Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates-a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus ( Branchiostoma lanceolatum ) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis -regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a f-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.The differentiation of B lymphocytes from progenitor cells to immunoglobulin-secreting plasma cells is a multistep process which is characterized by the sequential expression of specific cell surface markers involved in cell-cell interaction and signal transduction (29). Among them is the B-cellspecific antigen CD19. This protein is expressed throughout B-cell ontogeny from the early progenitor cell up to the mature B-cell stage and is lost from the cell surface only at the terminal stages of B-cell differentiation. The CD19 molecule is present on both normal and malignant B-lymphoid cells and hence is the most reliable diagnostic surface marker of the human B-cell lineage (23, 35). The CD19 gene codes for a 95-kDa protein with two extracellular immunoglobulinlike domains, a short transmembrane region, and a large cytoplasmic tail (33,34,38). These features suggest that the CD19 protein is a membrane receptor involved in signal transduction of B lymphocytes. No natural ligand for CD19 is as yet known; instead, monoclonal anti-CD19 antibodies have been used to study the physiological function of the CD19 molecule. In agreement with the receptor hypothesis, antibody binding leads to rapid internalization of CD19 (7,35) and to mobilization of free intracellular calcium ions (17,27,35). CD19 antibodies strongly inhibit proliferation of resting mature B cells in response to stimulation with antiimmunoglobulin M (IgM) antibodies (11,27,35 (14).Regulation of B-cell-specific gene expression is brought about by several distinct transcription factors (reference 3 and references therein). One of them is the B-cell lineagespecific activator protein (BSAP). We have identified this transcription factor as a mammalian homolog of the sea urchin protein TSAP, which is res...

Neurogenesis in the developing neocortex is a strictly regulated process of cell division and differentiation. Here we report that a gradual retreat of canonical Wnt signaling in the cortex from lateral-to-medial and anterior-to-posterior is a prerequisite of neurogenesis. Ectopic expression of a beta-catenin/LEF1 fusion protein maintains active canonical Wnt signaling in the developing cortex and delays the expression onset of the neurogenic factors Pax6, Ngn2 and Tbr2 and subsequent neurogenesis. Contrary to this, conditional ablation of beta-catenin accelerates expression of the same neurogenic genes. Furthermore, we show that a sustained canonical Wnt activity in the lateral cortex gives rise to cells with hippocampal characteristics in the cortical plate at the expense of the cortical fate, and to cells with dentate gyrus characteristics in the hippocampus. This suggests that the dose of canonical Wnt signaling determines cellular fate in the developing cortex and hippocampus, and that recession of Wnt signaling acts as a morphogenetic gradient regulating neurogenesis in the cortex.