Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology.

Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1.

Tissue stem cells divide to self-renew and generate differentiated cells to maintain homeostasis. Although influenced by both intrinsic and extrinsic factors, the genetic mechanisms coordinating the decision between self-renewal and initiation of differentiation remain poorly understood. The escargot (esg) gene encodes a transcription factor that is expressed in stem cells in multiple tissues in Drosophila melanogaster, including intestinal stem cells (ISCs). Here, we demonstrate that Esg plays a pivotal role in intestinal homeostasis, maintaining the stem cell pool while influencing fate decisions through modulation of Notch activity. Loss of esg induced ISC differentiation, a decline in Notch activity in daughter enteroblasts (EB), and an increase in differentiated enteroendocrine (EE) cells. Amun, an inhibitor of Notch in other systems, was identified as a target of Esg in the intestine. Decreased expression of esg resulted in upregulation of Amun, while downregulation of Amun rescued the ectopic EE cell phenotype resulting from loss of esg. Thus, our findings provide a framework for further comparative studies addressing the conserved roles of Snail factors in coordinating self-renewal and differentiation of stem cells across tissues and species.

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor-␣ (TGF-␣), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF-␣ exerts its antianoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-␣. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X L , another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Srcdependent manner. We conclude that TGF-␣ has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X L expression induced by detachment from the ECM.Most epithelia rest upon and are tightly bound to a thin extracellular matrix (ECM) 1 called basement membrane. Detachment of epithelial cells from the basement membrane results in apoptosis, a phenomenon termed anoikis (1-3). Solid tumors grow in vivo as multicellular masses in which at least a proportion of cells is deprived of normal contacts with the basement membrane and is anoikis-resistant. Acquisition of such resistance is, therefore, an essential prerequisite for invasion and metastases in most cancers of epithelial origin.Some of the molecular mechanisms by which epithelial cells become resistant to anoikis during tumor progression have been uncovered during the last few years (4 -8). Our laboratory, in particular, has investigated the mechanisms of anoikis resistance in the context of colorectal cancer, and we have demonstrated that one such mechanism is based on the mutation and activation of the ras proto-oncogene (4,6,8).A majority of colorectal cancers and cell lines established from these tumors express epidermal growth factor receptor (EGFR) and overproduce one or more of its ligands (9 -12). In addition, blockade of such ligands inhibits growth of human colorectal carcinoma xenografts (13). In principle, these EGFR ligands could, at least in part, exert their oncogenic effect through the induction of resistance to anoikis, since their antiapoptotic activity is well established (14 -16). However, it has been shown in several cell systems that efficient EGFR signaling requires cell-ECM attachment. For example, in fibroblasts the capaci...

Although vertebrate hematopoiesis is the focus of intense study, immunocyte development is well-characterized in only a few invertebrate groups. The sea urchin embryo provides a morphologically simple model for immune cell development in an organism that is phylogenetically allied to vertebrates. Larval immunocytes, including pigment cells and several blastocoelar cell subtypes, emerge from a population of non-skeletal mesodermal (NSM) precursors that is specified at the blastula stage. This ring of cells is first partitioned into oral and aboral fields with distinct blastocoelar and pigment cell gene regulatory programs. The oral field is subsequently specified into several distinct immune and non-immune cell types during gastrulation. Here we characterize the oral NSM expression and downstream function of two homologs of key vertebrate hematopoietic transcription factors: SpGatac, an ortholog of vertebrate Gata-1/2/3 and SpScl, an ortholog of Scl/Tal-2/Lyl-1. Perturbation of SpGatac affects blastocoelar cell migration at gastrulation and later expression of immune effector genes, whereas interference with SpScl function disrupts segregation of pigment and blastocoelar cell precursors. Homologs of several transcription regulators that interact with Gata-1/2/3 and Scl factors in vertebrate hematopoiesis are also co-expressed in the oral NSM, including SpE-protein, the sea urchin homolog of vertebrate E2A/HEB/E2-2 and SpLmo2, an ortholog of a dedicated cofactor of the Scl-GATA transcription complex. Regulatory analysis of SpGatac indicates that oral NSM identity is directly suppressed in presumptive pigment cells by the transcription factor SpGcm. These findings provide part of a comparative basis to understand the evolutionary origins and regulatory biology of deuterostome immune cell differentiation in the context of a tractable gene regulatory network model.

Summary Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behaviour. Somatic hub cells in the Drosophila testis regulate the behaviour of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually, CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the co-repressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.