Michael Schorpp

VLR-Based Adaptive Immunity

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

A thymus candidate in lampreys

Immunologists and evolutionary biologists have been debating the nature of the immune system of jawless vertebrates—lampreys and hagfish—since the nineteenth century. In the past 50 years, these fish were shown to have antibody-like responses and the capacity to reject allografts but were found to lack the immunoglobulin-based adaptive immune system of jawed vertebrates. Recent work has shown that lampreys have lymphocytes that instead express somatically diversified antigen receptors that contain leucine-rich-repeats, termed variable lymphocyte receptors (VLRs), and that the type of VLR expressed is specific to the lymphocyte lineage: T-like lymphocytes express type A VLR (VLRA) genes, and B-like lymphocytes express VLRB genes. These clonally diverse anticipatory antigen receptors are assembled from incomplete genomic fragments by gene conversion, which is thought to be initiated by either of two genes encoding cytosine deaminase, cytosine deaminase 1 (CDA1) in T-like cells and CDA2 in B-like cells. It is unknown whether jawless fish, like jawed vertebrates, have dedicated primary lymphoid organs, such as the thymus, where the development and selection of lymphocytes takes place. Here we identify discrete thymus-like lympho-epithelial structures, termed thymoids, in the tips of the gill filaments and the neighbouring secondary lamellae (both within the gill basket) of lamprey larvae. Only in the thymoids was expression of the orthologue of the gene encoding forkhead box N1 (FOXN1), a marker of the thymopoietic microenvironment in jawed vertebrates, accompanied by expression of CDA1 and VLRA. This expression pattern was unaffected by immunization of lampreys or by stimulation with a T-cell mitogen. Non-functional VLRA gene assemblies were found frequently in the thymoids but not elsewhere, further implicating the thymoid as the site of development of T-like cells in lampreys. These findings suggest that the similarities underlying the dual nature of the adaptive immune systems in the two sister groups of vertebrates extend to primary lymphoid organs.

Evolution of Genetic Networks Underlying the Emergence of Thymopoiesis in Vertebrates

About 500 million years ago, a new type of adaptive immune defense emerged in basal jawed vertebrates, accompanied by morphological innovations, including the thymus. Did these evolutionary novelties arise de novo or from elaboration of ancient genetic networks? We reconstructed the genetic changes underlying thymopoiesis by comparative genome and expression analyses in chordates and basal vertebrates. The derived models of genetic networks were experimentally verified in bony fishes. Ancestral networks defining circumscribed regions of the pharyngeal epithelium of jawless vertebrates expanded in cartilaginous fishes to incorporate novel genes, notably those encoding chemokines. Correspondingly, novel networks evolved in lymphocytes of jawed vertebrates to control the expression of additional chemokine receptors. These complementary changes enabled unprecedented Delta/Notch signaling between pharyngeal epithelium and lymphoid cells that was exploited for specification to the T cell lineage. Our results provide a framework elucidating the evolution of key features of the adaptive immune system in jawed vertebrates.

Evolutionary implications of a third lymphocyte lineage in lampreys

Jawed vertebrates (gnathostomes) and jawless vertebrates (cyclostomes) have different adaptive immune systems. Gnathostomes use T- and B-cell antigen receptors belonging to the immunoglobulin superfamily. Cyclostomes, the lampreys and hagfish, instead use leucine-rich repeat proteins to construct variable lymphocyte receptors (VLRs), two types of which, VLRA and VLRB, are reciprocally expressed by lymphocytes resembling gnathostome T and B cells. Here we define another lineage of T-cell-like lymphocytes that express the recently identified VLRC receptors. Both VLRC+ and VLRA+ lymphocytes express orthologues of genes that gnathostome γδ and αβ T cells use for their differentiation, undergo VLRC and VLRA assembly and repertoire diversification in the ‘thymoid’ gill region, and express their VLRs solely as cell-surface proteins. Our findings suggest that the genetic programmes for two primordial T-cell lineages and a prototypic B-cell lineage were already present in the last common vertebrate ancestor approximately 500 million years ago. We propose that functional specialization of distinct T-cell-like lineages was an ancient feature of a primordial immune system.

Conserved functions of Ikaros in vertebrate lymphocyte development: genetic evidence for distinct larval and adult phases of T cell development and two lineages of B cells in zebrafish.

Zebrafish has been advocated as an alternative animal model to study lymphocyte development, although the similarities in the genetic requirements of lymphopoiesis between fish and mammals have not yet been investigated. In this study, we examine the role of the transcription factor Ikaros in zebrafish lymphopoiesis. In fish larvae homozygous for an ikaros allele predicted to lack the C-terminal zinc fingers, T lymphopoiesis is absent; the presence of VHDμJμ rearrangements in adolescent fish is delayed in mutants. In adolescent mutant fish, T cells expressing tcrb and tcrd and B cells expressing igm are formed with low efficiency and display an oligoclonal Ag receptor repertoire. By contrast, B cells expressing the igz isotype do not develop, providing genetic evidence for two separate B cell lineages in zebrafish. Thus, Ikaros appears to play similar roles in fish and mammalian lymphopoiesis.

Essential role of c-myb in definitive hematopoiesis is evolutionarily conserved

The transcription factor c-myb has emerged as one of the key regulators of vertebrate hematopoiesis. In mice, it is dispensable for primitive stages of blood cell development but essentially required for definitive hematopoiesis. Using a conditional knock-out strategy, recent studies have indicated that c-myb is required for self-renewal of mouse hematopoietic stem cells. Here, we describe and characterize the c-myb mutant in a lower vertebrate, the zebrafish Danio rerio. The recessive loss-of-function allele of c-myb (c-mybt25127) was identified in a collection of N-ethyl-N-nitrosourea (ENU)-induced mutants exhibiting a failure of thymopoiesis. The sequence of the mutant allele predicts a missense mutation (I181N) in the middle of the DNA recognition helix of repeat 3 of the highly conserved DNA binding domain. In keeping with the findings in the mouse, primitive hematopoiesis is not affected in the c-myb mutant fish. By contrast, definitive hematopoiesis fails, resulting in the loss of all blood cells by day 20 of development. Thus, the mutant fish lack lymphocytes and other white and red blood cells; nonetheless, they survive for 2–3 mo but show stunted growth. Because the mutant fish survive into early adulthood, it was possible to directly show that their definitive hematopoiesis is permanently extinguished. Our results, therefore, suggest that the key role of c-myb in definitive hematopoiesis is similar to that in mammals and must have become established early in vertebrate evolution.

Forkhead/winged-helix transcription factor whn regulates hair keratin gene expression: Molecular analysis of the Nude skin phenotype

The molecular characteristics of the nude phenotype (alopecia and thymic aplasia) in humans and rodents are unknown. The nude locus encodes Whn, a transcription factor of the forkhead/winged‐helix class. Expression of Whn in HeLa cells induced expression of human hair keratin genes Ha3‐II and Hb5. Correspondingly, in nude mice, which are homozygous for a loss‐of‐function mutation of Whn, expression of mouse mHa3 and mHb5 hair keratin genes is severely reduced. Characterization of a previously identified nude allele, nuY, revealed a mis‐sense mutation (R320C) in the DNA binding domain of Whn. This mutant protein is unable to activate hair keratin gene expression in HeLa cells. When the Whn transcription factor was expressed in two parts, one containing the N‐terminal DNA binding domain and the other the C‐terminal activation domain, no activation of hair keratin genes in HeLa cells was observed. However, when these two proteins were noncovalently linked by means of synthetic dimerizers, hair keratin gene expression was induced. This finding suggests that target gene activation by Whn depends on the structural integrity and physical proximity of DNA binding and activation domains, providing a molecular framework to explain the loss‐of‐function phenotypes of all previously characterized nude mutations. Our results implicate Whn as a transcriptional regulator of hair keratin genes and reveal the nude phenotype as the first example of an inherited skin disorder that is caused by loss of expression rather than mutation of keratin genes. Dev Den;217:368–376.

Thymopoiesis requires Pax9 function in thymic epithelial cells.

The epithelial thymic anlage develops from the third pharyngeal pouch. Pax9 is expressed in the entire pharyngeal endoderm, and its function is required for normal development of organs derived from pharyngeal pouches. Here, we show that in Pax9 null mice, the thymic anlage develops as an ectopic polyp‐like structure in the larynx. It expresses Whn/Foxn1, a marker of thymic epithelium, but fails to perform the normal caudo‐ventral movement to the upper mediastinum. The thymic rudiment contains mesenchymal cells, blood vessels and is colonized by T cell progenitors. However, from embryonic day 14.5 onwards, the size of the Pax9 mutant thymus is severely reduced. Whereas expression of TCRβ chain genes is readily detectable in the mutant thymus, noexpression of the TCRγ chain was detectable. Our results identify a new genetically defined control point of thymopoiesis.

Genetic dissection of thymus development in mouse and zebrafish

Summary:  Lymphoid organs represent a specialized microenvironment for interaction of stromal and lymphoid cells. In primary lymphoid organs, these interactions are required to establish a self‐tolerant repertoire of lymphocytes. While detailed information is available about the genes that control lymphocyte differentiation, little is known about the genes that direct the establishment and differentiation of principal components of such microenvironments. Here, we discuss genetic studies addressing the role of thymic epithelial cells (TECs) during thymopoiesis. We have identifed an evolutionarily conserved key regulator of TEC differentiation, Foxn1, that is required for the immigration of prothymocytes into the thymic primordium. Because Foxn1 specifies the prospective endodermal domain that gives rise to thymic epithelial cells, it can be used to identify the evolutionary origins of this specialized cell type. In the course of these studies, we have found that early steps of thymus development in zebrafish are very similar to those in mice. Subsequently, we have used chemical mutagenesis to derive zebrafish lines with aberrant thymus development. Strengths and weaknesses of mouse and zebrafish models are largely complementary such that genetic analysis of mouse and zebrafish mutants may lead to a better understanding of thymus development.

A zebrafish orthologue (whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment

The cloning and characterization of the zebrafish orthologue of the mouse nude (Whn/Foxn1) gene, whnb are reported. A previously described Whn-like gene from zebrafish, now designated as whna, is shown to be the orthologue of the mouse Foxn4 gene. The whnb gene is specifically expressed in the thymic rudiment of zebrafish embryos at day 3 after fertilization, whereas the whna gene is expressed in eye and brain structures. Whnb expression is maintained in cloche mutants, where endothelial and haematopoietic cell differentiation is defective, but absent in casanova mutants where endoderm formation is impaired. In adult thymi, whnb is expressed throughout cortical and medullary areas, whereas whna expression is observed in rare cell clusters only. Our results provide the first specific marker for the epithelial compartment of the zebrafish thymus.