, Tom and Jeff.

This work was made possible by support from National Science Foundation

Graduate Research Fellowship, Cornell Presidential Life Sciences Fellowship, Sigma

Xi Cornell Chapter, the Cornell Center for Vertebrate Genomics Scholar’s Program,

American Society for Ichthyology and Herpetology Edward C. Raney Fund, McCune

Lab funds, and Cornell EEB Departmental Funds.

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TABLE OF CONTENTS BIOGRAPHICAL SKETCH...... v ACKNOWLEDGMENTS...... vi TABLE OF CONTENTS ...... viii LIST OF FIGURES ...... ix LIST OF TABLES...... xi PREFACE...... xii

CHAPTER 1...... 1 CHAPTER 2...... 48 CHAPTER 3...... 86 APPENDIX...... 119

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LIST OF FIGURES

Figure 1.1 : Basic morphology and phylogenetic distribution of lungs and the gas bladder…..4

Figure 1.2: Diagram of known gene interactions regulating lung development……………….8

Figure 1.3: Sox2 and Nkx2.1 expression in bichir lungs………………………………………20

Figure 1.4: Sox2 and Nkx2.1 expression in bowfin gas bladder………………………………21

Figure 1.5: Sox2 and Nkx2.1 expression in zebrafish gas bladder…………………………….22

Figure 1.6: Bmp4 expression and Smad phosphorylation in bichir lungs…………………….25

Figure 1.7: Bmp16 gene tree…………………………………………………………………..26

Figure 1.8: Bmp16 expression and Smad phosphorylation in bowfin gas bladder…………...29

Figure 1.9: Gene expression changes during the lung-to-gas bladder transition in ray-finned fishes………………………………………………………………………………………….32

Figure 2.1……………………………………………………………………………………..52

Figure 2.2: Principle Component Analysis (PCA) plots……………………………………...60

Figure 2.3: Differentially expressed genes between dorsal and ventral tissues of the foregut during gas bladder development………………………………………………………………63

Figure 2.4: Tissue-specific expression of known lung-development genes, differentially expressed at budding, during gas bladder development in bowfin……………………………68

Figure 2.5: Tissue-specific expression of known lung-development genes, differentially expressed at outgrowth, during gas bladder development in bowfin……………………...….69

Figure 2.6: Dorsoventral expression of Tbx5 during gas bladder development in bowfin……70

Figure 2.7: Expression of Tbx4, Wnt2ba, and Fgf10 during gas bladder development in bowfin………………………………………………………………………………………....72

Figure 3.1: Original and modified versions of the iconic 19th century morphological transformation series of lung and gasbladder morphology……………………………………90

Figure 3.2: Lung and gas bladder phenotypes arrayed in a modern phylogenetic framework..94

Figure 3.3: Location and morphology of the gas bladder in a juvenile bowfin……………….96

Figure 3.4: Paired photographs and nano-CT renderings of external morphology of developing bowfin larvae, stages 25 to 29……………………………………………………………….100

Figure 3.5: Morphology of the developing gas bladder in bowfin, stages 25, 26, and 27…..102

Figure 3.6: Morphology of the developing gas bladder in bowfin, stages 28 and 29……….104

Figure 3.7: Angular measurements of the right-twisted gas bladder bud……………………108

Appendix

Appendix Figure A1.1: Bowfin Bmp16 epitope for custom Bmp16 antibody design………121

Appendix Figure A1.2: Bmp4 expression in bowfin and zebrafish………………………….122

Appendix Figure A1.3: Bmp16 Immunofluorescence negative control in mouse…………..123

Appendix Figure A2.1: Bmp4 and Bmp16 expression during gasbladder development in bowfin…………………………………………………….………………………………….124

LIST OF TABLES

Table 1.1: Summary of expression patterns of four genes during early budding and organ outgrowth of lung or gas bladder development………………………………..34

Table 2.1: Top 10 Hallmark v7.0 gene sets enriched along the PCA……….………….……..62

Table 2.2: Dorsoventral differentially expressed (DE) genes…………………………………65

Table 3.1: Torsion of bowfin gasbladder development relative to vertical………………….107

PREFACE Key to understanding the evolutionary origin and modification of phenotypic traits is revealing the underlying developmental genetic mechanisms. An important morphological trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe- finned fishes. While gas bladders and lungs are similar in many ways, the distinguishing morphological difference between these organs is the general direction of budding from the foregut during development. Lungs bud ventrally and the gas bladder buds dorsally from the foregut endoderm. I investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction using immunohistochemistry and RNA sequencing to determine whether the gene expression patterns showed a dorsoventral inversion mirroring the morphological inversion in budding direction. I also characterized the morphological shift in a purported transitional form using nano-CT scanning. In particular, I investigated in detail three questions: (1) I asked whether several candidate genes known to influence the direction of lung budding in mice play a role in the budding direction of the gas bladder in fishes; (2) Using laser-capture microdissection and RNAseq I asked whether there were differentially expressed genes in dorsal and ventral tissue (both endoderm and mesoderm) associated with budding during gasbladder development; and (3) I deployed modern nano-CT technology to ask whether the gas bladder of bowfin, which had previously been purported to exhibit an intermediate form between lungs and the gas bladder of fishes, shows a a lateral direction of budding. I first investigated the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of the gas bladder and compared them to the patterns observed during the development of lungs. The relevant taxa for this comparison include bichir and bowfin. Bichir are the only living ray-finned fish that develops ventrally budding lungs. Bowfin are an early-diverging lineage, sister to teleosts, that develop a gas bladder and exhibit a number of ancestral characteristics. Additionally, I examined expression of these genes in zebrafish as a representative of the more recently derived clade of teleost fishes. During tetrapod lung development,

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Nkx2.1 and Sox2 are reciprocally expressed across the dorsoventral axis of the foregut, and both are important regulators of lung development. Bmp4 is expressed ventrally during lung development and inhibits the expression of Sox2. During bichir lung development, all three genes show similar expression patterns to those observed during mouse lung development. However, during bowfin and zebrafish development, none of these candidate genes show the dorsoventral inversion in expression pattern that I expected. However, I did discover a dorsal-ventral inversion of sorts. Bmp16, a paralogue of Bmp4, has been lost from the mammalian genome but is still present in the genomes of ray-finned fishes, including bowfin. During bowfin and zebrafish development, Bmp16 is expressed dorsally in the gas bladder compared to Bmp4, which is expressed ventrally during lung development in tetrapods and bichir. This work is the focus of Chapter 1. Rather than limiting my work to known candidate genes, I took advantage of relatively new technologies in order to discover new candidate genes that might be involved in the evolutionary transition from ventral lungs to a dorsal gas bladder. In Chapter 2, I report on my work to assay all genes expressed during gasbladder development in bowfin using laser-capture microdissection (LCM) and RNA sequencing. I used LCM to isolate the dorsal and ventral regions of the foregut and gas bladder and sequenced them separately to evaluate which genes are differentially expressed across the dorsoventral axis. I discovered a number of genes that were not previously known to be involved in lung development but are differentially expressed between dorsal and ventral foregut. Many of these genes are annotated to functions important for organ budding. Additionally, I found several known lung-regulatory genes that show dorsoventrally inverted expression patterns during gasbladder development compared to lung development. Specifically, Tbx5 is shows strong expression in the dorsal mesoderm surrounding the gasbladder bud, and several interacting genes, including Tbx4, Fgf10, and Wnt2, are co-expressed dorsally with Tbx5. In contrast, during lung development in mouse and bichir, Tbx5 is expressed in the ventral mesoderm surrounding the lung buds. These data demonstrate a dorsoventral inversion of expression of conserved genes during lung development

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compared to gasbladder development suggesting a mechanism underlying the lung-to- gas bladder evolutionary transition. Finally, in chapter 3, I resolved an early dispute about whether the gas bladder of bowfin represents an intermediate form between vertebrate lungs and the gas bladder of fishes. Historically, biologists sought to explain the shift from ventral lungs to a dorsal gas bladder by searching for intermediate morphological forms, in particular, a laterally budding gas bladder. In the early 20th century, it was suggested that the bowfin gas bladder buds laterally during development and represents this sought-after intermediate morphology. I used nano-CT scanning of bowfin developmental series to characterize the precise budding location and early development of the gas bladder in bowfin. I found that the bowfin gas bladder forms as a ridge budding dorsally from the foregut. Although the ridge is clearly dorsal, it does show a temporary, posterior right-hand twist during early stages of development. As development proceeds, the twist becomes shallower and the gas bladder itself is situated mid-dorsally in the body cavity. As such, the bowfin gas bladder does not represent an intermediate morphology between ventral lungs and a dorsal gas bladder. Given the results of this morphological study and the potentials of modern developmental genetic biology, I argue for a future focus on exploring what genes regulate the dorsal budding of the gas bladder rather searching for morphological intermediates. Taken together, the results of my studies of gene expression and the morphology of gasbladder development suggest that the inversion and modification of expression patterns of an ancestral lung-gene network underlies the evolution of a dorsal gas bladder from ventral lungs. A regulatory change producing the dorsoventral inversion of expression of Tbx5 in the foregut during gasbladder development may have facilitated the inversion of expression of downstream genes, including Tbx4, Wnt2, Fgf10, and Bmp-signaling. Furthermore, this gene network may have been modified during the evolution of the gas bladder by the expression of Bmp16, rather than the orthologous Bmp4, to regulate gasbladder development in ray-finned fishes.

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CHAPTER 1

Changes in Nkx2.1, Sox2, Bmp4 and Bmp16 expression underlying the lung-to-

gas bladder evolutionary transition in ray-finned fishes1

1 Funk, E.C., Breen, C., Sanketi, B. D, Kurpios, N. A, McCune, A.R. (2020). Changes in Nkx2.1, Sox2, Bmp4 and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes. Evolution and Development, 22 (5), 384-402.

Abstract

Key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to- dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gasbladder development, Nkx2.1 and Sox2 expression is inconsistent with the hypothesis that they regulate the direction of gasbladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gasbladder development,

Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of

Actinopteri (ray-finned fishes excluding bichir), but absent from mammalian genomes.

We hypothesize that Bmp16 was recruited to regulate gasbladder development in the

Actinopteri in place of Bmp4.

1. Introduction

The origin of phenotypic novelties, such as jaws and limbs is central to understanding the history of life, and it is now possible to expose the genetic underpinnings of such novel traits using modern developmental genetics. An important evolutionary novelty characterizing most of the ~30,000 species of living ray-finned fishes (including teleosts; Nelson et al., 2016) is the air-filled gas bladder, which is used primarily for buoyancy control in most species which have one (Steen,

1970; Helfman et al., 2009). Though Darwin (Darwin, 1859) argued that lungs were derived from the gas bladder, Sagemehl (1885) subsequently argued that the gas bladder is the more derived structure. This has been the dominant view since (Romer and Parsons, 1970; Liem, 1988; Graham, 1997) and is consistent with the phylogenetic distribution of these traits across a well-supported phylogeny of bony vertebrates

(Near et al., 2012; Hughes et al., 2018). The homology of lungs and gas bladders as air-filled organs is well supported. Both develop from the anterior foregut endoderm, are supplied by the pulmonary artery, produce surfactant proteins, and express a suite of regulatory genes with known lung-specific function (Goodrich, 1958; Daniels et al.,

2004; Cass et al., 2013; Longo et al., 2013). The defining difference between gas bladders and lungs is the direction of budding from the anterior foregut. Gas bladders lie just ventral to the spine (Figure 1.1a) and bud from the dorsal (or dorsolateral) wall, while lungs bud from the ventral (or ventrolateral) wall (Wilder, 1877; Graham, 1997;

Cass et al., 2013).

Figure 1.1 : Basic morphology and phylogenetic distribution of lungs and the gas bladder. a) Diagram of a typical teleost fish showing the dorsal location of the gas bladder situated above the gut and below the spine (Pough et al., 1996). b) A highly pruned phylogeny of bony vertebrates showing taxa of interest. The two major lineages of the bony vertebrate clade are the lobe-finned fishes (about 30,500 species), which includes tetrapods (e.g. mouse), and the ray-finned fishes (over 30,000 species ; Nelson et al., 2016). Within the ray-finned fishes, bichirs are the only ray-finned fish that possess lungs (blue), while bowfin and teleosts possess a gas bladder (orange). Based on phylogenetic distribution of air-filled organs, the lungs are the ancestral state for the bony vertebrates and the gas bladder originated in the lineage containing bowfin and teleosts (e.g. zebrafish). c) Dorsal versus ventral outgrowth of lungs and gas bladders in key taxa. Taxa are illustrated in the left column. To the right of each organism, in the middle column, is a line drawing showing a lateral view of the dissected foregut and lungs/gas bladder (after Dean, 1895). In the right-most column, a transverse- sectional schematic of the foregut and lungs or gas bladder is shown (after Dean, 1895). As shown, zebrafish and bowfin have a dorsally budding gas bladder. Bichir, Australian lungfish and mouse have ventrally budding lungs. Abbreviations: df, dorsal fin; fg, foregut; GB, gas bladder; in, intenstine; L: lungs; pd, pneumatic duct; pe, pectoral fin; sc, spinal cord; t, trachea.

To probe the underlying differences in gene expression between the dorsal budding of gas bladders and ventral budding of lungs, we focus on three important representative fish taxa, bichir, bowfin, and zebrafish. Bichir and bowfin are living ray-finned fish taxa that diverged before and after the origin of the gas bladder, respectively (Figure 1.1b). Bichirs are the only living ray-finned fish retaining ventrally budding lungs (Figure 1.1b, c) and thus represent the ancestral condition of bony vertebrates. Within the actinopterygian fishes, the bichirs are the sister-group to all other ray-finned fishes (Actinopteri) including sturgeon, bowfin, and zebrafish

(Figure 1.1b). Bowfin diverged after gas bladder origination and represent the sister clade to teleosts (including zebrafish), the dominant group of living fishes. In bowfin, the connection between the gut and gas bladder, the pneumatic duct, persists, and the gas bladder has elaborated surface area and vascularization, important for its respiratory function (Graham, 1997). Zebrafish is a more recently diverged teleost species with a two-chambered gas bladder and a persistent pneumatic duct (Finney et al., 2006).

Here we investigate whether the morphological inversion of the budding site is associated with ventral-to-dorsal inversion in the expression pattern of genes known to regulate lung development. We used immunofluorescence to compare the expression patterns of key candidate genes in our three focal taxa, bichir, bowfin, and zebrafish.

Given the homology between the lungs and the gas bladder, we hypothesized that the genes regulating lung development might also regulate gas bladder formation but via different patterns of spatial and temporal expression. We first focused on two essential transcription factors, Sox2 and Nkx2.1, because they show opposing dorsal-ventral

expression patterns in developing mouse lungs (Figure 1.2). We predicted that 1) in bichir, the only living ray-finned fish that retains lungs, Nkx2.1 and Sox2 would exhibit the same foregut expression pattern seen in mouse, and 2) in bowfin and zebrafish which have gas bladders, we would observe an inverted expression pattern of Nkx2.1 and Sox2 relative to mouse lungs. We also examined the expression patterns of Bmp4, known to play a role in lung budding and branching, and Bmp16, a paralog of Bmp4 present in ray-finned fishes but absent from mammalian genomes. We hypothesized that in bichirs, which possess ventrally budding lungs, Bmp4 would be expressed ventrally in the lung buds similar to the pattern during mouse lung development. In bowfin, which has a gas bladder, we hypothesized that Bmp16 (rather than Bmp4) would be expressed dorsally in the gas bladder bud.

Surprisingly, we learned that two key genes involved in regulating the ventral direction of lung budding do not appear to regulate the dorsal direction of gas bladder budding and thus do not underlie the morphological shift in budding site. Instead, we find a different gene, Bmp16, to be expressed dorsally in the gas bladder, while Bmp4 is expressed ventrally in taxa (mammals, bichirs) with lungs, suggesting Bmp signaling may play an important role during gasbladder evolution. Collectively, our results shed light on the possible molecular mechanisms responsible for the lung-to- gas bladder evolutionary transition.

Figure 1.2: Diagram of known gene interactions regulating lung development. a) At embryonic day 9 (E9) in mouse, the lung field is established. b) At E9.5, the nascent lungs bud ventrally from the anterior foregut. During these early stages of lung development, E9 and E9.5 (a, b), Nkx2.1 (yellow), the first marker of lung develoment, is expressed ventrally in the foregut endoderm and indicates the site of lung budding. Sox2 (blue) is expressed dorsally in the foregut endoderm and is mutually inhibitory with Nkx2.1. Bmp4 in the mesoderm interacts with its receptors, Bmpr1a and Bmpr1b, in the endoderm to inhibit the expression of Sox2 ventrally allowing for the expression of Nkx2.1. c) At E12.5, during lung branching, Bmp4 (dark green) becomes expressed at the distal tips of the branching lung buds, and Fgf10 (light green) is expressed in the mesoderm surrounding the distal tips. Bmp4 and Fgf10 interact to promote bud outgrowth and branching. Endo: endoderm, Meso: mesoderm

1.1 Background on Gas Bladder Function and Evolution

The gas bladder (also known as the swim bladder or air bladder) is located dorsal to the gut and ventral to the spine (Figure 1.1a). While a gas bladder persists as a respiratory organ in some bony fishes (e.g. bowfin, gar), in most ray-finned fishes, it functions primarily as a buoyancy organ (Steen, 1970; Helfman et al., 2009). By adding or eliminating oxygen from the gas bladder, a fish can maintain its vertical position in the water column without swimming continuously (Steen, 1970; Helfman et al., 2009). Presumably, the reduction in energy expenditure to maintain vertical position enables greater allocation of energy towards growth, predator avoidance, feeding, and reproduction (Marty et al., 1995). Indeed, the gas bladder characterizes the Actinopteri, which includes early diverging sturgeons, paddlefish, gars, and bowfin as well as the Teleostei, the dominant group of living fishes (Figure 1.1b;

Nelson et al., 2016). Within the Teleostei, there are taxa in which the gas bladder is further modified, enhancing varied functions, such as buoyancy in some groups, respiration in others, or even hearing and sound production (Helfman et al., 2009). In deep sea fishes or taxa that migrate vertically through great depths on daily cycles, an air-filled buoyancy organ would be a severe liability; in these taxa, the gas bladder may be lipid-filled, secondarily reduced, or lost (Wittenberg et al., 1980). The gas bladder, and its modified versions, are clearly important adaptations, critical to the lives of many fishes (Marshall, 1971; Helfman et al., 2009).

The common ancestor of bony vertebrates, i.e., ray-finned fishes and lobe-

finned fishes, which include tetrapods (Rosen et al., 1981; Zimmer and Emlen, 2013), possessed lungs that were used for aerial respiration (Bray et al., 1985; Clack, 2007).

Secondarily, air in these early lungs would have also provided buoyancy, keeping the fishes near the surface where they could breathe air (Alexander, 1966; Liem, 1988). In fully aquatic fishes, buoyancy control became an important function of the gas bladder

(Alexander, 1966; Liem, 1988). Conversely, in terrestrial bony vertebrates (i.e., tetrapods), the respiratory function of lungs was enhanced for life on land, while the buoyancy control function became largely irrelevant (Liem, 1988).

Lungs and gas bladders in living vertebrates may exhibit a number of morphological and functional differences. For example, mammalian lungs are paired and undergo branching morphogenesis producing bronchi and alveoli (Kardong,

2015). Gas bladders of teleost fishes are usually unpaired and do not branch (Helfman et al., 2009). Functionally, lungs are used for respiration while gas bladders in most fishes function in buoyancy control. However, there are exceptions to these generalizations. Gas bladders can be bilaterally paired (Rice and Bass, 2009), and some tetrapod taxa, such as snakes or caecilians have only a single lung (Wallach,

1998). Gas bladders can be used as supplementary respiratory organs, as in bowfin, gar and numerous teleosts (Liem, 1989; Graham, 1997). In the context of this diversity of form and function, the key distinguishing characteristic of lungs and gas bladder is the direction of budding from the anterior foregut during development; the gas bladder buds essentially dorsally and the lungs bud essentially ventrally (Figure 1.1c), though there is variation in the exact positioning of the budding site. For example, the lungs in

Australian lungfish (Neoceratodus) connect ventrolaterally to the foregut (Grigg, 1965) and the gas bladder in sturgeon and paddlefish connects dorsolaterally (Grom, 2015).

In an aquatic context, a shift of gas bladder budding site to a dorsal position may be functionally important by decreasing the tendency to roll when the fish is not actively swimming (Videler, 2012).

1.2 Background on Lung and Gas Bladder Development

Extensive research on lung development in the mouse model system has identified many genes involved in regulating development of the mouse respiratory system comprising the trachea and lungs (Morrisey and Hogan, 2010; Hines and Sun,

2014; Kim et al., 2019). Both the trachea and lungs develop from embryonic foregut endoderm. Septation divides the foregut tube into a ventral trachea and dorsal esophagus. Just posterior to the dividing foregut forming the trachea and esophagus, two primary lung buds evaginate from the ventral foregut endoderm (Morrisey and

Hogan, 2010; Ornitz and Yin, 2012).

Of the many genes known to regulate lung development (Mariani et al., 2002;

Morrisey and Hogan, 2010; Ornitz and Yin, 2012; Hines and Sun, 2014; Rankin et al.,

2015), Nkx2.1 is the first molecular marker of mouse lung development. Nkx2.1 is expressed in the ventral foregut endoderm, thus defining the position of future trachea and lung buds. Sox2 is reciprocally expressed relative to Nkx2.1 and is localized in the dorsal foregut endoderm defining the future esophagus (Minoo et al., 1999; Que et al.,

2007; Gontan et al., 2008; Domyan et al., 2011). This same pattern and timing of Sox2 and Nkx2.1 expression is evolutionarily conserved in chicken and Xenopus as well

(Ishii et al., 1998; Rankin et al., 2015). Deletion of Nkx2.1 in mice causes an expansion of Sox2 expression in the foregut and a failure of the foregut to divide into separate trachea and esophagus resulting in a tracheal-esophageal fistula (Minoo et al., 1999;

Que et al., 2007). This single tube exhibits strong Sox2 expression characteristic of a normal esophagus showing the loss of ventral identity of the foregut in Nkx2.1-null mice (Minoo et al., 1999; Que et al., 2007). While Nkx2.1-null mice do form ventral lungs, they are smaller in size and do not undergo normal branching morphogenesis resulting in the inability to perform gas exchange and thus postnatal death (Minoo et al., 1999). A conditional knock-out of Sox2 in mice also produces a tracheal- esophageal fistula; however, the single tube exhibits a ventralized foregut phenotype evidenced by strong Nkx2.1 expression characteristic of a normal trachea (Que et al.,

2007). Based on these mouse studies, Nkx2.1 and Sox2 are evidently important for determining dorsoventral identity in the foregut during development of the mouse respiratory system.

Bmp4, a member of the transforming growth factor-β protein superfamily, is involved in establishing the opposing dorsoventral patterns exhibited by Nkx2.1 and

Sox2 during lung development in mice (Li et al., 2008; Domyan et al., 2011). During early lung development at the initial budding stage, mouse embryonic (E) 9.5, Bmp4 is expressed in the ventral mesoderm surrounding the lung bud, where it represses Sox2 expression in the ventral foregut endoderm allowing for expression of Nkx2.1 (Figure

1.2a; Domyan et al., 2011). Thus, Bmp4 signaling restricts Sox2 expression to the dorsal foregut endoderm, while Nkx2.1 is expressed in the ventral endoderm. When

Bmp4 signaling is inhibited during mouse lung development, Sox2 expression expands ventrally and Nkx2.1 fails to be expressed, leading to an undivided foregut exhibiting expression patterns characteristic of the esophagus and extra ectopic lung buds (Li et al., 2008; Domyan et al., 2011). After initial lung budding, during branching morphogenesis, Bmp4 is expressed in the endoderm at the distal tips of the elongating lung branches (Figure 1.2c; Bellusci et al., 1996). Through interaction with Fgf10, expressed in the mesoderm surrounding the branch tips, Bmp4 helps regulate branch elongation and division (Figure 1.2c). Bmp4 acts to inhibit, while Fgf10 acts to promote branch elongation (Weaver et al., 2000). Bmp4 has been shown to play multiple and dynamic roles throughout mouse lung development, including interacting with Sox2 to establish a dorsoventral pattern across the anterior foregut and determine respiratory system identity, which includes proper lung budding and septation of the trachea and esophagus (Domyan et al., 2011).

To date, Bmp4 has not been shown to play a role in gas bladder development.

However, Feiner et al. (2009) discovered the existence of Bmp16, a paralogue of Bmp4 and Bmp2, in genomes of teleost fishes. Since then, Bmp16 has also been identified in coelacanths, lepidosaurs (e.g., snakes and lizards), and chondrichthyans (sharks, skates and rays). However, Bmp16 has not been found in any known mammalian genomes, including mouse and human (Feiner et al., 2009, 2019; Marques et al., 2016). During zebrafish development, at 3 days post fertilization (dpf), Bmp16 is expressed in the anterior foregut as well as in the gas bladder. Subsequently, at 5 dpf, Bmp16 is strongly expressed in the gas bladder (Feiner et al., 2009). Marques et al. (2016) also

showed that zebrafish Bmp16 is highly similar in both amino acid sequence and tertiary protein structure to Bmp2 and Bmp4 and can activate Bmp-signaling pathways, suggesting Bmp16 may play a similar role in gas bladder development as Bmp4 plays during lung development. Moreover, prdc, a known antagonist of both Bmp2 and

Bmp4, is also expressed during gas bladder development in zebrafish (Feiner et al.,

2009). Later in mouse lung development, during branching morphogenesis, gremlin, a

Bmp4 antagonist, interacts with Bmp4 to pattern the proximal-distal axis. The expression of antagonistic protein prdc in the gas bladder suggests a similar interaction between Bmp16 and prdc may promote gas bladder development and outgrowth (Feiner et al., 2009).

2. Materials and Methods

2.1 Egg Collection and Sampling

We collected bowfin eggs from Oneida Lake, NY during the spawning season in May. The eggs were immediately treated with methylene blue to prevent fungal growth and transported to our lab at Cornell University. The eggs and larvae were raised at 15oC in water from Oneida Lake. Water changes were done every other day, and the water was treated at each water change with methylene blue until hatching.

We sampled 20-30 larvae daily between days 0 to 12 post-hatching to capture bowfin development between stages 23 and 28 (Ballard, 1986). We followed Ballard’s (1986) staging series to track bowfin gas bladder development: stage 24 is just prior to gas bladder budding, stage 25 is at initial gas bladder budding, stage 26 is right after budding, and stages 27 and 28 are during gas bladder outgrowth.

Bichir and zebrafish were spawned in captivity. We received bichir eggs from

a private aquarist and reared the eggs and larvae in 26.6oC freshwater (0.05-0.1 parts per thousand) in the laboratory. Water was changed every other day. Until hatching, the water was treated with methylene blue to prevent fungal growth on the eggs. We sampled 10-20 larvae per day from 7 to 13 dpf, which spanned bichir stages 30 to 37 following Budgett (1902) to track bichir lung development: stage 32 is just prior to lung budding, stage 33 is at initial lung budding, stage 34 is right after lung budding, and stages 35 and 36 are during lung outgrowth.

Twenty zebrafish larvae were sampled per day between 2 and 5 dpf obtained from a laboratory stock of wild-type adults reared at standard conditions in the Fetcho

Lab at Cornell University. In zebrafish, the gas bladder bud forms at 2 dpf (pharyngula stage). Zebrafish were reared at standard conditions.

All sampled larvae were euthanized with an overdose of MS-222 and fixed in

4% paraformaldehyde (PFA) overnight at 4oC. The samples were then flash frozen in

2:1 Tissue Tek Optimal Cutting Temperature Compound: 30% sucrose embedding medium in a 2-Methylbutane bath with liquid nitrogen. Embedded samples were stored at -80oC. Samples were sectioned into 15 µm sections using a cryostat (Leica

CM 1950) and mounted on Fisher SuperFrost Plus microscope slides for immunofluorescence or RNAscope (Advanced Cell Diagnostics). All animal protocols and procedures were performed in accordance with IACUC (protocol #2006-0013).

2.2 Immunofluorescence

We used fluorescent immunofluorescence (IF) to observe the expression patterns of Sox2, Bmp4, and Bmp16 in all three species as well as the expression patterns of phospho-Smad1/Smad5/Smad9 (pSmad) to determine Bmp activity. We

followed standard protocol for IF on sections (Welsh et al., 2013). Primary antibodies include rabbit anti-Sox2 (1:500; My Bio Source MSB9127841), rabbit anti-Bmp4

(1:100; My Bio Source MSB9417271), rabbit anti-Bmp16 (Thermo Fisher Scientific,

Pierce Biotechnology), and phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/

Smad9 (Ser465/467) (D5B10) Rabbit mAb (Cell Signaling Technology). No commercial Bmp16 antibodies were available, so the rabbit anti-Bmp16 antibody was designed and produced by Pierce Biotechnology (Thermo Fisher Scientific) using a bowfin Bmp16 antigen (sequence: DQRGVDSSRLARLE). The Bmp16 antibody was designed not to cross react with Bmp4 or Bmp2 (Figure A1.1); to verify specificity, we performed IF with the rabbit anti-Bmp16 antibody in mouse. Because mammal genomes do not have a Bmp16 gene, we should not observe any fluorescent signal by rabbit anti-Bmp16 antibody in mouse tissues. Secondary antibodies used include

Alexafluor 568 goat anti-rabbit IgG or Alexafluor 568 goat anti-mouse IgG (1:500;

Life Technologies). pSmad samples were visualized with a biotinylated secondary

(1:500; Jackson ImmunoResearch), followed by a streptavidin HRP (1:500; Jackson

ImmunoResearch), and then tyramide amplification was performed following the TSA

Plus Cy3 protocol (1:50; Perkin-Elmer). We co-labeled with DAPI (1:1000) to visualize all cell nuclei to provide morphological context. To visualize the fluorescently labeled expression patterns, we used a Zeiss Observer.Z1/ApoTome.2 inverted microscope with AxiocamHRc camera.

As a negative control for the binding specificity of the rabbit anti-Sox2 antibody in bichir, bowfin, and zebrafish, we monitored signal in the pectoral fin tissue, in which Sox2 should not be expressed. We observed no non-specific binding

of the Sox2 antibody in the pectoral fin of bichir, bowfin, or zebrafish. As a positive control, we observed binding of the Sox2 antibody in the ventricular zone of the brain, a region of strong Sox2 expression (Bani-Yaghoub et al., 2006), in all study species.

The positive tissue-control for the rabbit anti-Bmp4 antibody included expression in the pectoral fins and inner ear.

2.3 RNAscope in situ hybridization

We performed RNAscope Multiplex Fluorescent Assay to observe the RNA expression patterns of Nkx2.1 during development of bowfin gas bladder and bichir lungs and of Nkx2.1b during development of zebrafish gas bladder. Nkx2.1b is one of two copies of the gene in zebrafish due to the teleost whole genome duplication event.

Previously, Cass et al. (2013) found both Nkx2.1 paralogues to be expressed during gas bladder development in zebrafish; however, Nkx2.1b was only expressed dorsally in the gas bladder bud. Advanced Cell Diagnostics designed species-specific RNA probes for Nkx2.1 for each of the three taxa. We used the RNAscope Multiplex

Fluorescent v2 Kit following the manufacturer’s protocol (Advanced Cell Diagnostics,

Newark, CA) with the Perkin Elmer Cy3 fluorophore. Fluorescent images were captured with a Zeiss Observed.Z1/ApoTome.2 inverted microscope with

AxiocamHRc camera. Nkx2.1 is expressed only in the foregut, the gas bladder, the thyroid, and the forebrain of zebrafish. As a negative control for the binding specificity of the RNAscope Nkx2.1 probes, we observed no fluorescent signal in any other tissues during the development of our three study species. We did observe binding of the Nkx2.1 probe, as expected, in the diencephalon and telencephalon of the forebrain (González et al., 2002).

2.4 Bmp16 Phylogeny

To document the paralogy and evolutionary history of bowfin Bmp2, Bmp4 and Bmp16, we reconstructed a gene tree for these three genes using sequences from

Feiner et al. (2019) plus the bowfin sequences. The outgroups used in the analysis were Xenopus, chicken, opossum, human, and sea lamprey. We aligned the protein sequences in MAAFT according to the default settings (Katoh et al., 2019). We then used IQ-TREE Web Service with ultrafast bootstrap and model selection to reconstruct the gene tree according to default settings and enabling the FreeRate heterogeneity model (Chernomor et al., 2016; Trifinopoulos et al., 2016;

Kalyaanamoorthy et al., 2017; D.T. Hoang et al., 2018). The resulting tree was viewed in FigTree v1.4.4 and rooted on sea lamprey bmp paralogues.

3. Results

3.1 Dorsal-Ventral expression of Sox2 and Nkx2.1

We hypothesized that in the developing lungs of bichir, Sox2 would be expressed dorsally, and Nkx2.1 would be expressed ventrally, as in mouse (Morrisey and Hogan, 2010; Domyan et al., 2011). As expected, we found that Sox2 was expressed in the dorsal foregut endoderm opposite the lung budding site and it was absent from the ventral lung bud and lungs proper at all stages (Figure 1.3a, b). As in mouse (Lazzaro et al., 1991; Morrisey and Hogan, 2010), Nkx2.1 is expressed strongly in the ventral foregut endoderm of bichir at all stages analyzed. However, unlike mouse, where Nkx2.1 is restricted ventrally, in bichir, Nkx2.1 is expressed in a gradient with weaker expression dorsally in the foregut (Figure 1.3c, d). During early lung development (stage 33 and 34) in bichirs, Nkx2.1 is also strongly expressed in the

lung bud (Figure 1.3c), but later in development, during outgrowth (stage 35 and 36),

Nkx2.1 expression weakens in the lungs compared to the ventral foregut (Figure 1.3d).

Given the dorsoventrally inverted location of budding of the gas bladder relative to lungs, we hypothesized an inverted pattern of gene expression relative to lung development. Thus, in taxa with gas bladders we predicted ventral expression of

Sox2 and dorsal expression of Nkx2.1. However, contrary to expectation, Sox2 is expressed throughout the foregut endoderm, both ventrally and dorsally, as well as in the gas bladder bud itself at all stages examined in both bowfin (Figure 1.4a, b) and zebrafish (Figure 1.5a, b). Sox2 expression in the gastrointestinal tract varies along the anteroposterior body axis differently in these two taxa with gas bladders. In bowfin,

Sox2 is expressed all along the anteroposterior axis of the gut and in the gas bladder proper at all stages examined (Figure 1.4a). In zebrafish, after the gas bladder has inflated (3 dpf), Sox2 expression occurs only in the anterior foregut endoderm continuing posteriorly up to the pneumatic duct, which connects the gas bladder to the foregut. Sox2 is also expressed in the pneumatic duct itself as well as in the walls of the gas bladder (Figure 1.5a). At all stages examined during bowfin gas bladder development, Nkx2.1 is highly expressed in the ventral foregut endoderm (Figure 1.4c, d), as observed in mouse lung development. Nkx2.1 is not expressed in the dorsal foregut or pneumatic duct but shows weak expression in the gas bladder itself during outgrowth (stage 27; Figure 1.4d). In zebrafish, Nkx2.1b is expressed in the gas bladder bud and throughout the foregut endoderm at budding and outgrowth stages

(Figure 1.5c, d).

Figure 1.3: Sox2 and Nkx2.1 expression in bichir lungs: For each pair of panels, the top panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom panels show gene expression alone in bright red. Compasses indicate orientation of sections. a) Sagittal section of Sox2 expression in the foregut and lungs at stage 35 during outgrowth. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval bichir. b) Sox2 expression in the lungs at stage 35 is shown in transverse section. These sections show that Sox2 is expressed throughout the foregut (fg) but absent from the lung buds (L). c) Nkx2.1 expression at stage 34, right after lung budding. At stage 34, Nkx2.1 is expressed throughout the foregut (fg) and lung bud (L) but strongest expression is in the ventral foregut and lung bud. d) Nkx2.1 expression at stage 35, during lung outgrowth. At stage 35, Nkx2.1 continues to be expressed throughout the foregut and lungs with strongest expression in the ventral foregut and weaker expression in the dorsal foregut and lungs. Abbreviations are: n, notochord; D, dorsal, V, ventral; A, anterior; P, posterior; R, right; L, left.

Figure 1.4: Sox2 and Nkx2.1 expression in bowfin gas bladder: For each pair of panels, the top or left panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom or right panels show gene expression alone in bright red. Compasses indicate orientation of sections. a) Sox2 expression in sagittal view in bowfin at stage 26, right after gas bladder budding. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval bowfin. b) Sox2 expression at stage 26 displayed in transverse section. As shown, Sox2 is expressed throughout the foregut (fg) and the gas bladder bud (GB), and expression extends beyond the pneumatic duct (pd) connection to the foregut. c) Nkx2.1 expression at stage 26, right after gas bladder budding shows clearly the ventrally restricted expression of Nkx2.1 in the foregut and absence of Nkx2.1 expression in the gas bladder bud. d) Nkx2.1 expression at stage 27, during gas bladder outgrowth showing strong Nkx2.1 expression ventrally in the foregut as well as very weak expression in the gas bladder. Abbreviations are: n, notochord; D, dorsal, V, ventral; A, anterior; P, posterior; R, right; L, left.

Figure 1.5: Sox2 and Nkx2.1 expression in zebrafish gas bladder. For each pair of panels, the top or left panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom or right panels show gene expression alone in bright red. Compasses indicate orientation of sections. a) Sagittal view of Sox2 expression in zebrafish at 3 dpf, right after gas bladder budding. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval zebrafish. b) Sox2 expression at 3 dpf shown in transverse section. As shown, Sox2 is expressed throughout the foregut (fg) and the gas bladder bud (GB). However, Sox2 expression is absent from the gut beyond the pneumatic duct (pd). c) Nkx2.1b expression in zebrafish at 2 dpf, initial gas bladder budding. d) Nkx2.1 expression at 3 dpf, right after budding. Nkx2.1b expression is expanded to the entire foregut and the gas bladder bud. Abbreviations are: n, notochord; D, dorsal, V, ventral; A, anterior; P, posterior; R, right; L, left.

3.2 Roles of Bmp genes in bichir, bowfin and zebrafish

In mouse, Bmp4 is expressed in the mesenchyme surrounding the ventral foregut before and during initial lung budding and also in the distal epithelium during lung outgrowth and bifurcation (Que et al., 2006; Morrisey and Hogan, 2010; Ornitz and Yin, 2012). Thus, we hypothesized that Bmp4 expression in bichir lungs would be similar to its expression in mouse lungs. We find the temporal expression of Bmp4 in bichir is indeed similar to that in mice, but the spatial expression differs. Bmp4 is expressed at the time of initial lung budding in both bichir (stage 33) and mice.

However, in bichir, Bmp4 is expressed strongly in the endoderm of the lung bud rather than the surrounding mesenchyme. In bichir, Bmp4 continues to be expressed in the lung epithelium during lung outgrowth (stage 35) but expression is not restricted to the distal tips as it is in mouse lungs (Figure 1.6a, b). Canonical BMP signaling results in intracellular phosphorylation of regulatory Smads 1/5/9. Confirming Bmp activity, we detected Smad phosphorylation in the ventral mesenchyme surrounding the lung buds, where Bmp4 is expressed, during bichir development (Figure 1.6c, d). In bichir,

Bmp4 also shows weak expression in the anterior foregut, ventrally and dorsally, from which the lungs bud, at all stages investigated (Figure 1.6a, b). Bmp4 expression has not been observed in the foregut or gas bladder during zebrafish development, and likewise, we do not find Bmp4 expression in the foregut or gas bladder of bowfin.

However, Bmp4 expression is observed in the inner ear and fin of zebrafish, confirming cross-reaction of the anti-Bmp4 polyclonal antibody (Figure A1.2c). In bowfin, we observe Bmp4 expression in the pharynx, anterior to the location of gas bladder budding (Figure A1.2a). However, neither in the foregut at the site of budding

nor in the gas bladder of bowfin do we find any Bmp4 expression (Figure A1.2b).

Recently, a Bmp4 paralog, Bmp16, present in multiple ray-finned fish genomes but absent mammalian genomes has been shown to be expressed in the zebrafish gas bladder (Feiner et al., 2009, 2019). Using sequence alignment, we identified a putative

Bmp16 gene in the bowfin genome. This putative Bmp16 contained all amino acid residues conserved among previously annotated Bmp16 genes in ray-finned fishes. To confirm that the bowfin Bmp16 we found was an ortholog, we constructed a gene tree for Bmp16, Bmp4, and Bmp2 as described above. Overall, our gene tree (Figure 1.7) agreed with the tree built by Feiner et al (2019). The putative bowfin Bmp16 clustered with the other known Bmp16 sequences of various ray-finned fishes (gar, zebrafish, stickleback, tetraodon, and tilapia) as well as with Bmp16 from coelacanth confirming that the gene we identified as Bmp16 in the bowfin genome is an ortholog of Bmp16.

Using the protein sequence for bowfin Bmp16 as an antigen, we designed a polyclonal antibody with Pierce Biotechnology (Thermo Fisher Scientific). To test the specificity of this antibody, we performed an IF in mouse using the custom Bmp16 antibody. Our

Bmp16 antibody did not react in mouse tissues and showed no expression (Figure

A1.3). Because Bmp16 is absent from mammal genomes, this confirms the specificity of our antibody, and that it does not cross-react with either Bmp4 or Bmp2 (Figure

A1.3).

Figure 1.6: Bmp4 expression and Smad phosphorylation in bichir lungs: For each pair of panels, top panels shown protein expression in bright red overlain with DAPI in blue. Bottom panels show protein expression alone in bright red. All panels are shown in transverse section, with the compass indicating orientation shown in upper right panel. Inset in panel a shows a diagram of a larval bichir with position of anterior and posterior sections indicated. a) Bmp4 expression in the anterior region of bichir lungs at stage 36 during lung outgrowth. b) Bmp4 expression in the posterior region of the lungs at stage 36. Bmp4 is expressed in the foregut (fg) and lung endoderm with strong expression in the lungs. c) Smad phosphorylation, shown in bright red, is detected in anterior region of bichir lungs at stage 36 during lung outgrowth. d) Smad phosphorylation is detected in posterior region of the lungs at stage 36. Smad phosphorylation, indicating Bmp4 activity, is detected strongly in the ventral mesenchyme surrounding the lung buds. Abbreviations are: rL, right lung; lL, left lung; D, dorsal, V, ventral; R, right; L, left.

Figure 1.7: Bmp16 gene tree: Reconstructed gene tree of relationships of Bmp16, Bmp4, and Bmp2 among bony vertebrates. The Bmp2 cluster is shown in blue. The Bmp4 cluster is shown in orange. The Bmp16 cluster is shown in purple. The sea lamprey Bmp2/4 genes (black) are used as the outgroup. Note that the putative bowfin Bmp16 is nested within the Bmp16 clade.

Because Bmp4 is expressed ventrally in the developing mouse lung, and

Bmp16 (not Bmp4) is expressed in the developing gas bladder of zebrafish (Feiner et al., 2009), we hypothesized that Bmp16 would be expressed dorsally in the developing gas bladder during bowfin development. Our IF assays first detect Bmp16 expression in the bowfin foregut and gas bladder bud right after initial gas bladder budding (stage

26). During this developmental stage (stage 26), Bmp16 is expressed dorsally in the anterior gas bladder, but absent posteriorly (Figure 1.8a, b). Bmp16 expression appears to be strongest during gas bladder outgrowth (stages 27 and 28) as shown by very bright fluorescent staining in the anterior gas bladder and pneumatic duct. Bmp16 is also expressed in the ventral folds of the foregut at this stage of development

(Figure 1.8a). However, in the posterior regions of the gas bladder, where lateral growth of the gas bladder occurs, Bmp16 expression is absent (Figure 1.8b).

Concurrent with Bmp16 expression, during gas bladder outgrowth, strong Smad phosphorylation, which demonstrates Bmp activity, is detected in the dorsal mesenchyme surrounding the gas bladder (Figure 1.8c, d). However, unlike Bmp16, which is not expressed posteriorly in the gas bladder, strong Smad phosphorylation is still detected in the dorsal mesenchyme surrounding the posterior region of the gas bladder (Figure 1.8d). Although, we cannot rule out the possibility of additional active

Bmp’s (Bmp4 or Bmp2) in the gut or gas bladder, the expression of Bmp16 in the endoderm and Smad phosphorylation in the surrounding mesenchyme suggests that

Bmp16-signaling plays a role in regulating gasbladder development in bowfin.

Overall, expression of Bmp16 during bowfin gasbladder development suggests that

Bmp16 was recruited to regulate development during the lung-to-gasbladder

evolutionary transition.

Figure 1.8: Bmp16 expression and Smad phosphorylation in bowfin gas bladder. For each column, top panels show gene expression in bright red overlaid with DAPI in blue. Middle panels show gene expression alone in bright red. The bottom panels depict graphically the gene expression patterns shown as photomicrographs above for Bmp16 (dark green) and pSmad (orange). All panels are transverse sections at stage 27 during outgrowth. The anterior- posterior location of the transverse sections is indicated on the larval bowfin diagrams shown above the panels. a) Bmp16 expression in the anterior region of bowfin gas bladder. b) Smad phosphorylation is detected in the anterior region of the gas bladder. c) Bmp16 expression in the posterior region of the gas bladder in bowfin. d) Smad phosphorylation is detected in the posterior region of the gas bladder. Bmp16 is expressed strongly in the anterior gas bladder (GB); however, more posteriorly, where the gas bladder expands laterally, Bmp16 expression is absent. Bmp16 activity, as indicated by Smad phosphorylation, is strong in the dorsal mesenchyme surrounding the gas bladder, both anteriorly and posteriorly. Abbreviations are: n, notochord; pd, pneumatic duct.

3.3 Summary of the changes in gene expression during gas bladder evolution

Expression patterns during lung budding and gas bladder budding for the four genes of interest are mapped on a phylogeny in Figure 1.9 to show the evolutionary changes in dorsoventral expression of these genes in four critical taxa representing major groups of bony vertebrates. Details of the expression patterns of each gene in all taxa are summarized in Table 1.1. Mouse (representing tetrapods) and bichirs develop ventral lungs, and in both taxa, Sox2 is expressed dorsally in the foregut, opposite the lung budding site. In contrast, in both bowfin and zebrafish, which develop dorsal gas bladders, Sox2 expression is expanded to the entire foregut, dorsally and ventrally. As expected, Nkx2.1 is expressed in the ventral lungs in both mouse and bichir.

Surprisingly, in both bowfin and bichir, both early-diverging ray-finned fishes, Nkx2.1 shows strong expression in the ventral wall of the foregut despite that bowfin have a gas bladder and bichir have lungs. In zebrafish, which are nested within the teleost clade, Nkx2.1b expression is expanded beyond the ventral wall of the associated foregut dorsally into the gas bladder bud. The spatial expression patterns of Nkx2.1 and Sox2 during gas bladder development do not support the hypothesis that these genes contribute to regulating the direction of gas bladder budding.

We found that Bmp4 is highly expressed ventrally in the lungs during lung outgrowth in bichir as well as in mouse. Our Bmp expression data for bowfin are consistent with findings in zebrafish (Feiner et al., 2009). In both bowfin and zebrafish, Bmp4 is absent, but its paralogue, Bmp16 was found to be highly expressed dorsally in the gas bladder during outgrowth. This inverted expression pattern of paralogous Bmp genes suggests that Bmp expression plays a significant role in the

morphological inversion of budding direction. As yet, Bmp16 has not been observed during lung development due to the loss of Bmp16 from mammal genomes. Because the bichir genome has not been sequenced, the presence or absence of the Bmp16 gene is unknown.

Figure 1.9: Gene expression changes during the lung-to-gas bladder transition in ray- finned fishes. Using a pruned bony-vertebrate phylogeny as an evolutionary framework, expression patterns of Sox2 (red), Nkx2.1 (purple), Bmp4 (light green), and Bmp16 (dark green) are shown for transverse sections of the foregut and either lungs or gas bladder of each taxon. Expression of Nkx2.1 and Sox2 are conserved for mouse and bichir. Bmp4 is expressed in the lung endoderm during outgrowth of both lungs and gas bladder, but Bmp4 is restricted to the distal tips of lung branch in mouse and not bichir. Expression of Sox2 is expanded in taxa with gas bladders (bowfin, zebrafish) relative to taxa with lungs (mouse, bichir). Expression of Nkx2.1 is expanded in taxa with gas bladders (bowfin, zebrafish) relative to taxa with lungs (mouse, bichir). Bmp16, rather than Bmp4 is expressed in taxa with gas bladders. Hypothesized expression patterns in the common ancestor of bony vertebrates, inferred from shared patterns between bichir and tetrapods, are indicated at the base of the phylogeny. The zebrafish gas bladder is connected to the foregut, but the pneumatic duct is not shown in the Bmp16 image because the section is posterior to the pneumatic duct. Abbreviations are: fg, foregut; GB, gas bladder; L, lungs

Table 1.1: Summary of expression patterns of four genes during early budding and organ outgrowth of lung or gas bladder development.

Early Development (budding)

Sox2 Nkx2.1 Bmp4 Bmp16 dorsal foregut ventral foregut ventral mesoderm absent from endoderm; absent endoderm; lung surrounding lung genome mouse from lung bud bud buds strong expression in

dorsal foregut ventral foregut unknown, genome endoderm; absent endo; lung bud; lung bud endoderm

bichir not sequenced from lung bud weaker in dorsal foregut endoderm

entire foregut ventral foregut endoderm; gas not expressed not expressed endoderm bowfin bladder bud

entire foregut entire foregut endoderm; gas endoderm; gas not expressed not expressed bladder bud bladder bud

zebrafish

Later Development (outgrowth)

Sox2 Nkx2.1 Bmp4 Bmp16

proximal stalks of distal tips of lung absent from lung epithelial cells lung branches branches genome

mouse

dorsal foregut strong in ventral lung endoderm; unknown, genome endoderm; absent foregut endoderm; activity in ventral

bichir not sequenced from lungs weaker in the lungs mesoderm (pSmad) entire foregut

ventral foregut anterior gas endoderm; endoderm; very bladder; activity in pneumatic duct; gas not expressed weakly in gas dorsal mesoderm bowfin bladder; posterior bladder (pSmad) gut entire foregut endoderm up to entire foregut and including endoderm; gas not expressed gas bladder pneumatic duct; in bladder bud zebrafish gas bladder but not posterior gut

4. Discussion

During the lung-to-gas bladder evolutionary transition along the ray-finned fish lineage, the developmental budding direction shifted from ventral to dorsal. We hypothesized that the underlying gene expression patterns regulating gas bladder development would also show a dorsoventral inversion compared to the patterns regulating lung development. Instead, we found a more complicated pattern of conserved gene expression in taxa with lungs, modified (but not inverted) expression of Nkx2.1 and Sox2 in taxa with gas bladders, and a newly discovered role in gas bladder development for Bmp16, a gene not present in tetrapods.

4.1 Conserved gene expression patterns between bichir and tetrapod lung development

Our result showing similar dorsoventral patterning of key regulatory genes,

Sox2, Nkx2.1, and Bmp4, in the foregut and lungs of both a fish (bichir) a mouse may seem surprising from a developmental perspective. However, this similarity is expected from an evolutionary perspective. The bichir has retained the primitive lung condition shared by the common ancestors of fishes and tetrapods (including mouse), and it is not surprising that the underlying regulatory networks would be conserved to some degree. Experimental evidence shows that dorsal Sox2 expression functions to restrict the budding location of lungs to the ventral wall of the foregut in mice

(Domyan et al., 2011); therefore, the strong expression of Sox2 in the dorsal foregut endoderm in bichir suggests Sox2 may also be involved in restricting the ventral location of lung budding in bichir. Having a dorsally restricted Sox2 expression pattern in the foregut during lung development is consistent across a wide-range of bony

vertebrates with lungs, including mouse, chicken, Xenopus, and bichirs (Ishii et al.,

1998; Que et al., 2007; Rankin et al., 2015). During mouse lung development, Nkx2.1 has a reciprocal expression pattern compared to Sox2 and is expressed in the ventral foregut endoderm and lung buds. Though Nkx2.1 is not completely restricted to the ventral foregut endoderm during bichir lung development as it is in mouse, our data show Nkx2.1 to be expressed in a gradient with strong expression ventrally and weaker expression dorsally across the foregut. Additionally, we find Nkx2.1 to be expressed during early lung development in bichir, right after initial budding (stage 34). Our results show earlier expression of Nkx2.1 than found by Tatsumi et al (2016), who observed Nkx2.1 to be weakly expressed throughout the bichir foregut and in the lungs no earlier than 12 dpf. This corresponds to lung outgrowth (stage 36) by our calibration to Budgett’s staging series (1902) based on the morphology of transverse sections. Conserved expression of Sox2 and Nkx2.1 across the dorsal-ventral axis of the foregut during early lung development in bichir and mouse suggest these genes may have regulated development of primitive lungs in the common ancestor of bony vertebrates.

During development of the respiratory system in mouse, BarX1 expression regulates the dorsoventral expression of Sox2 and Nkx2.1 across the foregut by repressing Wnt siganling. When BarX1 is knocked out in mouse, Nkx2.1 expression expands and Sox2 expression is reduced in the foregut, while morphologically, the foregut fails to separate into the esophagus and trachea but the lungs appear to bud normally (Woo et al., 2011). During normal bichir development, the foregut does not separate into an esophagus and trachea but remains a single tube. Given the role of

BarX1 in mouse lung development, it would be interesting to know what role it plays in gasbladder development. Perhaps, BarX1 is not expressed in the mesenchyme surrounding the dorsal foregut of bichir allowing Nkx2.1 expression to expand dorsally.

In bichir, Bmp4 shows the expected ventral expression pattern in the developing lung buds similar to the observed pattern in mice. Lung development in mice involves a complex interaction of genes both within and between the foregut endoderm and surrounding mesenchymal tissues (Ornitz and Yin, 2012; Hines and Sun,

2014), and it would be interesting to know to what extent these gene interactions are also conserved. During early lung development in mice, Bmp4 contributes to establishing the ventral budding site of the lung buds. Bmp4 is expressed in the ventral mesenchyme surrounding the future trachea and lung buds in mice and appears to regulate Nkx2.1 expression in the ventral foregut by signaling to the endoderm and repressing Sox2 expression (Figure 1.2; Li et al., 2008; Domyan et al., 2011; Herriges and Morrisey, 2014; Hines and Sun, 2014). During early lung development in bichir,

Bmp4 is expressed ventrally in the lung epithelium rather than in the surrounding mesenchyme as in mouse. Additionally, Smad phosphorylation is detected in the ventral mesenchyme surrounding the lung buds in bichir providing evidence that though Bmp4 is expressed in the lung epithelium, it may be signaling to the surrounding mesenchyme. Based on the expression patterns, the genes involved in bichir and tetrapod lung development may be conserved. However, Bmp-signaling appears to occur in opposite directions during bichir compared to mouse lung development suggesting that the interactions between Bmp4, Sox2, and Nkx2.1 may

differ.

4.2 Inversion of Sox2 and Nkx2.1 expression does not underlie the inversion of budding direction

We did not find an inverted pattern of Sox2 and Nkx2.1 expression in gas bladders relative to the dorsal Sox2 and ventral Nkx2.1 expression in lungs. Instead, the dorsal-ventral expression pattern of these two genes in the bowfin gas bladder was similar to the pattern in mouse but with Sox2 showing an expanded expression pattern in the foregut (Figure 1.4). In zebrafish, expression of both Sox2 and Nkx2.1b are expanded dorso-ventrally in the foregut during gas bladder development relative to lung development (Figure 1.5). Previously, using in situ hybridization, Cass et al.

(2013) found Nkx2.1b expression to be restricted dorsally to the gas bladder bud during zebrafish development. However, expression of Nkx2.1b was not detected until

4 dpf, two days after gas bladder budding at 2 dpf (Cass et al., 2013). Using

RNAscope in situ hybridization, we were able to detect expression of Nkx2.1b in the foregut and gas bladder bud of zebrafish as early as 2 dpf, the period of initial gas bladder budding. Thus, Nkx2.1 is expressed ventrally during the earliest stages of gas bladder development similar to both the spatial and temporal expression of Nkx2.1 during lung development.

In mice, Nkx2.1 plays a significant role in branching morphogenesis as evidenced by Nkx2.1-null mutant mice, which still develop lungs albeit with greatly reduced airway branching (Minoo et al., 1999; Que et al., 2007). This sac-like morphology of mutant lungs is reminiscent of a normal gas bladder (Cass et al., 2013).

The expression of Nkx2.1 in the ventral foregut during both early lung and early gas

bladder development suggests that ventral Nkx2.1 expression was also present in the foregut of the common ancestor of bony vertebrates, though it’s function in regulating the development of primitive lungs is unknown. Neither the lungs of bichir (a proxy for primitive bony vertebrate lungs) nor gas bladders of ray-finned fishes undergo branching, but perhaps the presence of Nkx2.1 expression allowed for co-option of

Nkx2.1 to regulate lung branching morphogenesis in early terrestrial vertebrates which relied on increasingly branching structures advantageous for aerial respiration.

4.3 Role of Bmp16 in gas bladder development

Bmp16, rather than its paralogue Bmp4, is expressed during gas bladder development in zebrafish (Feiner et al., 2009). Bmp16 is also present in the genomes of all other ray-finned fishes investigated thus far (including spotted gar, stickleback, tilapia, green spotted pufferfish, and Takifugu rubripes), but it has been lost from mammalian genomes (Feiner et al., 2009, 2019). In this study, we identified the

Bmp16 ortholog in the bowfin genome and found it to be expressed in the epithelium of the anterior gas bladder with strongest expression at outgrowth, stages 27 and 28.

Additionally, Smad phosphorylation detected in the dorsal mesenchyme surrounding the gas bladder bud suggests that Bmp16 ligands are active in the dorsal mesenchyme.

This dorsal pattern of Smad phosphorylation and Bmp16 activity is inverted compared to the ventral Bmp4 expression during lung development in mouse and bichir. When compared to the temporal pattern of Bmp4 expression during lung development

(Weaver et al., 2000; Domyan et al., 2011), the expression of Bmp16 during gas bladder development in bowfin is delayed. This delay in Bmp16 expression suggests that unlike Bmp4, Bmp16 is not acting upstream to regulate the dorsoventral patterns

of Nkx2.1 and Sox2 by repressing Sox2 expression dorsally during gas bladder development. Unlike the spatial pattern of Bmp4 during lung branching morphogenesis (Morrisey and Hogan, 2010), in the bowfin gas bladder, which grows distally and laterally into a “T” shape rather than branching, Bmp16 expression does not become restricted distally during gas bladder outgrowth. However, Bmp16 activity in the mesenchyme surrounding the gas bladder may contribute to the overall expansion of the organ. Protein structure is conserved between Bmp16, Bmp4, and

Bmp2 and all three significantly activate the bmp-signaling pathway (Marques et al.,

2016) suggesting Bmp16 may be regulating gas bladder outgrowth similarly to how

Bmp4 regulates lung outgrowth. However, without a bichir genome we do not know whether and where Bmp16 is expressed during bichir lung development. If Bmp16 is expressed in the dorsal foregut during lung development in bichir, then we may conclude that dorsal expression of Bmp16 arose either during the evolution of ray- finned fishes or was the ancestral bony vertebrate pattern, but it did not contribute to the evolution of a dorsal gas bladder. On the other hand, if Bmp16 is not expressed during bichir lung development or is expressed ventrally in the lungs, then Bmp16 expression may have arisen in concert with the evolution of the gas bladder, providing further evidence for its role in regulating gas bladder development. Given what we have found in this study, Bmp4 expression appears to have been lost during the evolution of the gas bladder from ancestral lungs, and Bmp16 expression appears to have been gained in gas bladder development at least for the Actinopteri. Whether

Bmp16 is involved in lung development of bichir, representing the sistergroup to

Actinopteri, remains an intriguing question.

4.4 Conclusion

Of the genes investigated, Nkx2.1 and Sox2 show conserved spatial and temporal expression patterns between taxa that develop lungs, including bichirs, the only ray-finned fish lineage that has lungs. Based on these shared gene expression patterns in the lungs of bichir and tetrapods, we can infer that Nkx2.1 was likely expressed in the ventral foregut and lung endoderm while Sox2 was likely expressed in the dorsal foregut endoderm during lung development in the common ancestor of bony vertebrates (Figure 1.9).

Interestingly, during the evolutionary transition from lungs to a gas bladder along the ray-finned fish lineage, Bmp4 expression appears to have been lost and

Bmp16 expression appears to have been gained in gas bladder development.

Alternatively, it is possible that Bmp16 was lost during the evolution of mammalian lungs. Further study of the patterns of Bmp16 expression in a variety of taxa could distinguish between these possibilities. The dorsal expression of Bmp16 during gas bladder development, inverted compared to Bmp4 expression during lung development, suggests that Bmp16 may have been involved in the shift from ventral to dorsal budding. While the reproductive biology of bowfin makes functional experiments extremely difficult, we hypothesize that CRISPR-Cas9 knockout experiments in zebrafish will reveal important roles of Bmp16 in gas bladder development.

Author Contributions

E. C. F. and A. R. M. designed the study and wrote the paper with input from co- authors. E. C. F. collected bowfin eggs and sampled developmental series of all fish

species used in this study. E. C. F and C. B. performed immunohistochemistry, and E.

C. F. carried out the RNAscope in situ hybridization assays. B. D. S. and E. C. F. designed the Bmp and pSmad expression assays and optimized RNAscope methods. N.

A. K. provided advice on methods development and use of lab space and equipment.

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CHAPTER 2

Dorsoventral inversion of the air-filled organ (lungs, gas bladder) in vertebrates:

RNA-sequencing of laser capture microdissected embryonic tissue2

2 Funk, E. C., Lencer, E. S., McCune, A. R. (2020) Dorsoventral inversion of the air-filled organ (lungs, gas bladder) in vertebrates: RNA-sequencing of laser capture microdissected embryonic tissue. Journal of Experimental Zoology Part B. DOI:10.1002/jez.b.22998

Abstract

How modification of gene expression generates novel traits is key to understanding the evolutionary process. We investigated the genetic basis for the origin of the piscine gas bladder from lungs of ancestral bony vertebrates. Distinguishing these homologous organs is the direction of budding from the foregut during development; lungs bud ventrally and the gas bladder buds dorsally.

We investigated whether this morphological inversion is associated with molecular inversion of conserved genes regulating lung and gasbladder development.

Using laser-capture microdissection and RNA-seq, we assayed transcript abundance and compared expression patterns between dorsal and ventral foregut tissues during gasbladder development. Our focal taxon, bowfin (Amia calva), representing the sistergroup to teleosts, is an early diverging ray-finned fish with a gas bladder. We discovered a number of genes with unknown function during lung development that are differentially expressed during gasbladder development and annotated to functions relevant for organ budding. We also identified several known lung-regulatory genes exhibiting inverted dorsoventral expression during gasbladder relative to lung development. Specifically, Tbx5 is strongly expressed in the dorsal mesoderm surrounding the gas bladder during bowfin development, and several interacting genes are co-expressed dorsally with Tbx5. In contrast, in mouse and bichir (Polypterus senegalus), the only ray-finned fish that have lungs, Tbx5 is expressed in the ventral lung mesoderm during development. Our data demonstrating dorsoventral inversion of conserved genes suggests that these genes may have contributed to the evolutionary transition between ventral lungs and a dorsal gas bladder in ray-finned fishes.

INTRODUCTION

Phenotypic evolution depends on the generation of variation through the modification of development. How that variation is generated is fundamental to understanding the evolutionary process. Increasing evidence points towards the existence of conserved genes across distantly related taxa regulating major aspects of the body plan (Averof and Patel, 1997; Carroll et al., 2013). Modification to the temporal and spatial expression patterns of those conserved genes during development produces novel traits and body plans (Carroll et al., 2013). For example, recent molecular studies have supported the controversial 19th-century hypothesis that the dorsoventral body axis in chordates is inverted relative to that in arthropods (Geoffroy

St-Hilaire, E., 1822; Arendt and Nubler-Jung, 1994; Holley, et al., 1995; Ferguson,

1996; De Robertis and Sasai, 2000). The expression patterns and function of orthologous genes important for dorsoventral axis development in both arthropods and chordates parallel the morphological inversion of the body axis.

Here we investigate another example of dorsal-ventral patterning, leading either to the development of ventral lungs in tetrapods or a dorsal gas bladder in ray- finned fishes. These two air-filled organs are homologous (Darwin, 1859; Romer and

Parsons, 1970; Cass et al., 2013; Longo et al., 2013) with the common ancestor of tetrapods and ray-finned fishes characterized by ventral paired lungs (Figure 2.1B).

The gas bladder is an important innovation originating in the Actinopteri, a clade of more than 30,000 species of ray-finned fishes, including all teleosts, sturgeon, paddlefish, gar, and bowfin (Nelson et al., 2016; Betancur-R et al., 2017). The gas

bladder lies dorsal to the gut and ventral to the spine (Figure 2.1A) and in most fishes, functions to control buoyancy, but in some groups, it serves as a respiratory organ or aids in hearing and sound production (Alexander, 1966; Helfman et al., 2009). The evolution of the gas bladder from lungs involved a shift in the direction of budding from ventral (lungs) to dorsal (gas bladder). Thus, we investigate whether this inverted morphology is associated with the molecular inversion of conserved gene regulatory networks specific to both lungs and gas bladders.

Reflecting their homology, lungs and gas bladders are similar in many ways.

Morphologically, both develop from the anterior foregut endoderm, and both are supplied by the pulmonary artery. At a molecular level, both lungs and gas bladders express a suite of orthologous genes known to have lung-specific interactions, and both produce pulmonary surfactant proteins (Goodrich, 1958; Daniels et al., 2004; Cass et al., 2013; Longo et al., 2013). Despite these commonalities, several differences exist between the lungs and the gas bladder. The lungs are usually paired and branching

(Kardong, 2015), while the gas bladder is usually single and non-branching (Helfman et al., 2009). However, exceptions include snakes and caecilians, which can have a single lung (Wallach, 1998), and toadfishes, which can have bilaterally paired gas bladders (Rice and Bass, 2009). The defining difference between the lungs and the gas bladder is the direction of budding from the anterior foregut during development; lungs bud ventrally and gas bladders bud dorsally (Figure 2.1B; Wilder, 1877;

Graham, 1997; Cass et al., 2013). Thus, similar to arthropod versus chordate body plans, the evolution of the gas bladder from lungs represents a dorsoventral inversion.

Figure 2.1: A) Lateral view of a cutaway of a schematic teleost fish showing the location of the gas bladder (GB), which is located dorsal to the foregut (fg) and ventral to the spinal column (sc). The gas bladder is an outgrowth of the foregut, and in many species, it remains connected to the gut via the pneumatic duct (pd). B) Highly-pruned bony vertebrate phylogeny showing the relationship of bichir, bowfin, and teleosts (phylogeny after Betancur et al., 2017), the clade containing the majority of extant ray-finned fishes (at least 30,000 extant species; Nelson et al., 2016). Lobed-finned fishes, represented here by tetrapods and lungfishes, develop ventral lungs (magenta lineages). The dorsal gas bladder arose after the divergence of bichirs (green lineages), which have lungs. The gas bladder is characteristic of the Actinopteri, including bowfin and teleosts. That bichirs, sister to Actinopteri, have lungs as do tetrapods and lobed-finned fishes, indicating that lungs are the ancestral state for bony vertebrates. Transverse and lateral sectional drawings of either lungs or gas bladders (modified from Romer 1970) are shown above each taxon. Note that members of the Actionpteri have dorsal gasbladders (gb) and this clade is nested within the bony vertebrates, the common ancestor of which had ventral lungs (L). The teleost whole-genome duplication (WGD) is also shown on the phylogeny. C) Diagram of a transverse section of a bowfin embryo showing the tissue regions of the foregut dissected using laser capture microscopy (LCM). Dorsal mesoderm, dM, pictured in light green; dorsal endoderm, dE, in dark green; ventral mesoderm, vM, in pink; and ventral endoderm, vE, in purple. Tissue was sampled between the dotted lines, which were intentionally placed well within the actual boundaries of dorsal and ventral regions to avoid sample contamination from adjacent tissue. Three individuals and 4 tissues were sampled at three developmental stages.

Development of the tetrapod lungs, based on mouse and chick, is well characterized with the earliest stages beginning as a ventral evagination from the foregut endoderm. This ventral budding is known to depend on the expression of a gene regulatory network (GRN) that includes Nkx2.1, Sox2, and Bmp4. We previously investigated the expression of these candidate genes during gasbladder development in bowfin and zebrafish (Funk et al., in review), hypothesizing that the expression of

Nkx2.1 and Sox2 would show an inverted expression pattern relative to the pattern during tetrapod lung development. We found that dorsal and ventral spatial expression of these two genes was similar during lung and gasbladder development. In both tetrapod lungs and bowfin gas bladder, Nkx2.1 is expressed ventrally. In tetrapod lungs, Sox2 is expressed dorsally, while in bowfin gas bladder, Sox2 expression is expanded to the entire foregut. However, we made the unexpected discovery that paralogous genes Bmp16 and Bmp4 show an inverted expression pattern during later gas bladder development in bowfin compared to lung development in mouse, respectively (Funk et al., in review), suggesting that Bmp-signaling played a role in the lung-to-gas bladder evolutionary transition. Known core genes patterning the dorsal- ventral axis during lung budding appear not to exhibit an inverted expression pattern associated with the evolution of a dorsally budding gas bladder. To discover additional genes that may be driving the evolutionary transition from ventrally budding lungs to a dorsally budding gas bladder in ray-finned fishes, we took a genome-wide approach.

We sequenced the transcriptome at three developmental stages to screen for the suite of genes differentially expressed between dorsal and ventral foregut tissue during gasbladder development. We found many genes with unknown function in lung

development that show differential expression dorsoventrally during gas bladder development. A number of these novel genes are annotated to functions (GO terms) relevant to the process of organ budding, such as extracellular matrix, cell polarity, membrane trafficking, growth factors and patterning, and angiogenesis. Additionally, we identified several lung-development genes that are also differentially expression during gas bladder development. In particular, we found the expression pattern of

Tbx5 during gas bladder development is inverted dorsoventrally compared to the that during lung development.

MATERIALS & METHODS

Taxon selection

To identify genes potentially involved in the ventral-to-dorsal shift in budding location underlying the lung-to-gas bladder transition in ray-finned fishes, we chose bowfin as our focal species. Bowfin are an early-diverging ray-finned fish within the sister group to teleosts, the clade containing the majority of extant fishes (over 30,000 species; Figure 2.1B). Bowfin develop a gas bladder that exhibits ancestral characteristics, including maintaining a connection to the foregut, the pneumatic duct, and being highly vascularized for respiration (Liem, 1989; Graham, 1997).

Conveniently, bowfin diverged prior to the whole genome duplication that occurred at the base of the teleost clade (Figure 2.1B; Hoegg et al., 2004). After identifying specific genes-of-interest from the tissue expression profiles in bowfin, we investigated those expression patterns in the bichir, Polypterus senegalus. Bichirs are the sister group to all other ray-finned fishes, and they are the only living ray-finned fishes which develop ventral lungs, making them the most pertinent species to study

lung development in comparison to gas bladder development in bowfin.

Tissue sampling

Recently fertilized bowfin eggs were collected from nests in Oneida Lake NY during May of 2018 ( for details, see Funk et al., in review). Eggs were reared at 12.8 degrees Celsius in water collected from Oneida Lake in the animal care facility at

Cornell University. Bowfin embryos were sampled just before gas bladder budding, at budding, and during outgrowth at stages 24, 25, and 27 respectively (Ballard, 1986).

Sampled embryos were euthanized with a lethal dose of MS-222, rinsed in deionized water, and equilibrated in TissueTek optimal cutting temperature (O.C.T.) embedding medium for 2 minutes (IACUC Protocol 2006-0013). Following equilibration, the embryos were transferred to individual cryomolds with fresh O.C.T. and flash frozen in a 2-Methylbutane bath with liquid nitrogen. Additionally, a number of samples were fixed in 4% paraformaldehyde overnight and flash frozen in 2:1 O.C.T:30%- sucrose embedding medium for RNAscope in situ hybridization assays. All embedded samples were stored at -80oC until further processing.

Bichir eggs were obtained from a private aquarist. Eggs were shipped overnight in water treated with methylene blue to prevent fungal growth. We reared the embryos in freshwater at 24 degrees Celsius. Bichir embryos were sampled between 2 and 10 days post hatching to capture a developmental series spanning lung budding ( IACUC Protocol 2006-0013; Budgett, 1902). Sampled embryos were euthanized with a lethal dose of MS-222, rinsed in PBS, and fixed overnight in 4% paraformaldehyde (PFA). After fixing, the samples were flash frozen in 2:1

O.C.T:30%-sucrose embedding medium. Embedded samples were stored at -80oC

until further processing.

Laser capture microdissection and RNA-seq

Using a cryostat (Leica CM 1950), embedded bowfin embryos were sectioned transversely at 15 µm from the anterior to posterior ends of the gas bladder bud and mounted on polyethylene naphthalate (PEN) membrane slides. For stage 24 embryos, we sectioned the entire foregut along the anterior-posterior axis. For stage 25, we sectioned the entire gas bladder bud and corresponding foregut tissue along the anterior-posterior axis. For stage 27, we ended sectioning of the gas bladder and foregut just anterior to the stomach to avoid contamination. From each transverse section, the dorsal and ventral regions of the foregut endoderm and mesoderm were excised using the Zeiss Palm Microbeam LCM System at the Cornell University

Biotechnology Resource Center. When dissecting the dorsal and ventral regions of the foregut, we sampled well within the actual dorsal-ventral boundary to avoid contamination from adjacent tissue regions (Figure 2.1C). LCM was completed in 2 hours or less for each slide to minimize RNA degradation in the samples. Immediately following LCM, we added buffer RLT with β-Mercaptoethanol (Qiagen RNeasy

Micro) to the tube containing dissected tissue to stabilize the RNA and homogenized the sample by vortexing for 2 minutes and then storing at -20oC. We sampled 3 developmental stages x 4 tissue regions x 3 replicates for 36 samples total.

RNA extraction was performed using the Qiagen RNeasy Micro Kit following the manufacturer’s instructions. RNA amplification and cDNA reverse transcription was completed using the NuGEN Ovation RNA-seq System V2. We used NEBNext

Ultra II DNA Library Prep Kit to prepare cDNA libraries for sequencing. All libraries

were quality checked on a Tape Station by the Cornell BRC Genomics Facility and showed a single peak of between 170 and 240 base pairs. The individually barcoded libraries (36 total) were pooled and sequenced in one lane of the Illumina NextSeq500

(single end, 150 bp) by the Cornell BRC Genomics Facility.

Bioinformatics

Trimmomatic (version 0.38; Bolger et al., 2014) was used to remove adaptor sequences and low quality regions from the reads. Reads were aligned to the bowfin genome (Braasch pers. comm.) using STAR aligner (version 2.6; Dobin et al., 2013), and read counts per gene were quantified using STAR quant mode. We generated

PCA plots in R (version 3.6.0) with the gene expression profiles of all tissue regions for each stage separately. For principle component axes 1-3 at each stage, we ranked the loadings from positive to negative. Using the loadings as pre-ranked lists, we performed Gene Set Enrichment Analysis (GSEA, version 4.0.3) as implemented in the Broad Institute Java script software (Mootha et al., 2003; Subramanian et al.,

2005). We tested for enrichment in the GSEA Hallmarks v7.0 gene sets and performed

1000 permutations to establish significance.

To determine differential expression between dorsal and ventral tissue regions, we analyzed each developmental stage separately (12 samples per stage) by building generalized linear models (GLM) in edgeR (Robinson et al., 2010) carried out in R

(version 3.6.0). We filtered low expressed genes and included only those genes expressed at minimum of 1 counts per million (cpm) in at least half of the samples or genes expressed at 0.5 cpm in at least three quarters of the samples. We manually curated the functional categories related to tissue budding for our differentially

expressed genes based on biological process GO (gene ontology) terms and gene descriptions from UniProt (Anon, 2008).

Tbx5 RNAscope

Fixed and embedded bowfin and bichir embryos were cryosectioned transversely at 15 µm and mounted onto Superfrost Plus slides (Fisher Scientific). We performed RNAscope Multiplex Fluorescent Assays to validate Tbx5 expression patterns during gas bladder development in bowfin and to determine the expression patterns these two genes during lung development in bichir. Species-specific RNA probes were designed for both species and genes by Advanced Cell Diagnostics. We used the RNAscope Multiplex Fluorescent v2 Kit (Advanced Cell Diagnostics,

Newark, CA) and Perkin Elmer Cy3 fluorophore following the manufacturer’s protocol. To visualize the fluorescently labeled expression patterns, we used a Zeiss

Observer.Z1/ApoTome.2 inverted microscope with AxiocamHRc camera.

Results

Functional categories of genes expressed during bowfin gasbladder development

At each stage of development, gene expression patterns distinctly separated foregut-tissue samples by tissue type, endoderm or mesoderm, along the first two principle component (PC) axes (Figure 2.2). At pre-budding (stage 24), gene expression patterns show no separation between dorsal and ventral regions of the foregut and gas bladder (Figure 2.2A). Some separation between dorsal and ventral regions of the mesoderm is evident during budding (stage 25) along PC 3 (Figure

2.2B) and during outgrowth along PC 1 (Figure 2.2C). Dorsal versus ventral identity does not explain much of the variation in gene expression patterns between

foregut/gas bladder tissue regions implying few genes are differentially expressed dorsoventrally. This is consistent with our results from the differential expression analysis between dorsal and ventral tissue regions (see below).

Figure 2.2: Principle Component Analysis (PCA) plots showing the main axes of variation

in gene expression patterns among foregut/gas bladder tissue samples at each stage.

Figure 2.2: Principle Component Analysis (PCA) plots showing the main axes of variation in gene expression patterns among foregut/gas bladder tissue samples at each stage. Top row of plots depicts PC1 and PC2, and bottom row depicts PC2 and PC3. Principal components reflect the expression level of all genes in the sampled tissue regions. Endoderm samples are colored in orange, and mesoderm samples are colored in blue. Dorsal samples are labeled D, and ventral samples are labeled V. A) At pre-budding (stage 24), gene expression patterns separate samples by tissue type, endoderm and mesoderm, along PC2. B) During budding (stage 25), gene expression levels also separate by tissue type along PC2. Along PC3, gene expression in the mesoderm shows some separation between dorsal and ventral tissue regions. C) During outgrowth (stage 27), gene expression patterns distinguish endoderm and mesoderm samples along PC1. Gene expression patterns in mesoderm samples show separation along PC1 as well.

To identify conserved gene functions enriched along the PC axes, we ranked the genes contributing to each PC based on their loadings (Table 2.1; Table A2.1). The

PC axes that discriminate endoderm and mesoderm samples at each stage show similar functional enrichment. At pre-budding and outgrowth stages, genes with positive loadings along these PC axes showed highest enrichment, based on the normalized enrichment score (NES), in epithelial to mesenchymal transition (Table 2.1). Epithelial to mesenchymal transition was also one of the top enriched functions for genes with negative loadings along PC 2 at budding stage. Additionally, several processes related to cell cycle are enriched along the PC axes at each stage including G2M checkpoint,

E2F targets, mitotic spindle, and DNA repair (Table 2.1, Table A2.1). Several signaling pathways, including Kras signaling, Notch signaling, Wnt/Beta-catenin signaling, Hedgehog signaling, and Il6/Jak/Stat3 signaling, are enriched as well.

(Table 2.1, Table A2.1)

As development progresses and the gas bladder becomes more differentiated, more genes become differentially expressed between dorsal and ventral foregut tissue.

Twelve, 45, and 160 genes are differentially expressed during pre-budding (stage 24), budding (stage 25), and outgrowth (stage 27) stages, respectively, at a false discovery rate (FDR) of 0.1 (Figure 2.3). By manual curation, we found many of the genes differentially expressed between dorsal and ventral tissues are annotated to functions relevant to the process of gas bladder budding, including extracellular matrix and cell adhesion, cell polarity and actin cytoskeleton, cell migration, membrane trafficking, growth factors and patterning, and angiogenesis (Table 2.2).

Table 2.1: Top 10 Hallmark v7.0 gene sets enriched along the principle component axis that distinguishes endoderm (endo) and mesoderm (meso) at each stage of gas bladder

development.

1.95 1.78 1.51 1.19 1.18 1.16 1.15 1.13 1.11 1.06

1.37 NES 2.05 1.74 1.61 1.56 1.49 1.49 1.42 1.40 1.30 NES ------

catenin signaling catenin

Outgrowth (stage 27) (stage Outgrowth

junction

Hedgehog signaling Hedgehog PC1: Separates endo and meso and endo PC1:Separates loading Positive NAME transition mesenchymal Epithelial Dn response UV Myogenesis Apical Coagulation Angiogenesis Hypoxia Complement homeostasis Cholesterol loading Negative NAME phosphorylation Oxidative V2 targets Myc V1 targets Myc response alpha Interferon Up Uvresponse metabolism Fattyacid checkpoint G2M Dn signaling Kras response Androgen Beta Wnt

1.35 1.29 1.27 1.23 1.10 0.97 0.83 0.81 0.72 0.65

1.24 NES 1.50 1.38 1.32 1.30 1.29 1.26 1.26 1.24 1.23 NES ------

catenin signaling catenin

Budding (stage 25) (stage Budding

Jak Stat3 signaling Stat3 Jak

DNA repair DNA PC2: Separates endo and meso and endo PC2:Separates loading Positive NAME E2Ftargets checkpoint G2M response Androgen metabolism Xenobiotic Complement late response Estrogen response protein Unfolding pathway P53 spindle Mitotic loading Negative NAME Il6 Beta Wnt junction Apical Hypoxia rejection Allograft transition mesenchymal Epithelial signaling Notch via Nfkb signaling Tnfa metabolism acid Bile secretion Protein

1.48 1.42 1.34 1.33 1.30 1.27 1.17 1.16 1.11 1.06

1.10 NES 1.81 1.35 1.34 1.25 1.24 1.19 1.19 1.13 1.09 NES ------

transition

budding (stage 24) (stage budding

Pre

Glycolosis PC2: Separates endo and meso and endo PC2:Separates loading Positive NAME mesenchymal Epithelial checkpoint G2M Spermatogenesis junction Apical E2Ftargets rejection Allograft Peroxisome via Nfkb signaling Tnfa V1 targets Myc loading Negative NAME response alpha Interferon response gamma Interferon pathway P53 late response Estrogen Complement surface Apical homeostasis Cholesterol Dn signaling Kras early response Estrogen metabolism Xenobiotic

Figure 2.3: Differentially expressed genes between dorsal and ventral tissues of the foregut during gas bladder development. Heat maps showing differentially expressed genes between dorsal and ventral foregut tissue at 3 stages of gasbladder development in bowfin. Each column is a sample and each row is a gene; gene names are listed along the right hand side. Below each heatmap, samples (columns) are labelled dorsal (D) or ventral (V) and endoderm or mesoderm. Warmer colors indicate higher relative gene expression, and cooler colors indicate lower relative expression. A) Twelve genes are differentially expressed at pre- budding (stage 24). B) At budding (stage 25), 45 genes are differentially expressed bewteen dorsal and ventral tissues. C) During outgrowth, 160 genes show differential expression dorsoventrally. Note that as gas bladder development proceeds, more genes are differentially expressed between dorsal and ventral foregut tissues.

Table 2.2: Dorsoventral differentially expressed (DE) genes manually curated by function based on GO terms and gene descriptions from UniProt (Anon, 2008). FUNCTION Stage 24 DE Genes Stage 25 DE Genes Stage 27 DE Genes postn, itgb5, tnc, mmp11, matn4, itga3, ddr2, col3a1, Extracellular Matrix col6a3, dcn, adam19, rgcc, st3gal5 Cell Adhesion pcdhgc5 pkp3, loxla, cdh31, epcam, mcam, pkp1, megf10, nectin1, sorbs3 thbs2a, loxla, gata6, mcam, ptgis, Angiogenesis N/A dcn, map3k3 plcg1, prox1, rgcc, notch3 limch1, prex1, Cell polarity actr2, was, fnbp1l, rab11fip2, actc1a, N/A Actin fryl, arhgef25 cotl1, ttn, acta2, tpm3, ripor1 hgfb, limch1, itgb5, loxla, spata13, epcam, igfbp5, Cell migration itga3, plcg1, ddr2, insr, brd9 dcn, map3k3 Cancer megf10, ripor1, prox1, rgcc, arid2, tbx5, tead3, kiaa1549, vwce hgfb, igfbp5, gata6, Growth factors tead3, esrp2, foxa2, mid1, fzd7a, plcg1, arl6 Patterning tbx5, rab7 fibl3, c3, igf2, pitx3, rgcc, olfml, irx2 Membrane Trafficking fnbp1l, tmem111, pkp3, krt18, itga3, Membrane arl6 actr2, stx16, rab7, tlcd1, gpd1l, mrap, Organization aftph sfxn2, abcc8

Expression of lung development genes during bowfin gasbladder development

We manually curated the differentially expressed genes to identify those known to be involved in lung development in mice. During gasbladder budding (stage

25), these include Tbx5 and FoxA2 (Figure 2.4; Weidenfeld et al., 2002; Arora et al.,

2012), and during outgrowth (stage 27), these include Fzd7a, Gata4, Gata5, Gata6,

Irx2, and Notch3 (Figure 2.5; Becker et al., 2001; Weidenfeld et al., 2002; Wang et al.,

2005; Ackerman et al., 2007; Hussain et al., 2017), all of which are significantly expressed dorsoventrally in our datasets. Of these 8 genes, Tbx5 is particularly interesting because its early expression in the gas bladder is inverted relative to its spatial expression during mouse lung development. In the gas bladder during pre- budding, Tbx5 is expressed at similar levels in both dorsal and ventral mesoderm.

However, at budding and outgrowth, Tbx5 expression increases in the dorsal mesoderm while decreasing in the ventral mesoderm (Figure 2.6A). During gasbladder development, Tbx5 is expressed in a gradient across the dorsoventral axis of the foregut with strong expression dorsally and weak expression ventrally. This is in contrast to mouse lung development, during which Tbx5 is expressed strongly in the ventral mesoderm.

We used RNAscope in situ hybridization to confirm the dorsoventral expression patterns of Tbx5 during bowfin gas bladder development. In addition, we also investigated the expression patterns of Tbx5 during bichir lung development to better rule out the possibility that the change in expression pattern is associated with the evolution of ray-finned fishes (Figure 2.1B), and determine whether changes to

Tbx5 expression are associated with the clade within ray-finned fishes that has a

dorsally budding gas bladder. Bichir are ray-finned fish more closely related to bowfin than to tetrapods; however, importantly, bichir retain ventrally budding lungs (Figure

2.1B). During the outgrowth stage of gasbladder development, Tbx5 shows a clear dorsoventral expression gradient across the foregut, in which Tbx5 is expressed strongly in the dorsal mesoderm surrounding the gasbladder bud (Figure 2.6B). In contrast, during lung outgrowth in mouse and bichir (Figure 2.6B), Tbx5 is expressed strongly in the ventral mesoderm surrounding the lung buds and is absent from the dorsal mesoderm.

In mouse, Tbx5 is known to interact with a number of genes during lung development including Tbx4, Fgf10, and Wnt2/Wnt2b (Morrisey and Hogan, 2010;

Arora et al., 2012; Hines and Sun, 2014). None of these genes were found to be significantly differentially expressed across the dorsoventral axis during gasbladder development in bowfin. However, all were expressed in foregut and gasbladder tissue and showed supporting trends in expression pattern differences dorsoventrally. Tbx4 is strongly expressed in the dorsal and ventral mesoderm during gasbladder budding

(stage 25). During gasbladder outgrowth (stage 27), Tbx4 expression increases in the dorsal mesoderm while it remains the same in the ventral mesoderm creating a dorsoventral gradient similar to Tbx5 (Figure 2.7A). Wnt2ba, a paralogue of Wnt2b, shows increasing expression in the dorsal mesoderm between pre-budding (stage 24) and outgrowth (stage 27) during gasbladder development. Expression of Wnt2ba also increases in the ventral mesoderm from budding (stage 25) to outgrowth (stage 27); however, expression is weaker than that observed in the dorsal mesoderm (Figure

2.7B). Fgf10 expression is weak or absent during gasbladder pre-budding (stage 24)

and budding (stage 25). But, during gasbladder outgrowth (stage 27), Fgf10 is strongly expressed in the ventral and dorsal mesoderm (Figure 2.7C).

Figure 2.4: Tissue-specific expression of known lung-development genes, differentially expressed at budding, during gas bladder development in bowfin. Average level of expression of lung-development genes (FoxA2 and Tbx5) differentially expressed during gasbladder budding (stage 25) in the A) dorsal mesoderm, B) dorsal endoderm, C) ventral mesoderm, and D) ventral endoderm, at all stages of gasbladder development in bowfin. From these line plots, the pattern of Tbx5 expression is particularly interesting because expression is stronger in the dorsal mesoderm than the ventral, which is opposite of the expression pattern during lung development. Sample size of 3 for each stage and tissue region. Error bars show the standard error of the mean.

Figure 2.5: Tissue-specific expression of known lung-development genes, differentially expressed at outgrowth, during gas bladder development in bowfin. Average level of expression of lung-development genes (Fzd7a, Gata4, Gata5, Gata6, Irx2, and notch3) differentially expressed during outgrowth (stage 27) in the A) dorsal mesoderm, B) dorsal endoderm, C) ventral mesoderm, and D) ventral endoderm at all stages of gasbladder development in bowfin. These genes, all with known roles in lung development, are differentially expressed across the dorsoventral axis during gasbladder development in bowfin. Sample size of 3 for each stage and tissue region. Error bars show the standard error of the mean.

Figure 2.6: Dorsoventral expression of Tbx5 during gas bladder development in bowfin. A) Boxplot of Tbx5 expression at pre-budding, budding, and outgrowth stages of gasbladder development. Tbx5 expression in the dorsal and ventral mesoderm is equally high during pre- budding (pink and light green), but as development proceeds through budding and outgrowth, dorsal expression increases while ventral expression decreases. B) RNAscope in situ hybridization of Tbx5 during gas bladder development in bowfin compared with lung development in bichir. During gas bladder outgrowth (stage 27) in bowfin, Tbx5 is expressed most strongly in the dorsal mesoderm surrounding the gas bladder (top panels). During lung outgrowth (stage 35) in bichir, Tbx5 is expressed in the ventral mesoderm surrounding the developing lungs (bottom panels). The left side of panels show Tbx5 expression in bright red overlaid with DAPI (bright blue), which stains nuclei, and the right panels show Tbx5 expression alone in bright red. Abbreviations: fg, foregut; gb, gas bladder; lL, left lung; rL, right lung.

Figure 2.7: Expression of Tbx4, Wnt2ba, and Fgf10 during gas bladder development in bowfin. Tbx4, Wnt2ab, and Fgf10 are all expressed during gas bladder development; however, none were found to be significantly differentially expressed between dorsal and ventral tissue regions. A) Tbx4 is expressed in the dorsal mesoderm (dM) during pre-budding and budding stages. During outgrowth, expression of Tbx4 increases in the dorsal mesoderm. Though Tbx4 is also expressed in the ventral mesoderm (vM) at budding and outgrowth stages, expression is weaker than in the dorsal mesoderm. B) Wnt2ba is expressed in the dorsal mesoderm during gas bladder budding and shows an increase in expression in the dorsal mesoderm as development proceeds to the outgrowth stage. C) Fgf10 shows weak or no expression in any foregut tissue regions during pre-budding and budding; however, during gas bladder outgrowth, expression in both the dorsal and ventral mesoderm increases. Abbreviations: dE, dorsal endoderm; dM, dorsal mesoderm; vE, ventral endoderm; vM, ventral mesoderm.

Discussion

RNA-seq identifies novel genes associated with gas bladder budding in bowfin

Using RNA-seq, we aimed to discover genes that are differentially expressed across the dorsoventral axis of the foregut during gas bladder development and thus may play a role in regulating the dorsal direction of budding. By manual curation, we discovered many of the differentially expressed dorsoventral genes are annotated to

GO terms that are related to organ development, e.g. gas bladder budding (Table 2).

The extracellular matrix (ECM) is an interface between the epithelium and the mesenchyme such that during organ budding, the ECM is continuously remodeled through both chemical and mechanical signals (Kim and Nelson, 2012). Thus, the

ECM plays a role in regulating cell proliferation, cell differentiation, tissue shape change, and the establishment of cell polarity, all of which are happening during the budding process. Specifically, during lung branching morphogenesis, the thinning of the ECM adjacent to the tip of nascent lung buds in mice occurs simultaneously with high rates of epithelial cell proliferation (Kim and Nelson, 2012). Both ECM-cell and cell-cell adhesion can be regulated by the actin cytoskeleton producing contractile and tensile forces to cause shape changes to tissue and organs, such as budding (Kim and

Nelson, 2012). During lung budding, the epithelial cells begin migrating out into the surrounding mesenchyme before cell proliferation occurs (Nogawa et al., 1998; Lü et al., 2005), and Fgf10, a growth factor expressed in the mesenchyme, regulates the direction of this cell migration (Park et al., 1998). The epithelial cells of the lungs or gas bladder form a monolayer over the surface of the organ serving as a barrier

between extracellular environments (Drubin and Nelson, 1996; Mostov et al., 2000).

To function properly as a barrier and regulate membrane trafficking of proteins, these epithelial cells are polarized, meaning the distribution of cellular components inside a single cell is asymmetrical (Drubin and Nelson, 1996; El‐Hashash and Warburton,

2011). As cells proliferate and differentiate during budding, the polarity of new epithelial cells must be established. The key players stimulating cell proliferation, differentiation, and migration are growth factors.

Lung-gas bladder inversion

Of the genes differentially expressed between the dorsal and ventral foregut during gas bladder development, we identified those known to regulate lung development and asked whether they show an inverted expression pattern during gas bladder development relative to lungs. Tbx5 is an early marker of a dorsally budding gas bladder and is strongly expressed in the dorsal mesenchyme surrounding the gas bladder both at budding and outgrowth stages in bowfin. In addition, the paralogous gene, Tbx4, is also strongly expressed in the dorsal mesenchyme during gas bladder outgrowth. The dorsal expression of these genes during gasbladder development contrasts with the ventral expression patterns observed during bichir and tetrapod

(Sakiyama et al., 2003; Arora et al., 2012; Tatsumi et al., 2016) lung development. We confirmed the early expression of Tbx5 in the ventral mesenchyme surrounding the bichir lung at budding and outgrowth stages of development using RNAscope in situ hybridization assays. Notably, in mouse and chicken, Tbx5 and Tbx4 are known to contribute to the budding and elongation of the lung. Specifically, Tbx5 alone is important for bilateral specification during initial lung budding in mouse. When Tbx5

expression is reduced in mouse foregut tissue cultures, Nkx2.1 expression, the first genetic marker of tetrapod lung development, is lost in one lung bud (Arora et al.,

2012). Later in development, during tetrapod lung elongation, both Tbx5 and Tbx4 are important for proper branching morphogenesis (Sakiyama et al., 2003; Arora et al.,

2012). Thus, the expression pattern of Tbx5 and Tbx4 during development of the dorsal gas bladder in bowfin is inverted relative to that during development of the ventral lungs in bichir and tetrapods. These data suggest Tbx5 may be involved in regulating the direction of budding of both gas bladders and lungs.

Resulting from the teleost whole genome duplication (WGD), zebrafish have two copies of Tbx5: Tbx5a and Tbx5b (Albalat et al., 2010; Boyle Anderson and Ho,

2018). Although spatial expression of either Tbx5a or Tbx5b in the gas bladder has not yet been examined in zebrafish, in Tbx5b-knockdown embryos, the gas bladder does not inflate (Boyle Anderson and Ho, 2018) providing evidence supporting a role for

Tbx5 in gas bladder development. However, non-inflation of the gas bladder is common and known to be the result of many distinct genetic mutations (McCune and

Carlson, 2004). Both the dorsal expression pattern of Tbx5 during gas bladder development in bowfin and the functional role that Tbx5b plays in zebrafish gas bladder development are consistent with the hypothesis that inverted spatial expression of Tbx5 contributed to the morphological inversion of budding location from ventral lungs to dorsal gas bladder in ray-finned fishes.

Genes interacting with Tbx5

After revealing the inverted expression pattern of Tbx5 mirroring the inverted

budding direction during lung versus gasbladder development, we examined whether genes known to interact with Tbx5 exhibit similar expression patterns to Tbx5 during development of the gas bladder. Tbx5 and Tbx4 expression overlaps with Fgf10 expression in the dorsal mesoderm during gas bladder development in bowfin suggesting that these genes may interact to regulate outgrowth as they do during tetrapod lung development. T-box genes have a known role as transcriptional regulators, and Tbx5 and Tbx4 have been shown to regulate Fgf10 expression during mouse and chick lung development, respectively (Ng et al., 2002; Cebra‐Thomas et al.,

2003; Sakiyama et al., 2003; Arora et al., 2012). Fgf10 plays an important role in lung budding as evidenced by the absence of lungs in Fgf10-knockdown mice (Min et al.,

1998; Sekine et al., 1999). Later during lung development, Fgf10 is also critical for regulating branching morphogenesis (Morrisey and Hogan, 2010). During lung development in mice, Tbx4 and Tbx5 show overlapping expression in the lung mesenchyme and may be functionally redundant (Chapman et al., 1996; Naiche and

Papaioannou, 2003; Cardoso and Lü, 2006). In fact, when Tbx4 and Tbx5 expression is reduced, the mutant mice show decreased Fgf10 expression during lung branching and accordingly, the lungs were smaller than wild type (Cebra‐Thomas et al., 2003; Arora et al., 2012). In chick, Tbx4 is is co-expressed with Fgf10 in the ventral lung mesenchyme (Sakiyama et al., 2003; Morrisey and Hogan, 2010). When Tbx4 is ectopically expressed in the visceral mesoderm of the foregut, Fgf10 expression is induced and ectopic lung buds form on the esophagus, whereas when Tbx4 function is disrupted in chick, Fgf10 expression is repressed and lung buds fail to form (Sakiyama

et al., 2003). During bowfin gasbladder development, both Tbx4 and Tbx5 are expressed most strongly in the dorsal mesenchyme surrounding the gasbladder bud at all stages. Fgf10 expression also overlaps with Tbx4 and Tbx5 in the dorsal mesenchyme but not until the outgrowth stage. Compared to lung development, the delayed onset of Fgf10 expression suggests it is not involved in initiating gasbladder budding. However, the overlapping expression patterns between Tbx4, Tbx5, and

Fgf10 during outgrowth make apparent the possibility of gene interactions regulating gasbladder development.

During bowfin gas bladder development, Wnt2ba, whose ortholog is known to act upstream of Tbx4 and Tbx5 during lung development, is co-expressed with Tbx4,

Tbx5, and Fgf10 in the dorsal mesenchyme. In mouse, Wnt2 and Wnt2b are expressed ventrally in the lung mesenchyme during lung specification and outgrowth (Goss et al., 2009), and when both are lost in mice, the lung buds fail to develop. Additionally, the loss of Wnt2 leads to reduced expression of Fgf10 (Goss et al., 2009). Though the exact interactions between Tbx5, Fgf10, and Wnt2/2b during lung development have not yet been determined, the interaction of these genes is established in other contexts, such as fin bud initiation and outgrowth in zebrafish (Ng et al., 2002). These comparative data suggest that Wnt2ba could be regulating Tbx5 and Tbx4 expression, which in turn, are interacting with Fgf10 to promote gasbladder development.

Interactions between genes expressed in the mesenchyme and those expressed the in epithelium are also critical. Specifically, Fgf10 expression in the mesenchyme and Bmp4 expression in the epithelium interact to regulate lung outgrowth and branching (Weaver et al., 2000; Morrisey and Hogan, 2010; Hines and Sun, 2014). In

this study, all tissues, dorsal and ventral endoderm and mesoderm, show equal RNA expression of Bmp4 at all stages of gas bladder development in bowfin (Figure

A2.1A). However, previously, we showed that Bmp4 protein expression appears absent from the foregut and gas bladder during both bowfin and zebrafish development (Funk et al., in review). Instead, Bmp16, a paralogue of Bmp4 that has been lost from the mammalian genome but retained in ray-finned fishes, is expressed strongly in the gas bladder epithelium during outgrowth (Funk et al., in review; Feiner et al., 2009). Bmp16 RNA expression, on the other hand, is weak or absent at all developmental stages (Figure A2.1B). The discrepancy between RNA and protein expression is not unusual for signaling molecules like Bmps, which are not necessarily active in the same location that they are transcribed. Because the protein, translated from RNA, is the molecule that carries out biological processes, protein expression is more reliable for studying Bmp patterns. Bmp16 protein expression during outgrowth

(stage 27; Funk et al., in review), the same stage at which Fgf10 expression increases, suggests an interaction between Bmp16 and Fgf10 may contribute to the regulation of gas bladder expansion.

Conclusion

Here we show that Tbx5, known to be important for lung development, is expressed in the dorsal mesenchyme surrounding the gas bladder. Spatial expression of Tbx5 is inverted in the bowfin gas bladder relative to its expression during mouse lung development. Additionally, we identified Tbx4, Wnt2, and Fgf10, all of which interact with Tbx5 during mouse lung development, as potential players in gas bladder development based on their overlapping expression patterns with Tbx5. Functional

experiments are not yet possible to perform on bowfin because we cannot collect single-cell stage embryos from the wild and we are unable to breed them in the lab.

However, zebrafish could provide the opportunity to test the function of these candidate genes during gasbladder development.

Data Availability

The data supporting the findings of this publication are openly available on NCBI’s

Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE152992

(https://www.ncbi.nim.nih.gov/geo/query/acc.cgi?acc=GSE152992).

Author Contributions

E. C. F. and A. R. M. conceived of the study and wrote the article with input from co- authors. E.C.F. collected eggs and sampled the developmental series. E. C. F. performed the laser-capture microdissection and library prep for RNA sequencing. E.

C. F. and E. S. L. analyzed differential gene expression.

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[dataset]Funk, EC, Lencer, ES, McCune, AR; 2020; Dorso-ventral inversion in the air- filled organ (lungs, gas bladder) of vertebrates: RNA-sequencing of laser capture microdissected embryonic tissue; NCBI Gene Expression Omnibus; Accession #GSE152992; https://www.ncbi.nim.nih.gov/geo/query/acc.cgi?acc=GSE15299

CHAPTER 3 Does the bowfin gas bladder represent an intermediate stage during the lung-to- gas bladder evolutionary transition?3

3 Funk, E. C., Birol, E., McCune, A. R. (in review) Does the bowfin gas bladder represent an intermediate stage during the lung-to-gas bladder evolutionary transition? Journal of Morphology.

Abstract

Whether phenotypic evolution occurs gradually through time has prompted the search for forms showing intermediate stages between the ancestral and derived states of morphological features, especially when there appears to be a discontinuous origin of a feature. The gas bladder, a derived character of the Actinopteri, is a modification of lungs, which characterize the common ancestor of bony vertebrates. While gas bladders and lungs are similar in many ways, the key morphological difference between these organs is the direction of budding from the foregut during development; essentially, the gas bladder buds dorsally and the lungs bud ventrally from the foregut.

Did the shift from ventral lungs to a dorsal gas bladder transition through a lateral- budding stage? To answer this question, the precise location of budding during gasbladder development in bowfin, representing the sister lineage to teleost fishes, has been debated. In the early 20th-century, it was suggested that the bowfin gas bladder buds laterally from the right wall of the foregut. We used nano-CT scanning to visualize the development of the bowfin gas bladder in 3D to verify historical studies of gasbladder developmental morphology and determine whether the direction of gasbladder budding in bowfin could be intermediate between ventrally-budding lungs and dorsally-budding gas bladders. We found the bowfin gas bladder buds dorsally from the foregut; however, during early outgrowth, the posterior gas bladder twists right, perhaps due to crowding in the body cavity by the yolk and stomach. As development progresses, the posterior, right-hand twist of the gas bladder becomes shallower, and the gas bladder shifts towards a mid-dorsal position. The budding site is definitively dorsal, despite the temporary lateral twist of the posterior gas bladder.

Instead, recent studies of genes expressed during the development of both lungs and gas bladders suggest that modifications of the ancestral lung-gene network, including

Tbx5, Tbx4, Wnt2, Fgf10, and Bmp-signaling, led to the ventral-to-dorsal shift in budding direction associated with the gasbladder evolution.

Introduction

Since publication of the Origin of Species, evolutionary biologists have sought intermediate forms, especially when the origin of a novel feature appears to be discontinuous in the fossil record. Among ray-finned fishes, an important phenotypic novelty with an apparent discontinuous origin is the gas bladder (or swim bladder), functioning primarily in buoyancy control or respiration. The gas bladder has long been considered to be a modification of the lungs found in the common ancestor of bony vertebrates (Sagemehl, 1885; Dean, 1895; Romer and Parsons, 1970; Graham, 1997), and an iconic figure depicting a morphological transformation series of lungs and gas bladders in vertebrates (Figure 3.1A,B; Wilder, 1877; Dean, 1895) has been reproduced many times in monographs and textbooks over the last 150 years (see Figure 3.1).

How gas bladders might have evolved from lungs (or the reverse) and the significance of this transition has attracted the attention of a number of evolutionary biologists.

For example, Ernst Mayr used the lung-gas bladder transition to illustrate how pre- adaptation can lead to evolutionary novelty (Mayr, 1960). And earlier, Darwin himself used gas bladders and lungs as an example of the power of natural selection, although he mistakenly assumed the gas bladder was the ancestral state when he wrote “there is no reason to doubt that the swim bladder has actually been converted into lungs”

(Darwin, 1859, p. 191).

Figure 3.1: Original and modified versions of the iconic 19th century morphological transformation series of lung and gasbladder morphology. In all versions of the figure, for each taxon, a transverse section of lungs or gas bladder is given at the left and a lateral view at the right. The top left panel is the original illustration by Burt Green Wilder (1877), the first to propose a morphological transformation series of lung and gasbladder morphology. In this original figure, Wilder groups the ray-finned fishes at the top, positions the “intermediate” lungfish in the middle, and places tetrapods, along with the enigmatic bichir (Polypterus), at the bottom. At the time, some regarded bichir, which has lungs, as the missing link between fishes and amphibians (Hall, 2001). B) For his book, Fishes, Living and Fossil, Bashford Dean (1895) co-opted Wilder’s illustration, modifying it to the form that was subsequently reproduced (with variations) in many important vertebrate textbooks for a century (e.g., Goodrich, 1930; Young, 1962; Romer and Parsons, 1970; Hyman and Wake, 1979; Graham, 1997). C) Hyman & Wake (1979) illustrated only “fishes” and D) Graham (1997) updated the morphology represented in the illustration. All iterations show the ventral connection of lungs to the foregut (reptiles/birds/mammals, and Bichir/Ropefish), the dorsal connection of the gas bladder to the foregut (gar/bowfin, sturgeon/teleosts); and taxa in which the gas bladder has a more lateral connection to the foregut, the Australian lungfish and the characin, Erythrinus, neither of which can be considered transitional taxa in a phylogenetic sense. Original figures used a mixture of scientific and common names. We have shown common names, where possible, with scientific equivalents given in the text, or for the largest clades, on the figure itself.

Gas bladders and lungs share many characteristics, including budding from the foregut, pulmonary artery supply, surfactant protein production, and a lung-specific gene regulatory network (Graham, 1997; Cass et al., 2013; Longo et al., 2013), but they are distinguished by one key feature, the general direction of budding from the foregut during development. Though both lungs and the gas bladder evaginate from the anterior foregut, the lungs bud from the ventral wall, while the gas bladder buds from the dorsal wall (Wilder, 1877; Graham, 1997; Cass et al., 2013). Thus, the search for intermediates between lungs and gas bladders has focused on the shift from ventral-to- dorsal budding. Does an intermediate form exist, in which the gas bladder buds laterally from the foregut?

Darwin’s highlighting of the significance of the gas bladder and lungs led many 19th century zoologists to study the variation in form, function, and development of these air-filled organs. In 1877, Burt Green Wilder (1877) proposed and illustrated the first morphological transformation series of air-filled organs showing how the ventral lungs of tetrapods and a dorsal gas bladder in fishes might have evolved from the same ancestral organ (Figure 3.1A) and Sagemehl (1885) argued convincingly that lungs were the ancestral condition. Subsequently, Wilder’s illustration was co-opted and popularized by Bashford Dean in his text, Fishes, Living and Fossil (Figure 3.1B;

1895) and since then, modified versions of the figure have been reproduced in many of the major textbooks on vertebrate biology published during the 20th century (Figure

3.1C-D; e.g., Kerr, 1921; Goodrich, 1930; Romer, 1959; Jollie, 1962; Young, 1962; Romer and

Parsons, 1970, 1986; Hyman and Wake, 1979; Torrey and Feduccia, 1979; Graham, 1997;

Liem et al., 2001). Notably, this familiar figure and its variations illustrate a morphological progression in the direction of budding from ventral to lateral to dorsal reflecting mainly the direction of budding without regard to a phylogenetic framework or the relative recency of common ancestry (Figure 3.1A).

Although lungs bud from the ventral foregut and gas bladders bud from the dorsal foregut, Dean’s illustration (Figure 3.1A) does show that the connection site varies somewhat among taxa. For example, in Neoceratodus forsteri (Australian lungfish), the pneumatic duct connecting the lung to the foregut is slightly offset from the right of ventral midline (Grigg, 1965). In Polyodon spathula (American paddlefish) and Acipenser gueldenstaedtii (Russian sturgeon) the pneumatic duct from the gas bladder connects slightly offset of the left of dorsal midline of the foregut (Grom,

2015). And, intriguingly, the teleost, Erythrinus (a member of the characin family), which originated long after the first species with a gas bladder, has a lateral connection from the gas bladder to the foregut (Wilder, 1877; Dean, 1895). Though this lateral connection must have been inspiring to 19th century zoologists searching for transitional forms, it cannot be an intermediate because Erythrinus is nested within the

Teleostei, which arose well after the first taxa with gas bladders (Figure 3.2).

Figure 3.2: Lung and gas bladder phenotypes arrayed in a modern phylogenetic framework. A highly pruned phylogeny of bony vertebrates with the same cross-sectional drawings from Dean’s original figure mapped onto the tree. Note that in a phylogenetic context, the taxa with somewhat lateral connections do not precede origin of the gas bladder, associated with the Actinopteri. The morphological transformation series is thus misleading because Australian lungfish, with a ventro-lateral connection, is nested within the fleshy- finned fishes; and Erythrinus, the taxon with a lateral connection between the gas bladder and foregut, is nested within the teleosts, most of which possess dorsal gas bladders. There appears to be no progression of ventral lungs to a lateral organ and then to a dorsal gas bladder. L, lungs; GB, gas bladder

To look for possible stages documenting an evolutionary transformation, taxa like Amia calva, the bowfin (see Figure 3.2), which possess a gas bladder and which diverged prior to most living taxa with gas bladders are clearly of greater interest than a teleost such as Erythrinus. In fact, the bowfin, representing the sistergroup to teleosts, has often been used as a model for early bony fishes (Graham, 1997). Like lungs, the bowfin gas bladder is respiratory with a complex internal structure, which increases surface area and extensive vascularization. But like other ray-finned fishes, the gas bladder in bowfin is positioned dorsally in the body cavity, above the gastrointestinal tract and below the spine (Figure 3.3B, C), and it retains the developmental connection to the foregut, the pneumatic duct.

During bowfin development, the precise budding location of the gas bladder from the foregut has been debated. In a histological study, Piper (1902) detailed the development of the bowfin gas bladder and concluded that the gas bladder buds mid- dorsally from the foregut. However, Ballantyne (Ballantyne, 1927) contested Piper’s conclusion, arguing that the gas bladder actually buds dorsolaterally from the right side of the dorsal foregut during bowfin development, suggesting that bowfin might represent a transitional stage to a more definitively dorsal gas bladder. To resolve this dispute, we take advantage of modern nano-CT imaging, to investigate whether the gas bladder originates from the lateral or dorsal wall of the foregut during development.

Figure 3.3: Location and morphology of the gas bladder in a juvenile bowfin. Micro-CT volume renderings and digital sections of a juvenile bowfin (175 mm standard length; CUMV 97349), which illustrates the morphology of a gas bladder in context. Orientation compass for all images is in the lower right corner of panel A. A) Micro-CT volume rendering of the external morphology of a juvenile bowfin. Only the mid-section, where the gas bladder is located, is shown. B) Digital longitudinal section of the juvenile bowfin with the gas bladder outlined in red. C) Volume rendering of only the spine (white) and gas bladder (red). The gas bladder is positioned dorsally in the body cavity ventral to the spine and dorsal to the gut. Abbreviations: f, fin; GB, gas bladder; op, operculum; s, stomach; sp, spine.

Materials and Methods

Egg Collection & Sampling

Bowfin eggs were collected from Oneida Lake, Brewerton, NY in May 2014 and 2015. The eggs were immediately treated with methylene blue (1/4 tsp per 2.5 gallons of water) to prevent fungal growth. The eggs were transported to the fish facility in Corson-Mudd Hall at Cornell University and kept at 14oC. Embryos were reared for 3 weeks, during which 20 individuals were sampled daily to generate a reference developmental series spanning early gas bladder development, from Ballard stages 25 to 29 (Figure 3.4; Ballard, 1986). Fish care was performed according to

IACUC protocol #2006-0013. Samples were fixed overnight in 4% paraformaldehyde

(PFA) in phosphate buffered saline (PBS) then transferred to 100% MeOH by stepping through 25%, 50%, and 75% MeOH dilutions in PBS. Specimens were stored in 100% MeOH at -20oC. Before CT-scanning, the sampled individuals were viewed and photographed using a stereo microscrope (Olympus America Inc., model SZX16) fitted with a DP25 camera.

CT Scanning

For reference, a juvenile bowfin with a standard length of 175 mm (CUMV

97349, Cornell University Museum of Vertebrates) was stained in iodine (1% iodine metal in 100% ethanol) and scanned on the GE CT 120 micro-CT scanner at 80 kV.

For a final resolution of 50 µm, 1200 fluoroscopy images were collected at 0.3 degree intervals over 360 degrees and 100 ms exposure time. The fluoroscopy dataset was reconstructed and imported into either Avizo (version 2019.3) or Osirix software programs. The 3D volume rendering of external morphology and the digital section

images were created in Avizo, while the 3D volume rendering of only the spine and gas bladder was created in Osirix.

A growth series of larval specimens (CUMV 99738, Cornell University

Museum of Vertebrates) were stained in 30% PTA (phosphotungstic acid) /70% ethanol solution overnight to increase tissue contrast for nano-CT scanning. The PTA solution was prepared as 1% (weight/volume) PTA in water (Metscher, 2009). Stained samples were scanned by the Cornell Imaging Facility on an Xradia Zeiss VersaXRM-

520 system at 80kV and 7W. For a final resolution of between 2 and 5 µm/pixel, 801

X-ray fluoroscopy images were collected over 360 degrees with a 4x objective and 1-

2 second exposure time. The detector was binned at 1000x1000 pixels. The fluoroscopy data was reconstructed using the standard Zeiss reconstruction software and imported into Avizo software program (version 2019.1 and version 2019.3), where we created 3D volume-rendered images of the entire specimen. These 3D volume renderings of the whole fish body were longitudinally sliced down the midline. We also manually selected the 2D gas bladder ROI (region of interest) for every third digital transverse section (dicom) and interpolated between them to create a surface view of the gas bladder alone in 3D. The whole body volume renderings and gas bladder surface views were combined in Photoshop. At each developmental stage, we measured the angle of twist from vertical of the gas bladder at the anterior, the middle, and the posterior regions using the ruler tool in Photoshop.

Figure 3.4: Paired photographs and nano-CT renderings of external morphology of developing bowfin larvae, stages 25 to 29. Staging of developing bowfin was determined using external morphological characters following Ballard (1986). Gas bladder budding occurs at stage 25. Caudal and lateral outgrowth of the gas bladder starts at stage 27. Abbreviations: a, adhesive organ; h, heart; pf, pectoral fin; y, yolk.

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Results

The bowfin gas bladder begins developing at stage 25 (Ballard, 1986) as a twisted longitudinal ridge budding from the dorsal to dorsolateral wall of the foregut (Figure

3.5A). At its most anterior end, the gas bladder ridge buds mid-dorsally from the foregut. More posteriorly, the gas bladder ridge twists such that the ridge is evaginating dorsolaterally on the right side of the foregut (Figure 3.5B). At stage 26, just after initial budding, the gas bladder still forms a longitudinal ridge along the dorsal side of the foregut extending mid-dorsally at the anterior and dorsolaterally at the posterior end (Figure 3.5B). The ridge has grown taller and the posterior right- hand twist is greater. At stage 27, the posterior ridge of the gas bladder is shifting towards a mid-dorsal position above the foregut as the right-hand twist of the posterior region of the gas bladder becomes shallower. Also during stage 27, the top of the ridge begins to expand laterally, to both right and left forming a “T” shape (Figure 3.5C) and the gas bladder is lengthening caudally. At stage 28, the gas bladder continues to grow laterally, and the “T” shape becomes more pronounced. As the gas bladder continues to lengthen caudally, it also begins to constrict at the base and separate posteriorly-to-anteriorly from the foregut (Figure 3.6A). At stage 29, the separation of the gas bladder from the foregut continues, foreshortening the pneumatic duct and reducing the connection between the gas bladder and foregut from a longitudinal ridge to a more tubular-like opening. The gas bladder grows laterally and caudally, and no longer appears as a ridge but rather begins to take on a more sac-like form (Figure

3.6B). As the gas bladder develops through both stages 28 and 29, posteriorly, it continues to shift towards the mid-dorsal position.

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Figure 3.5: Morphology of the developing gas bladder in bowfin, stages 25, 26, and 27. Top panels display two-dimensional digital transverse sections with 3-dimensional rendering of the gasbladder bud (gb) and the gut inserted to show context. Bottom panels show two views: a 45-degree oblique view of a nano-CT volume rendering sliced digitally through the midline and, below that, an enlarged image of the gas bladder bud (pink) and gut (white). These 3-dimensional views correspond to the transverse sections of the same stages shown in top panels. Abbreviations are as follows: fg, foregut; nc, notochord; nt, neural tube; pd, pneumatic duct; s, stomach; y, yolk. Image view is from the right side of specimens (contrary to ichthyological convention of photographing the left side) because the gas bladder is situated towards the right side of the body cavity and the liver and stomach are situated towards the left. A) Stage 25 shows earliest budding. At stage 25, initial gas bladder budding occurs, and the bud appears as a longitudinal ridge on the dorsal side of the foregut. The most anterior point of the gas bladder bud grows dorsally from the mid-dorsal side of the foregut, more posteriorly, the bud is twisted somewhat to the right side of the embryo and grows from the right lateral- dorsal side of the foregut. Twisting angles are summarized in Table 1. B) In stage 26, the region of budding elongates and begins to push outward. At stage 26, the gas bladder still appears as a longitudinal ridge but has grown higher dorsally compared to stage 25. At the most anterior point, the gas bladder bud extends dorsally from the mid-dorsal side of the foregut. More posteriorly, the bud is twisted to the right side of embryo and appears to be extending from the right lateral-dorsal side of the foregut; however, the twist is less pronounced than at stage 25. C) In stage 27, the gas bladder bud is an elongated ridge. At stage 27, the gas bladder begins to grow caudally and ‘T’ out laterally to both right and left. By this stage, the gas bladder is situated mid-dorsally and symmetrically above the foregut.

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Figure 3.6: Morphology of the developing gas bladder in bowfin, stages 28 and 29. Top panels display two-dimensional digital transverse sections with 3-dimensional rendering of the gas bladder bud (gb) and the gut inserted. Bottom panels show two views: a 45-degree oblique view of a nano-CT volume rendering sliced digitally through the midline and, below that, an enlarged image of the gas bladder bud (pink) and gut (white). These 3-dimensional views correspond to the transverse sections of the same stages shown in top panels. Abbreviations are as follows: fg, foregut; nc, notochord; pd, pneumatic duct; s, stomach. Photos were taken of the right side of specimens (contrary to ichthyological convention of photographing the left side) because the gas bladder is situated towards the right side of the body cavity with the liver and stomach are towards the left. A) At stage 28, the gas bladder continues to grow caudally and the connection between the gut and the gas bladder, the pneumatic duct, starts to become constricted. The gas bladder also keeps growing laterally into a more defined ‘T’ shape. B) At stage 29, lateral and caudal growth continues. The gas bladder becomes increasingly constricted and separates from the gut making the pneumatic duct (pd) more narrow.

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Based on 3D volume renderings, the budding site of the bowfin gas bladder does not appear to represent a transitional phenotype between ventral lungs and a dorsal gas bladder. It forms as a dorsal to dorsal-lateral ridge, and through development, the gas bladder shifts progressively closer to a mid-dorsal position in the body cavity. However, the twisting topology of the developing gasbladder ridge is complicated and its position relative to the foregut changes during development as well as from anterior to posterior in each stage. To quantify, we measured the angle of the gasbladder outgrowth from vertical in an anterior, middle and posterior section of the developing gas bladder from stages 25-29 (Table 3.1). These angular measurements are illustrated for a series of three transverse sections at budding (stage

26) in Figure 7. At all stages, the anterior end of the gas bladder extends mid-dorsally with negligible twist, between 1.2 and 2.5 degrees (Table 3.1), and the right-hand twist progressively increases towards the middle and then the posterior end. At stage 25, the posterior of the gasbladder ridge is twisted at an angle of 51.3 degrees, and at stage 26, the posterior twist increases to an angle of 63.4 degrees (Table 3.1). From stage 27 onward, the angle of twist gradually becomes shallower from 56.4 degrees at stage 27 to 47.6 degrees at stage 28 to 34.6 degrees at stage 29. It is clear from the cross- sectional images, however, that even when the twist angle of the posterior gas bladder is at its maximum of 63.4 degrees (Figure 3.7D), the gas bladder is still evaginating from the dorsal foregut.

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Table 3.1: Torsion of bowfin gasbladder development relative to vertical. The angle of right-twist of the budding gasbladder varies from anterior to posterior and through developmental stages 25 to 29. Angles were measured on the most anterior section, a middle section, and the most posterior section (see Figure 3.7). At all the stages, the gas bladder buds dorsally with negligible twisting, and moving posteriorly, the angle of twist increases. As the gas bladder develops, the angle of twisting decreases, and the gas bladder ridge becomes more dorsally positioned in the body cavity.

Stage Stage Stage Stage 25 Stage 26 27 28 29 Anterior Angle 1.2 1.2 2.5 2.3 2.2 Middle Angle 33.7 27.9 25.9 38.1 16.7 Posterior Angle 51.3 63.4 56.4 47.6 34.6

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Figure 3.7: Angular measurements of the right-twisted gas bladder bud. Nano-CT scan of a bowfin at stage 26 is used to illustrate how the angle of twist changes from anterior to posterior and how the degree of twist was measured during gasbladder development. A) 3D volume rendering of the foregut (white) and gasbladder bud (pink). The rectangles indicate the location of the following transverse images. Shown are the digital cross-sections of the whole fish at the B) anterior end, C) the middle, D) and the posterior end of the gas bladder. For each stage, we measured the angle of rightward twist from vertical (red) of the gas bladder at the anterior, middle, and posterior regions. At all developmental stages (25-29), the angle of twist increases from anterior to posterior. As gasbladder development progresses, the angle of twist gradually decreases (Table 1), and the gas bladder shifts closer to a mid-dorsal position in the body cavity. Abbreviations: b, brain; f, fin; fg, foregut; GB, gasbladder bud; n, notochord; y, yolk.

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Discussion

With the benefit of high-resolution nano-CT scanning, we find evidence supporting aspects of both arguments about the direction of gasbladder budding by early 20th century zoologists. The gas bladder does indeed bud mid-dorsally from the anterior foregut as described by Piper (1902). However, early during gasbladder development in bowfin, the posterior dorsal ridge is twisted to the right, as proposed by Ballantyne

(1927). Given this torsion of the early gasbladder ridge, we can understand the differing interpretations. However, the fact remains that, at the initiation of gasbladder development, the gasbladder bud evaginates dorsally, despite the dorsolateral twist of the gasbladder ridge more posteriorly.

As in bowfin, several other species of ray-finned fish also show temporary lateral positioning of the gas bladder during development (Griel, 1905). Within teleosts, certain species from the cyprinid and salmonid families exhibit lateral positioning of the gas bladder organ during development, even though budding occurs from the mid- dorsal wall of the foregut (Makuschock, 1913; Graham, 1997). In gar, the sistergroup to bowfin, the gas bladder forms mid-dorsally as a ridge from the foregut, but this ridge is temporarily displaced laterally to the right (Makuschock, 1913). Then, as development progresses, the gut rotates left and the stomach swings downward, and the gas bladder is situated dorsally and continues to grow caudally (Makuschock, 1913).

The one known instance of a gas bladder with a lateral connection to the foregut is in

Erythrinus, a genus within the Characiformes, embedded in the teleost clade. Though

Wilder (1877) and Dean (1895) depicted the evolution of the gas bladder as a graded

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morphological series with Erythrinus representing the intermediate phenotype, the argument falls apart when taking the phylogeny into account (Figure 3.2). What is the relevance of an “intermediate form” in a taxon that arose much later than the transition? Instead, the relevant species to investigate are the early-diverging lineages that exhibit ancestral characteristics, such as bowfin. As yet, lateral budding, a transitional phenotype between ventrally budding lungs and a dorsally budding gas bladder, has not been observed among any extant early-diverging ray-finned fishes.

Historically, some have proposed that the ventral-to-dorsal shift in budding site during gas bladder evolution is a consequence of a differing degrees of foregut rotation, referred to here as the rotation hypothesis (Griel, 1905; Ballantyne, 1927;

Graham, 1997). Griel (1905; translated excerpt Appendix 3.1) posited that though the direction of gasbladder budding in bowfin is dorsal, the gas bladder actually develops from what was originally the right lateral side of the foregut. He argues that due to the rotation of the gut, the original dorsal wall of the foregut is rotated to the left and the original right wall of the foregut is rotated dorsally at the time of initial gas bladder budding (Griel, 1905). This could imply lateral budding that, with the rotation of the foregut, appears dorsal. Similarly, Ballantyne (1927) stated that the bowfin gas bladder buds laterodorsally from the foregut, and during development, the leftward rotation of the foregut brings the gas bladder and connecting pneumatic duct to a mid-dorsal location. The degree of gut rotation may also be affected by the amount of connective tissue. The bowfin foregut hangs relatively free in the body cavity so that gut rotation is relatively unconstrained. In contrast, in the Australian lungfish, the gut is anchored by connective tissue and the dorsally-located lung retains a ventral connection to the

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foregut, with the pneumatic duct wrapping around the right side of the gut.

Variation in the degree of gut rotation during development could also lead to the subtle differences observed between species in the exact location of the foregut connection, mid-dorsal or dorsolateral, in adults (Grigg, 1965; Grom, 2015). During bowfin development, the posterior lateral twist of the gas bladder ridge may be due to the gut not yet being fully rotated consistent with the hypothesis that the shift to a dorsal connection of the gas bladder to the foregut was achieved via increased gut rotation. However, with further gut rotation, one might expect to observe changes in the relative positioning in the body cavity of other organs, e.g. pancreas, liver, and stomach, that also bud from the gut during development. To investigate further, gut development and rotation of other organs would need to be documented in bowfin and bichir, the only early-diverging actinopterygian lineage with lungs.

Perhaps a more interesting question than whether the morphological shift from ventral lungs to a dorsal gas bladder was gradual or discontinuous, is what underlying genes specify the shift in direction of budding? Recent work in developmental genetics suggests that genes involved in early lung budding are also involved in early gas bladder budding (Feiner et al., 2009; Winata et al., 2009; Cass et al., 2013; Funk et al.,

2020a; b). Two genes, Tbx5 and Bmp-signaling show ventral-dorsal inverted expression patterns in lungs and gas bladders and thus appear to be good candidates for having played a critical role in driving the evolutionary transition from ventral lungs to a dorsal gas bladder. Tbx5 is a transcription factor essential for lung budding and branching in mouse (Arora et al., 2012). During lung development in tetrapods and bichirs, the only ray-finned fish lineage that possess ventral lungs, Tbx5 is expressed

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in the ventral mesoderm surrounding the lung buds (Arora et al., 2012; Tatsumi et al.,

2016; Funk et al., 2020b). In contrast, during gas bladder development in bowfin, Tbx5 is expressed in the dorsal mesoderm surrounding the gas bladder bud, an inverted expression pattern relative to that in lungs (Funk et al., 2020b). Tbx5 is part of a network of genes, including Tbx4, Wnt2, and Fgf10, known to interact during lung development (Ng et al., 2002; Sakiyama et al., 2003; Lü et al., 2005), all of which appear to show an inverted expression pattern across the dorsoventral axis of the foregut during gasbladder development in bowfin (Funk et al., 2020b). In addition, there may be an inversion of expression pattern of paralogous Bmp genes. Bmp4 is expressed ventrally during lung development in mouse, and not at all in gas bladder development, while Bmp16, a paralog of Bmp4, is expressed dorsally during gas bladder development in bowfin and zebrafish (Feiner et al., 2009; Funk et al., 2020a).

Thus, the dorsoventral inversion of gene expression patterns of Tbx5 and

Bmp4/Bmp16 parallels the morphological shift from ventral to dorsal budding from the foregut during the lung-to-gas bladder evolutionary transition.

When considering the expression patterns of conserved genes in both lung and gasbladder development, a dorsoventral inversion of expression appears to underlie the morphological inversion of budding direction, making the gut rotation hypothesis less attractive. If the budding direction was inverted via increased gut rotation, one would expect that most gene expression patterns would have been inverted. However, most conserved genes are not inverted; rather, they show similar patterns of expression between gasbladder and lung development. For example, Nkx2.1, the first known marker of lung development, is expressed in the ventral foregut during both mouse

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lung development and bowfin gasbladder development (Funk et al., 2020a). The dorsoventral spatial expression of several genes, identified from an RNA-seq study, is conserved (Funk et al., 2020b), while other genes (Tbx5, Bmp) show inverted expression.

Given the differential patterns of gene expression revealed in our RNAseq study, an attractive alternative to the gut rotation hypothesis is that the expression of an ancestral lung-gene network has been inverted for induction of dorsal budding during gasbladder development. A regulatory change causing the dorsoventral inversion of Tbx5 expression during gasbladder development may have facilitated the inversion of expression in downstream genes, such as Tbx4, Wnt2, Fgf10, and Bmp- signaling, within the larger lung-gene network. Further modification of the lung-gene network may have led to the observed difference in Bmp signaling between lung and gasbladder development: ventral Bmp4 expression during lung development and dorsal Bmp16 expression during gasbladder development (Funk et al., 2020a). Without a bichir genome, we do not know whether Bmp16 is present in bichir or expressed during bichir lung development; thus, we cannot infer the ancestral state of Bmp4 or

Bmp16 expression in the lungs of the common ancestor to bony vertebrates. However, we can hypothesize two evolutionary scenarios. If Bmp16 is not expressed during bichir lung development, then we hypothesize that Bmp16 expression arose during the origination of the gas bladder. Further modification of the lung-gene network during gasbladder evolution led to the loss of Bmp4 and gain of Bmp16 expression to regulate organ outgrowth. Alternatively, if Bmp16 is expressed during lung development in bichir, then we hypothesize that Bmp16 was also expressed during lung development

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in the common ancestor to bony vertebrates. Subsequently, modification of the lung- gene network might have led to the loss of Bmp4 expression during gasbladder evolution, possibly as a result of redundant gene function. The induction of dorsal budding during gas bladder development may have evolved via the inversion and modification of the ancestral lung-gene network initiated by the dorsoventral inversion of Tbx5 expression.

We conclude that available morphological evidence does not support the view that the gas bladder in bowfin represents a transitional phenotype between ventral lungs and a dorsal gas bladder. Furthermore, taken together, the morphological observations of budding direction and the dorsoventral gene expression patterns suggest that rotation of the gut alone does not explain the ventral-to-dorsal shift during the evolutionary transition from lungs to a gas bladder. Given the patterns of gene expression described thus far, along with morphology, it seems more plausible that modest diversity of exact budding locations among living fishes, whether dorsolateral or mid-dorsal, is more indicative of variation in gut rotation occurring after gasbladder budding than the remnant of a graded transition from ventral to dorsal. We suggest that modifications of the ancestral lung gene network led to the shift in direction of budding associated with the evolution of the gas bladder.

Author Contributions

E. C. F. and A. R. M. designed and carried out this study.

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APPENDIX

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BMP16 ------0 BMP4 ------0 BMP2 MSFRCLITYCVDVKSSMSIVKPTAVLLTLLFCVACGGQVGDVPKQERLFLSALGLSSRPK 60

BMP16 ------0 BMP4 ------0 BMP2 AEGRFPVPSLLWKIFKRKTIGKEAVDPEHDPCTVSEFKVRGNIVRFIQDQGRLLPVFHRQ 120

BMP16 ------0 BMP4 ------0 BMP2 CPSCIERHLYFNMSVLQDVEQLTLAQLEVTFNQDLHRLPRHADLFTMHLYKVMKTTLKGV 180

BMP16 ------0 BMP4 ------0 BMP2 KHQSNRKLLLSQSFQLIPGSITLNLTGMAEIWRKPGKNFGLVLTIQRSAPIDNTDPVRLM 240

BMP16 ------0 BMP4 ------0 BMP2 NLQNDVEHSLVGHFPHFYASLVVVSLNPLQCRSRRRRSAYYFPVTPSNVCKARRLYIDFK 300

BMP16 ------0 BMP4 ------0 BMP2 DVGWQDWIIAPQGYMANYCKGECPFPLSESLNGTNHAILQTLVHSFDPTGTPQPCCVPVR 360

BMP16 ------0 BMP4 ------0 BMP2 LSPISMLYYDNNDNVVLRHYEDMIKEDCPETCLSWKNALSYSSSQKNPTRKSVKKSYSKD 420

BMP16 ------MFPANLLVLMVLLLPQVFSGDQRGVD------SSR------LAR 32 BMP4 ------MIPHNRMLMVILLCQVLLGESSHASLIPEEGKKKVSELQGRRSG 44 BMP2 AEHDHDRPGQEALALIMVAGVRALLVLLLCQVLLGGS--AGLIPEVGRRKFSESGKESPQ 478 : *:*:** **: * . .. :

BMP16 LEPSLAQSIQNLLLTRLGLRNQPAPQPGAPVPQYLLDLYRFHSQEPHLI----QDRDFSF 88 BMP4 QSHELLRDFEATLLQMFGLQRRPRPSHSAVVPHYMLDLYRLQSGEAEEA--GAHDANFEY 102 BMP2 QSEDILNEFELRLLNMFGLKRRPSPSKGAVVPQYMVDLYHMHAGSGDQDHRRARAGPGQH 538 . .: ..:: ** :**:.:* *. .* **:*::***:::: . . : ..

BMP16 PVQHTQSANTIRSFHHLESPGHLYPSSLGKANNFQIIFNISSLPDDEQVTSAELRLYRVQ 148 BMP4 PERSASRANTVRGFHHEEHLERVEGVQEEPDSPLQFLFNLSSIPEDEVLSSAELRLYRQQ 162 BMP2 QERAASRANTIRSFHHEESLESLSSISGK--TTQQFFFNLTSIPGEELITSAEMRIFRDQ 596 : :. ***:*.*** * : . . *::**::*:* :* ::***:*::* *

BMP16 CSG-----DSRGGQRVNLYHFPNPGSPLAAARPKLMESRLLPQDPDAPSWESFSLSTDLF 203 BMP4 IEDAGLYRDHEGLHRINVYEVLKPPRKG-QLITRLLDTRLVRH--NISRWESFDVSPAVL 219 BMP2 VLGA-ITNNSSGNHRINIYEVIKPSVSSMEPITRLLDTRLVHH--SQSKWESFDVSPAVM 653 . : * :*:*:*.. :* :*:::**: : . ****.:* ::

BMP16 TQSSG---LLVFVLEAVPLNTSSRQPREHLRVRRAPKQD-DPSWAQERPLLVTYSHDGRG 259 BMP4 RWTQDRLPNHGLAVEVLHLNHTTSQQGRHVRVSRSLHQVPGEDWAQLRPLLVTFGHDGKG 279 BMP2 RWTMEGLTNHGFVVEVVHLDNERSDSKRHVRISRSLHED-EETWPQIRPLLVTFSHDGKG 712 : :.:*.: *: : .*:*: *: :: * * ******:.***:*

BMP16 QSLKLRPPTITSTTLGREGRTRGPGRDWRYGRRQGRERRVKRNGRNHKLKKLAKARCKRH 319 BMP4 HPLTRRVKRG------TKPR-GRKRNRNCRRH 304 BMP2 HVLHKREKRQ------ARSKQKRKHKSSCRRH 738 : * * . : : : *:**

BMP16 PLYVDFKDVGWNKWIVAPSGYHAFFCLGECRFPLTDHMNSSSHAMVQTLVNSVNGKVPRA 379 BMP4 ALYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNTNIPKA 364 BMP2 ALYVDFSDVGWNDWIVAPPGYHAFYCQGECPFPLADHLNSTNHAIVQTLVNSVNTNIPKA 798 *****.*****.***** **:**:* *:* ***:**:**:.**:********* ::*:*

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BMP16 CCVPTSLSPIAMLYLDQHDRVVLKNYQDMVVEGCGCR 416 BMP4 CCVPTELSAISMLYLDEHDKVVLKNYQEMVVEGCGCR 401 BMP2 CCVPTELSAISLLYLDEYEKVILKNYQDMAVEGCGCR 835 *****.** *::****::::*:*****:*.*******

Appendix Figure A1.1: Bowfin Bmp16 epitope for custom Bmp16 antibody design. The alignment of bowfin Bmp16, Bmp4, and Bmp2 proteins shows conservation between the protein sequences only at the C-terminal. The Bmp16 epitope (highlighted in yellow) used to design the Bmp16 antibody is in a region of the protein that is distinct from Bmp4 and Bmp2. Therefore, the bowfin Bmp16 antibody should not cross-react with either Bmp4 or Bmp2.

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Appendix Figure A1.2: Bmp4 expression in bowfin and zebrafish: For each column, top panels show Bmp4 expression alone in bright red, and bottom panels show Bmp4 expression in bright red overlain with DAPI in blue. All panels are transverse sections. a) Bmp4 expression in bowfin at stage 26 in the foregut (fg), anterior to the gas bladder bud. Bmp4 is expressed throughout the foregut anterior to the site of gas bladder budding. b) Bmp4 expression in the gas bladder (GB) of bowfin at stage 26, right after budding. At the site of budding, Bmp4 expression is absent from the foregut and the gas bladder bud. c) Bmp4 expression in zebrafish at 3 dpf. Bmp4 is not expressed in the foregut or the gas bladder but is expressed in the inner ear (e) and fin (fn). Abbreviations: b, brain; n, notochord.

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Appendix Figure A1.3: Bmp16 Immunofluorescence negative control in mouse. For each column, top panels show Bmp16 staining alone in bright red. Middle panels show Bmp16 expression in red overlain with autofluorescence in bright green. Bottom panels show Bmp16 expression in red overlain with DAPI staining of the nuclei in blue. All panels are transverse sections of mouse at E12.5. a) Bmp16 expression in an anterior region of the esophagus (e) and trachea (t). b) Bmp16 expression in a posterior region of the esophagus and trachea. The absence of bright red staining illustrates the absence of Bmp16 expression in mouse tissues, as expected given that the Bmp16 gene is absent in mammalian genomes. The absence of bright red staining also indicates that the bowfin specific anti-Bmp16 antibody is not cross reacting with Bmp4 or Bmp2. Abbreviations: e, esophagus; t, trachea; gr, laryngo- tracheal groove.

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Appendix Figure A2.1: Bmp4 and Bmp16 expression during gasbladder development in bowfin. A) Boxplot of average Bmp4 expression at pre-budding (24), budding (25), and outgrowth (27) stages of gas bladder development. Bmp4 is expressed at all stages. During outgrowth, Bmp4 is expressed strongest dorsally, in both endoderm and mesoderm. B) Boxplot of average Bmp16 expression at all stages. No or weak expression of Bmp16 is detected at all stages in all tissue regions. Abbreviations: dE, dorsal endoderm; dM, dorsal mesoderm; vE, ventral endoderm; vM, ventral mesoderm.

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Appendix 3.1 Remarks on the Question of the Origin of the Lungs (Griel, 1905, p.628-632) Excerpt of text translated from German to English by Eda Birol.

This insight also makes the so-often discussed relationships of the lungs to the gas bladder of fishes more comprehensible. At this point, I do not want to go into more detail about the hypotheses set out above, but instead use a specific case, the gas bladder of Amia calva, for comparison. I choose this form because on the one hand, it is well known, and on the other hand, the purely dorsal location of this structure, in comparison with the ventrally located lungs, has hitherto caused great difficulties. After the excellent illustrations of the Piper models, I have made some sketches (Fig. 5 Ia-IVa), which should show how the findings of Piper are to be interpreted. Piper sums up his own findings: “Amia gas bladder is formed from a dorsal, long epithelial fold of the dorsal esophagus and stomach wall; it extends in a caudal-cranial direction from (Last sentence of p. 628) and grows into a wide bag, whose lumen communicates with that of the esophagus through a short, cranial (anterior), longitudinal canal.” We want to start from a stage in the recapitulation of Piper’s findings, which Piper does not depict but clearly describes. At this stage, the endoderm tract is still stretched in the craniocaudal direction and is thus arranged bilaterally symmetrically. This primitive behavior is illustrated by sketch (section?) Ia, which lies between the gill and the anterior intestine. (In this and following figures, the median plane is indicated and the dorsomedian zone of the intestinal wall is shown as dotted line, the intestine is shown from the dorsal side.) Now, there is a shift of the front intestinal opening to the left (Fig. 5, IIa). Therefore, the middle part of the endodermal (gastrointestinal) tract, the stomach and duodenum primordium, bends to the left. Piper does not give any details as to the cause of this development. However, in my opinion, there can be no doubt that this phenomenon is caused by the fact that the intestine is forced, as it grows for a long time by the powerful yolk, to wind itself against its surface, that is, in the frontal plane, while the original dorsomedian zone of the intestinal wall deviates to the left (Sections Ib, IIb). In a third phase (Shema IIa and IIb, After Piper’s Figure 1, Table 1), this expansion or prolongation of the intestinal tract in question has increased considerably. The area of the anterior intestinal connection appears separated from the median plane by a considerable gap. The stomach begins to bulge caudally. The former right margin of this intestinal tract now crosses the median plane, moving from the right and cranial sides to the left and caudally. At the same time, the approach of the mesentery of the stomach has shifted from its dorsomedian zone towards its right border, which now appears connected to the posterior body wall by a dorsal mesentery (Shema III). In older embryos (stage 5 Piper), this process is further progressed (Fig 13). The gastro-duodenal loop now extends far to the left of the anterior intestinal aperture; the region of the pylorus is located completely off the median plane. The discharge of the stomach has increased in volume. A longitudinal fold has now appeared on the right wall of the stomach, which spreads over against the dorsal wall of the esophagus. This fold is the first primordium of the gas bladder (S), the right wall of the stomach. It then constricts itself from the stomach on the caudal side (at the place marked with a * in the picture and remains in contact with the wall of the

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esophagus only in its anterior section. – However, this fold lies almost exactly median and grows into the dorsal mesentery of the stomach and esophagus. This impression is particularly noticeable when the stomach has become free after the partial absorption of the yolk and has folded down (vgl. Schema IV c). However, according to what has been said, this behavior is to be regarded as secondary; it was brought about by the fact that the right wall of the stomach, as a result of the bend, respectively, extension of this intestinal section is moved to the left in the region of the median plane and at the same time, the point of attachment of the mesentery was shifted against the right margin of the stomach. The original, dorsomedian zone of the intestinal wall is thus to the left and front of the gas bladder primordium, which actually arises on the right wall of the esophagus and the stomach. With these findings, the conditions, which have resulted in the lung primordium (e.g. Bombinator), can be settled informally and without difficulty. In fact, even in Bombinator, the expansion and elongation of the intestinal tracts in question would have been impeded by similar obstacle in the sagittal plane, as in Amia, and the intestine is forced to develop and extend in a frontal plane, so Bombinator’s corresponding right lung primordium would have been quite similar to the gas bladder of Amia. Extremely important is the fact that the lung primordia are cut off from the intestine in much the same way as the gas bladder fold. Quite similar conditions as with Amia can be found, as is apparent from the investigations of Moser and others, in most Teleosts with gas bladders, e.g. Rhodeus and Cyprinus, except that in these forms, the gas bladder is not created as a fold but as a diverticulum of the intestinal wall. It is interesting that with other teleosts (e.g. Salmo trutta), the deflection of the intestine does not seem to take place to the left, as in Amia, but to the right side, and accordingly, the gas bladder does not originate on the right but on the left side of the intestine. After all, it also comes to lie dorsally in these forms, except, as with the Erythrinus, it remains on the left side. This unilateral formation of the gas bladder may be related to the fact that the liver and the ventral pancreas are on the opposite side of the system and therefore, the gas bladder appears to be hindered in its formation. It seems the primordium has been suppressed on this side. One would presume that then at least rudimentary such formations would be found, but there are no observations on this, they have not paid much attention to them so far. – Unfortunately, the paired gas bladder of Polypterus, as well as the lungs of Lepidosiren and Protopterus have not yet been investigated evolutionarily in detail. Specifically, Polypterus has a more original behavior with respect to the presence of paired gas bladders than forms with only one gas bladder. The ventral connection of the Polypterus gas bladder, that is, the formation of the ventrally-mounted aisle, is, however, decidedly conceived as a secondary process; because also with the lungs, the bilaterally symmetrical primordium is primary and the ventral connection secondary. Special attention should be paid to the findings that Semon and Neumayer have recently made about Ceratodus. In this form, the lungs or gas bladder emerge as a blind-ended pocket on the ventral wall of the intestine just to the right of the median plane. Accordingly, the lung of Ceratodus is to be regarded as an organ of the right half of the body, which appears suppressed on the left side. It is very understandable that Neumayer has searched unsuccessfully for a ventral-median groove of the intestinal wall as the first primordium of the lung. According to our current views, the

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first primordium of the gas bladder or the lungs are not to be found on the dorsal nor the ventral side, respectively, but on the lateral side of the intestine. As with the gas bladder of teleosts, so do the lungs of Ceratodus originate as a simple diverticulum of the lateral intestinal wall. The comparative history of development thus shows that the gas bladder and the lungs arise at corresponding locations and in essentially the same way, and even in their further configuration they often follow the same paths, and we can therefore conclude with all justification that these structures are common in origin and homologous organs.

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