Amphioxus Immunity: Tracing the Origins of Human Immunity

Amphioxus Immunity

Tracing the Origins of Human Immunity

Edited by

Anlong Xu

Sun Yat-sen University, Guangzhou, China

Table of Contents

Cover

Title page

Copyright

Dedication

List of Contributors

About the Author

Foreword

Preface

Chapter 1: Amphioxus as a Model for Understanding the Evolution of Vertebrates

Abstract

1.1. Brief introduction of amphioxus

1.2. Biology of amphioxus

1.3. The story of amphioxus and early research studies in China

Chapter 2: Basic Knowledge of Immunology

Abstract

2.1. Immune organs

2.2. Immune cells

2.3. Innate immunity

2.4. Adaptive immunity

2.5. Complement system

Chapter 3: Immune Organs and Cells of Amphioxus

Abstract

3.1. Introduction

3.2. Organs of the amphioxus immune system

3.3. Cells of the amphioxus immune system

Chapter 4: Genomic and Transcriptomic View of Amphioxus Immunity

Abstract

4.1. Introduction

4.2. Trace evidence of adaptive immunity

4.3. Extraordinary gene expansion in amphioxus innate immunity

4.4. Regulation of the amphioxus immune system

4.5. Discussion

Chapter 5: Pattern Recognition System in Amphioxus

Abstract

5.1. TLR signaling pathway in amphioxus

5.2. Innate antiviral immunity in amphioxus

5.3. NLR signaling pathway

Chapter 6: Transcription Factors in Amphioxus

Abstract

6.1. NF-κB family members in amphioxus

6.2. The interferon regulatory factor family in amphioxus

6.3. The STATs in amphioxus

Chapter 7: The Complement System of Amphioxus

Abstract

7.1. Introduction

7.2. Tracing the evolutionary origin of the complement system

7.3. The complement system of amphioxus

Chapter 8: The Oxidative Burst System in Amphioxus

Abstract

8.1. Introduction

8.2. NADPH oxidase 2 and the other NOX families

8.3. NOX subunits and regulatory proteins

8.4. Functions of ROS generated by NADPH oxidases

8.5. Phagocytic respiratory burst (PRB) appears to couple with phagocytosis in the amphioxus gut epithelial lining cells

Chapter 9: Immune Effectors in Amphioxus

Abstract

9.1. Galectin

9.2. C-type lectin

9.3. Peptidoglycan recognition protein

9.4. Gram-negative bacteria-binding protein

9.5. Chitin-binding protein

9.6. Apextrin-like protein

9.7. Bactericidal/permeability-increasing protein

9.8. Other effector genes

Chapter 10: Evolution of Inflammation-Related Lipid Metabolism Pathway and Inflammatory Lipid Signaling in Amphioxus

Abstract

10.1. Introduction

10.2. The classification, structure, and function of fatty acids and eicosanoids

10.3. Origin of vertebrate eicosanoid production

Chapter 11: Apoptosis in Amphioxus

Abstract

11.1. Apoptosis in mammals

11.2. Apoptosis-related gene families in amphioxus

Chapter 12: Primitive Adaptive Immune System of Amphioxus

Abstract

12.1. Evidence of the emergence of adaptive immunity in amphioxus

12.2. The primary building blocks for MHC and Rag protein

12.3. Alternative forms of variable immune receptors

12.4. Closing remark

Chapter 13: Future Research Directions

Abstract

13.1. Ubiquitination of immune signaling in amphioxus

13.2. The microRNAs in amphioxus

13.3. Alternative 3’ UTRs in the amphioxus immune response

Abbreviation Index

Subject Index

Copyright

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Dedication

This book is dedicated to my PhD mentor Dr Harris Lewin and postdoctoral mentor Dr Helen M. Ranney.

List of Contributors

Xingsheng Cai,     South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China

Xiangru Dong,     Thermo Fisher Scientific, Shanghai, China

Yuchao Feng,     Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, China

Guangrui Huang,     Beijing University of Chinese Medicine, Beijing, China

Shengfeng Huang,     Sun Yat-sen University, Guangzhou, China

Jun Li,     Liaocheng University, Shandong, China

Rui Li,     Beijing Sinobioway Group Co., Ltd., Beijing, China

Yuxin Li,     Sun Yat-sen University, Guangzhou, China

Jian Peng,     Guizhou Medical University, Guizhou, China

Jie Ruan,     Sun Yat-sen University, Guangzhou, China

Xin Tao,     Sun Yat-sen University, Guangzhou, China

Ruihua Wang,     Guangdong Provincial Hospital of Traditional Chinese Medicine

Anlong Xu,     Sun Yat-sen University, Guangzhou, China

Ping Yang,     GenStar Biosolutions Co., Ltd., Beijing, China

Rirong Yang,     Guangxi Medical University, Guangxi, China

Dongjuan Yuan,     Sun Yat-sen University, Guangzhou, China

Shaochun Yuan,     Sun Yat-sen University, Guangzhou, China

Jie Zhang,     DiaSys Diagnostic Systems Greater China, Shanghai, China

Tingting Zheng,     Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

About the Author

Anlong Xu was awarded a government scholarship to study in the United States after his BS degree in Biology from Zhongshan University in 1985. He attended the University of Illinois at Urbana-Champaign (UIUC) in September 1986 to pursue graduate studies in immunogenetics under Dr Harris Lewin’s (member of the US National Academy of Science) supervision, and he obtained his PhD from UIUC in 1992. Dr Xu then did his postdoctoral research for 2 years under the supervision of Dr Helen M. Ranney, a professor in Medicine at the University of California, San Diego, and member of the US National Academy of Science and Institute of Medicine. Dr Xu worked at the San Diego–based Alliance Pharmaceutical Corporation from 1994 to 1996 researching new drugs. In 1996, after 10 years of study and research work in the United States, he joined the faculty of the Department of Biochemistry at the College of Life Sciences, Sun Yat-sen (Zhongshan) University, his alma mater. He served as Director of the State Key Laboratory of Biocontrol and Director of the National Engineering Center for Marine Biotechnology, South China Sea. Dr Xu was appointed as Vice President for Research and Development at Sun Yat-sen University in 2008 after serving as Dean of the College of Life Sciences for 8 years. He currently is a professor of Molecular Biology and Immunology and President of Beijing University of Chinese Medicine.

Dr Xu has published more than 150 papers in peer-reviewed international journals, such as the New England Journal of Medicine, Nature, Nature Immunology, Nature Communications, Science Signaling, Genome Research, Journal of Human Genetics, Proceedings of the National Academy of Sciences, Cell Research, Journal of Immunology, International Society of Microbial Ecology Journal, Journal of Biological Chemistry, Free Radical Biology and Medicine, European Journal of Immunology, Molecular Immunology Developmental and Comparative Immunology, Human Immunology, Immunogenetics, and Fish and Shellfish Immunology.

Dr Xu is currently a member of the American Association of Immunologists, American Society for Biochemistry and Molecular Biology, American Association for the Advancement of Science, Chinese Society of Biochemistry and Molecular Biology and Chinese Society of Immunology. He is currently President of the Guangdong Society of Biochemistry and Molecular Biology and Vice President of International Society for Developmental and Comparative Immunology. He currently serves on the Editorial Board for the following international journals: Annual Review of Animal Biosciences, BMC Genomics, and Animal Biotechnology. His main research focus is the study of the origin and evolution of the vertebrate immune system.

Foreword

In this book, Amphioxus Immunity, Anlong Xu covers a remarkable range of information about this small fishlike organism and its evolutionary context. This compilation of what is currently known about amphioxus, with a sharp focus on its immune system, is especially timely for many reasons. The recent availability of the amphioxus genomic sequence and its comparison with other metazoan genomes firmly establishes the phylogenetic position of amphioxus as the representative head of the chordates. The new phylogeny tree thus places amphioxus basal to the sister chordate lineages, tunicates, and vertebrates.

Amphioxus has single copies of the ancestral genes that originated through two rounds of genome-wide duplication to up to four identifiable paralogous genes in jawed vertebrates, including humans. There is still debate about whether the first round of genome-wide duplication occurred before or after divergence of the jawless and jawed vertebrate lineages. Nevertheless, the resultant gene redundancy undoubtedly facilitated the evolutionary selection of new gene functions and more complicated biological systems, including those devoted to immune defense.

The competitive struggle for survival during the evolution of living forms on our planet inevitably led to the development of a wide variety of mechanisms for recognizing and repelling invasion by neighboring organisms. The diversity and complexity of the immune defense systems that have been recognized in bacteria, plants, and animals defy simple classification, but they can be broadly categorized into innate and adaptive immune systems. An important distinction between the two types of immunity is that for innate immunity, the genes encoding recognition elements are inherited in a ready to use form by each individual organism, whereas for adaptive immunity, the genes for the recognition receptors are inherited in pieces that undergo combinatorial assembly during the differentiation of specialized lymphoid cells. This combinatorial assembly strategy results in the generation of a very large repertoire of clonally diverse lymphocytes, each of which has its own unique receptor as the basis for recognition and response to a specific pathogen. Members of the lymphocyte population are thus available to recognize and respond to specific pathogens at their first encounter and give rise to long-lived progeny to provide specific immunological memory.

The diversity and constraints of presently known mechanisms for innate immunity is described by Dr Xu, who also traces the evolution of the central genetic elements used for these heritable defense systems. The unifying theme for innate immunity is the use of germline-encoded pattern recognition receptors that can recognize molecular patterns shared by many potential pathogens. The expression of a limited spectrum of cell surface and intracellular pattern recognition receptors can thus be used to sense a wide variety of potential pathogens and trigger cell-signaling cascades that lead to the activation of genes responsible for effective defense responses.

Innate immune systems are universal, but adaptive immune systems based on clonally diverse lymphocytes have been defined only in vertebrates. Surprisingly, jawed and jawless vertebrates use very different genes to encode their antigen-specific receptors, although the lymphocytes that express them are very similar. In jawed vertebrates, combinatorial assembly of different immunoglobulin variable (V), diversity (D), and joining (J) gene segments during lymphocyte differentiation in the thymus or hematopoietic tissues results in the generation of highly diverse receptor repertoires for T and B lymphocytes. In jawless vertebrates, the combinatorial assembly of different leucine-rich-repeat (LRR) gene segments to complete variable lymphocyte receptor (VLR) genes during lymphocyte differentiation in thymus-equivalent or hematopoietic tissues results in the generation of clonally diverse T- and B-like lymphocytes. The similarity between these lymphocyte differentiation pathways in both vertebrate lineages suggests that bifurcated lymphocyte differentiation evolved in a common vertebrate ancestor, before different primordial genes were co-opted for modification to serve antigen recognition purposes in the alternative adaptive immune systems of jawless and jawed vertebrates.

In this context, amphioxus is not only the best available model to gain insight into the beginnings of vertebrate evolution, but it also provides a pivotal representative for the study of how an adaptive immune system might have gradually emerged. Obsessed with the daunting challenge of gaining insight into the evolutionary puzzle of how an adaptive immune system evolved to augment pre-existing mechanisms for innate immunity, Xu and his colleagues have sifted through the genomic sequences of two amphioxus species, Branchiostoma floridae and Branchiostoma belcheri, which they sequenced, to find an abundance of genes used in innate immunity. Although this exhaustive search does not reveal orthologous genes for those used in the combinatorial generation of antigen-specific receptors in vertebrates, ancestral gene candidates were found for immunoglobulin receptors and for many of the signaling elements used for activation of vertebrate lymphocytes. Most tantalizing is their identification of lymphocyte-like cells in the gill and intestinal regions. These lymphocyte-like cells may be immobile and, indeed, blood in the amphioxus circulatory system is acellular. Nevertheless, the tissue-based lymphocytes of amphioxus respond to bacterial pathogens with an increase in size and the upregulation of several genes characteristically used in vertebrate lymphocyte differentiation.

There are many interesting evolutionary principles yet to be learned from studies in the amphioxus model, not least among them being the unfinished story about how lymphocytes and their functions evolved. Amphioxus Immunity is filled with information that will be useful for anyone who wishes to learn more about the origin of vertebrates and adaptive immunity.

Max D. Cooper MD

Georgia Research Alliance Eminent Scholar, Professor of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA

Preface

I came to know the word immunity in Chinese for the first time when I was given a shot of vaccine during my childhood in my hometown, a small town called Poyang Town, named after the largest freshwater lake of China, Poyang Lake, near the middle of Yangtze River, in Jiangxi province, China. After elementary school, middle school, and high school in Poyang, I was admitted to Sun Yat-sen University (SYSU), my first alma mater. At university, I had a chance to learn the basic terms of immunology, such as antibody and antigen, with a major in biology. After graduating from SYSU, I was awarded a government scholarship to study in the United States in 1985, and was eventually admitted to the PhD program of Immunogenetics at the University of Illinois at Urbana-Champaign (UIUC) in 1986. Under Dr Harris Lewin’s supervision, I became fascinated by the complexity and diversity of animal and human immunity, which prompted me to question how this immunity originated and how it was formed during the evolutionary process. In addition to my scientific curiosity, my way of thinking about scientific questions and conducting scientific experiments was completely established by my PhD mentor’s hard training, which has influenced my scientific career ever since. After graduating from UIUC, my second alma mater, with both MS and PhD degrees, I was fortunate to work with a world-famous physician scientist, Dr Helen M. Ranney, a professor in the Department of Medicine, University of California San Diego (UCSD), in 1992 as her last postdoctoral student. I then went with Dr Ranney to work at a San Diego–based pharmaceutical company, Alliance Pharmaceutical Corporation, after completing my postdoctoral research. Dr Ranney has also had a great influence on my scientific career, like Dr Lewin. In 1996, in consultation with these two important persons in my scientific career, I decided to come back to China to join the faculty of the Department of Biochemistry, College of Life Sciences.

The first thing I decided to do for my scientific research after returning to China was to start something new that could use my scientific training in the United States and explore new avenues in the field of immune research. I had many ideas about my new research direction, and one of the most important for me was to understand the origin and evolution of human immunity. However, I had no idea about which model species to use in pursuit of this very important goal. It took me 1 year to figure out that amphioxus was the model organism to address my question best, and another 2 years to establish a laboratory-based aquaculture system of amphioxus and the infection model for understanding the host immune response to infection. This made my laboratory a global pioneer in the study of amphioxus immunity, although this species had been an iconic model for evolutionary biology for more than 200 years. Instead of using conventional immunological methods only, my laboratory combined traditional immune methods with cellular, biochemical, and molecular approaches, particularly a genomic approach, to conduct a comprehensive survey on the immune response of amphioxus to bacterial infection from the beginning. This gave us a quick opening for this brand new field. For last 15 years, my laboratory has contributed more than 40 papers related to amphioxus immunity. In summary, our contributions to the field of amphioxus immunity, with the aim of understanding the origin and evolution of vertebrate immunity, especially human immunity, can be briefly described as follows:

1. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity, suggesting that our chordate ancestors had a remarkably elaborate innate immune system, but this system was somehow reduced in the vertebrate lineage. This finding provides obvious evidence for the so-called immunological big bang to explain the origin of vertebrate immunity.

2. Functional analyses of important innate immune genes in amphioxus involved in two basic forms of innate immune signaling, TLR and TNF signaling, suggest that the basic frameworks for these two signaling pathways were established at the basal chordate, which have laid the foundation for the eventual formation of these two pathways in vertebrates.

3. Identification of lymphocyte-like cells, along with related transcription factors and signaling molecules for lymphoid proliferation and differentiation, indicates the emergence of adaptive immunity for vertebrates along with some basic components for adaptive immunity. The finding of an extrinsic apoptosis pathway in amphioxus further substantiates the claim for the beginning of adaptive immunity in the basal chordate, because the extrinsic apoptosis pathway is generally believed to have coevolved with adaptive immunity.

4. In addition to tracing the origin of the existing system for immune response and regulation in vertebrates, including humans, we may be able to reveal a novel mechanism involved in immune regulation, which has never been described in other organisms by studying this animal model. It includes finding novel molecules for immune recognition and novel mechanisms for immune regulation epigenetically by alternative 3’ UTRs.

5. In the immune signaling system, most proteins have characteristic and conserved multiple domains that exert specific functions in proteins so as to interact with specific molecular partners. Our studies on amphioxus immune signaling molecules indicate that different combinations of these domains (e.g., CARD, TIR, DFD, DEATH) are the evolutionary sources for the generation of new signaling molecules that can result in interaction specificity. Understanding the evolutionary mechanisms of these domains and their shuffling for the generation of new proteins, with new functions, should provide a novel vista for synthetic biology and insights that may help in the treatment of diseases associated with mutated protein activity.

Finally, I would like to thank all my students who have made significant contributions to our understanding of amphioxus immunity. I would not have come this far without their diligent and intelligent work on this research. I would also like to thank Professors Xu Xun of the Third Institute of Oceanography, State Oceanic Administration of China, Zhang Shicui of Ocean University of China, Zhang Hongwei of Shandong University, Zhang Peijun of Institute of Oceanology, Chinese Academy of Sciences (CAS), Wang Yiquan of Xiamen University, Chen Junyuan of Nanjing Institute of Geology and Palaeontology, CAS, Gao Fu of the Institute of Microbiology, CAS, Peng Xuanxian of Sun Yat-sen University, and Liu Xiaolong of Shanghai Institutes for Biological Sciences, CAS, for their scientific comments and academic discussions on my research. I would also like to thank Professors Linda and Nick Holland of the Institute of Oceanography, University of California, San Diego, and Hector Escriva of CNRS UMR, UPMC University of Paris, Banyuls, France, for their academic communication and exchanges about my research. In particular, I would like to thank Professor Max Cooper of Emory University School of Medicine, Atlanta, GA, USA, for his long-time enthusiastic support to my research; his important foreword to my book is deeply appreciated.

Anlong Xu

Sun Yat-sen University, Guangzhou, China

Chapter 1

Amphioxus as a Model for Understanding the Evolution of Vertebrates

Yuchao Feng*

Jun Li**

Anlong Xu†

*    Guangdong Provincial Hospital of Traditional Chinese Medicine, South China Sea Institute of Oceanology, Chinese Academy of Sciences

**    Liaocheng University

†    Sun Yat-sen University, Guangzhou, China

Abstract

This chapter introduces the biology of amphioxus and discusses the research history of this living fossil. Amphioxus was first discovered in Europe as a lancelet. Taxonomically, it belongs to the subphylum Cephalochordata, which means chordate in the head. The fossil evidence, as well as the modern molecular phylogenetic tree, suggests amphioxus as the most basal chordate. The body plan and embryonic development of amphioxus are close to its vertebrate ancestors. It was used as a model animal for developmental studies since the nineteenth century. The famous Chinese zoologist Tung Tichou made many contributions to the developmental research of amphioxus. Amphioxus can now be used as a model for the study of the origin of vertebrate immunity.

Keywords

protochordate

lancelet

Wenchang fish

living fossil

Tung Tichou

Contents

1.1 Brief introduction of amphioxus 1

1.2 Biology of amphioxus 3

1.2.1 Reproduction and embryogenesis of amphioxus 4

1.2.2 Anatomy of amphioxus 6

1.3 The story of amphioxus and early research studies in China 10

References 12

1.1. Brief introduction of amphioxus

The amphioxus was first described by Peter Simon Pallas (1774). Also, the Russian zoologist Alexander Kowalevsky (1867) did extensive pioneering work on amphioxus embryology. He noted that the late embryonic development of amphioxus has many features of vertebrates, such as notochords. This discovery supported the concept that protochordates were the closest living relatives of vertebrates.

The fossils of invertebrate chordates give additional support of amphioxus as the model for ancestral vertebrates (Holland et al., 2004). Haikouella, a 530-million-year-old fossil found in Haikou, Yunnan province, China (Mallatt and Chen, 2003), is known for the anatomy of protochordate and vertebrate features. It possesses the typical features of protochordate amphioxus, such as a notochord, pharynx, and dorsal nerve cord (Figure 1.1). But, more importantly, Haikouella seems to have a dorsal ventral aorta, heart, and neural crest (Chen, 2008). This indicates that amphioxus has the primitive body plan of ancestral vertebrates and is a living fossil close to the origin of vertebrates.

Figure 1.1   Comparison of Reconstructed Haikouella and Amphioxus.

(A) Showing the feature of Haikouella, which possesses similar structure such as dorsal nerve cord, notochord, postanal tail, which is also in (B) amphioxus. This indicates that the amphioxus is the most primitive chordates and has the features of common vertebrates ancestors. Adapted from Holland et al. (2004).

Amphioxus has long been regarded a key phylogenetic model animal in the research on the origin of vertebrates. Molecular phylogenetic analyses (Figure 1.2) have placed cephalochordates as the most basal chordate group (Blair and Hedges, 2005; Delsuc et al., 2006; Vienne and Pontarotti, 2006; Putnam et al., 2008). Unlike the much-reduced metamorphosis of the tunicates, cephalochordates have maintained their basic body organization for several hundred million years. Therefore, studies on cephalochordates can provide important insights into the understanding of the conserved patterning mechanisms for all chordates (Schubert et al., 2006).

Figure 1.2   The Phylogenetic Tree of Amphioxus.

This indicates that amphioxus is the most basal root of all chordate. Adapted from Putnam et al. (2008).

There are three cephalochordate genera: Branchiostoma, Asymmetron, and Epigonichthys (Nishikawa, 2004; Kon et al., 2007; Zhong et al., 2009). Currently, 32 amphioxus species have been found – 24 Branchiostoma species, 7 Asymmetron species, and 1 Epigonichthys species (Holland and Holland, 2010). The two genera, Epigonichthys and Asymmetron, differ from Branchiostoma in having gonads only on the right side.

In some areas, amphioxus populations can be quite large. Population density as high as several thousand individuals per square meter has been recorded for Branchiostoma floridae. It is even formed in fisheries when they were commercially harvested in Xiamen, China, before the 1950s. However, when it reached the industrial era, pollution and the exploitation of sea sand destroyed the harmonic living environment for the largest populations of amphioxus; thus, amphioxus has been considered as endangered since the 1980s (Zhou et al., 2005).

Amphioxus is mainly found in shallow tropical, subtropical, and temperate sand flats, where they burrow in clean gravel or sand (not silty sediments), with just the head exposed (Figure 1.3). Cephalochordates are passive feeders. When disturbed, amphioxus leaves its burrow, swims quickly for a short distance, and then rapidly burrows again, with its posterior end in the substrate.

Figure 1.3   Adult Amphioxus in the Substrate.

Using their special tentacles around the oral cavity, they filter small particles of planktons and diatoms as food. Water is driven into the mouth and pharynx and out through the pharyngeal gill slits into the surrounding atrium and exits the body through the ventral atriopore.

1.2. Biology of amphioxus

Amphioxus is an amazing animal and possesses a notochord, dorsal tubelike nerve cord, pharyngeal gill slits, postanal tail, and segmented somites, which are assumed to be present only in the common chordate ancestor. Its embryonic development has feature of invertebrates and vertebrates, as noted by Kowalevsky (1867). Thus, amphioxus is a helpful model for understanding the origin of vertebrates. However, detailed information about amphioxus biology will not be described here because it has been featured in numerous books and articles. Here, we will emphasize the embryonic development and anatomic features that indicate the role of amphioxus as a model for understanding the origin of vertebrates.

1.2.1. Reproduction and embryogenesis of amphioxus

Generally, cephalochordates are separate sexes (gonochoric) and have great reproductive ability. The females develop yellow gonads, whereas the males have pale white gonads (Figure 1.4). In the review of amphioxus by Garcia-Fernandez and Benito-Gutierrez (2009), the reproduction and development of amphioxus have been well described. Spawning of amphioxus occurs around or after sunset. Millions of eggs and sperm are released into the water flowing out of the atrium, and fertilization is external. After fertilization, the development of amphioxus zygotes proceeds through six stages – morula, blastula, gastrula, neural, larva, and adult (Figure 1.5).

Figure 1.4   The Morphism of two Sexes.

Upper one is a male, lower one is a female.

Figure 1.5   Embryonic Development of Amphioxus.

This shows the developmental stages of blastula, gastrula, and neurula. Adapted from Garcia-Fernandez and Benito-Gutierrez (2009).

The blastulation phase continues with enlargement of the blastocele and formation of a spherical zygote. The gastrulation phase starts with spherical zygote invagination at its vegetal pole, where it invades the blastocele and gives rise to the archenteron. The opening of the archenteron is a blastopore in the rear end of the embryo. From then on, the inner archenteron begins to rotate anticlockwise. However, instead of the autonomous development of ascidian embryos, there is a distinctly different organizing center that coordinates the development of the animal and vegetal poles in a vertebrate-like manner. This organizing center has been observed by Tung et al. (1965) and been verified by molecular data (Yu et al., 2007). The neurulation of amphioxus, similar to that of vertebrates (Figure 1.5), begins elongation in the dorsal region and forms a flat dorsal; later, the bottom of this dorsal flat becomes the neural plate. The neural tube is formed by the fusion of overgrowing epidermal tissue. The notochord and mesoderm develop from the underlying chordomesodermal plate, parallel to the neural plate. It has three separate parts – one central section, giving rise to the notochord primordia, and two lateral sections, giving rise to the first somite or future myomeres. The ventral epithelium of the archenteron develops into the digestive tract, pharynx, and other endodermal derivatives (Garcia-Fernandez and Benito-Gutierrez, 2009). Finally, the embryo hatches at the end of the neurula.

The time of development varies according to the species and environment (Garcia-Fernandez and Benito-Gutierrez, 2009). Amphioxus can have a lifespan of about 4–6 years, but differs among different species (Wu et al., 1995; Escriva et al., 2007). After fertilization, the embryo needs dozens of hours to develop to a ciliated neurula before hatching, about 10 h for B. floridae (Stokes and Holland, 1995). Then there is a free swim stage, where larvae begin their life as plankton. At the end of this stage, the larvae undergo metamorphosis and lose the epidermal cilia, gain a second row of gill slits, and transform into juveniles, which burrow into the substrate and slowly develop into adults (Figure 1.6).

Figure 1.6   Amphioxus Life Cycle and Reproduction. Adapted from Garcia-Fernandez and Benito-Gutierrez (2009).

1.2.2. Anatomy of amphioxus

The amphioxus is a slender, small, fishlike animal that is generally 3–6 cm in length, although some reach nearly 10 cm. Its slender body is very smooth and muscular, with a spindle-shaped anterior and a lancelet-shaped posterior, from which the specific name of Amphioxus lanceolatus is derived, meaning that both ends are sharp. Although it lacks an elaborate head or forebrain or primary sense organs, such as eyes, ears, and nose, which characterize most vertebrates, it still amazed biologists by having typical features such as a notochord, dorsal tubelike nerve cord, pharyngeal gill slits, postanal tail, and segmented somites, which had been assumed only to be present in the common chordate ancestor. An anatomic diagram of amphioxus is shown in Figure 1.7A. The body plan of amphioxus can help us visualize the ancestral chordates.

Figure 1.7   Anatomic Diagram of Amphioxus and its Dissection View.

(A) External anatomy diagram; (B) internal dissection view of diagram including guts, notochord, neural tube, etc.

The body is generally divided into three axial parts, the head, trunk, and tail. The head, at the anterior end, is small and poorly defined. The rostrum extends anteriorly and overhangs the mouth and buccal cavity. The large mouth lies under the rostrum and opens into a spacious buccal cavity. The mouth is surrounded by a ring of tentacle-like buccal cirri (oral cirri). These are involved in the preliminary mechanical sorting of food particles and are probably also chemoreceptive. The roof and walls of the buccal cavity form the oral hood. The whole body of amphioxus exhibits wide-range fluorescence, which is strongest in the oral cirri, so the popular tag using fluorescein cannot easily be used for amphioxus.

Most of the body is the trunk, which extends posteriorly from the head to the anus. The trunk contains most of the gut, including the large conspicuous pharynx and the musculature. The segmental and asymmetric arrangement of the axial musculature (body musculature) is readily apparent through the translucent integument and is a common feature of vertebrate body plan.

There are no paired appendages but, on either side of the trunk, there is a ventrolateral metapleural fold. The atriopore is located on the midventral margin where the two metapleural folds join the ventral margin (see Figure 1.7A).

Further posteriorly, the anus is beside the caudal fin, which is the