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.

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.

Time-lapse microscopy has advanced our understanding of yolk sac and early embryonic vascularization. However, it has been difficult to assess endothelial interactions during epithelial morphogenesis of internal organs. To address this issue we have developed the first time-lapse system to study vascularization of a mammalian organ in four dimensions. We show that vascularization of XX and XY gonads is a highly dynamic, sexually dimorphic process. The XX gonad recruits vasculature by a typical angiogenic process. In contrast, the XY gonad recruits and patterns vasculature by a novel remodeling mechanism beginning with breakdown of an existing mesonephric vessel. Subsequently, in XY organs individual endothelial cells migrate and reaggregate in the coelomic domain to form the major testicular artery. Migrating endothelial cells respect domain boundaries well before they are morphologically evident, subdividing the gonad into 10 avascular regions where testis cords form. This model of vascular development in an internal organ has a direct impact on the current dogma of vascular integration during organ development and presents important parallels with mechanisms of tumor vascularization.organogenesis ͉ ovary ͉ testis I nsights into vascular development and patterning in internal organs have historically relied on xenograft models and static analysis (1-7). However, this approach does not elucidate dynamic interactions between endothelial cells and other cells of developing organs. Advances in time-lapse imaging have improved understanding of vasculogenesis, flow-induced vascular remodeling, the genetic programming of angiogenesis, and many other facets of vascular development in the mouse and chick yolk sac and during establishment of the body plan in zebrafish (8-17). However, culturing internal organs throughout critical and prolonged periods of development has been difficult. The technical challenges of organ culture, compounded by simultaneous live imaging, is a major hurdle in understanding how endothelial cells remodel and integrate in an internal organ undergoing morphogenesis.For more than a decade the urogenital ridge (UGR) explant model has provided a unique system to analyze morphogenesis of an internal organ (18,19). The fact that the UGR can be successfully explanted to culture at the critical sex-determination stage [11.5 days postcoitum (dpc)], using conditions that maintain the normal morphological structure of the gonad, has been the driving force behind its broad adoption in the sexdetermination field. However, with respect to internal organ vascularization, the UGR has received little attention as a model system. In the UGR explant model the gonad retains contact with the mesonephros, the source of the endothelium, and vascularization ex vivo occurs similarly to vascularization in vivo (20). During the period of vascularization, development of the testis and ovary diverge morphologically. Whereas the XX gonad shows few morphological changes during this period, the XY gonad undergoes dramatic epi...

The organization of the genome in the three-dimensional space of the nucleus is coupled with cell type-specific gene expression. However, how nuclear architecture influences transcription that governs cell identity remains unknown. Here, we show that nuclear pore complex (NPC) components Nup93 and Nup153 bind superenhancers (SE), regulatory structures that drive the expression of key genes that specify cell identity. We found that nucleoporin-associated SEs localize preferentially to the nuclear periphery, and absence of Nup153 and Nup93 results in dramatic transcriptional changes of SEassociated genes. Our results reveal a crucial role of NPC components in the regulation of cell type-specifying genes and highlight nuclear architecture as a regulatory layer of genome functions in cell fate.

The initiation of de novo testis cord organization in the fetal gonad is poorly understood. Endothelial cell migration into XY gonads initiates testis morphogenesis. However, neither the signals that regulate vascularization of the gonad nor the mechanisms through which vessels affect tissue morphogenesis are known. Here, we show that Vegf signaling is required for gonad vascularization and cord morphogenesis. We establish that interstitial cells express Vegfa and respond, by proliferation, to endothelial migration. In the absence of vasculature, four-dimensional imaging of whole organs revealed that interstitial proliferation is reduced and prevents formation of wedge-like structures that partition the gonad into cord-forming domains. Antagonizing vessel maturation also reduced proliferation. However, proliferation of mesenchymal cells was rescued by the addition of PDGF-BB. These results suggest a pathway that integrates initiation of vascular development and testis cord morphogenesis, and lead to a model in which undifferentiated mesenchyme recruits blood vessels, proliferates in response, and performs a primary function in the morphogenesis and patterning of the developing organ.organogenesis | sex determination | testis formation E ndothelium-derived signals are required for the development and maintenance of many vertebrate organs. Information about vascular influences on organ budding, tissue-specific celltype specification, and generation of progenitor niches have come from seminal work on the liver, pancreas, and nervous system (1-3). Despite the broad implications of this research, the mechanisms through which endothelial cells influence tissues have been difficult to identify. In several organs, specialized progenitor cells associate with the vasculature and maintain their proliferative status through contact with the ECM that shrouds vessels (4). However, the dynamics and cellular response of less specialized cells that mediate organ morphogenesis are not understood with the same clarity.Endothelial cells influence testis cord morphogenesis in the embryonic mouse gonad (5, 6). The gonad is a uniquely powerful model in which to study the role of vasculature during organ morphogenesis because of the ability to culture and image whole organs during the coincident process of vascularization and epithelial morphogenesis. In the lung, the endothelium is reported to interact with the airway epithelium to induce septae formation in the distal airways (7). However, the potential importance of the mesenchyme was not investigated in that, despite the extensive literature supporting mesenchymal-epithelial interactions as a primary force during lung morphogenesis. In the gonad, Sertoli cells are currently assumed to attract migrating endothelial cells and initiate the hallmark patterning of testis cords, although there is no direct evidence that this is the case.Here we report that cross-talk between the endothelium and nonspecialized mesenchymal cells drives testis morphogenesis. Vegfa is expressed specifically ...

Cell migration is one of the earliest events required for development of the testis. Migration occurs only in XY gonads downstream of Sry expression and is required for the subsequent epithelialization of testis cords. Using organ culture experiments and tissue recombination, we and others speculated that peritubular myoid (PTM) cells were among the migratory cells and were likely the cell type required for cord formation. However, because no unique marker was found for PTM cells, their positive identification during or after migration remained unclear. α-Smooth Muscle Actin (αSma; approved gene symbol Acta2), a classic marker of adult PTM cells,is expressed broadly in testis interstitial cells at E12.5, and becomes highly enriched in PTM cells by E15.5–16.5. We used a novel transgenic line expressingEYFP under the control of an αSma promoter to determine whether αSma-EYFP positive cellsmigrate into the gonad. Surprisingly, mesonephroi expressing αSma-EYFP do not contribute any EYFP positive cells to XY gonads when used as donors in recombination cultures. These results indicate that αSma-EYFP cells do not migrate into the gonad during the critical window of sex determination and cannot be the migrating cell type required for testis cord formation. Our results suggest that PTM cells, and most other interstitial lineages, with the exception of endothelial cells, are induced within the gonad. These experiments suggest that endothelial cells are the migrating cell type required for epithelialization of testis cords.

Soon after Sry initiates male sex determination, cells in XY gonads undergo an unusual process of de novo cord formation that results in the organization of Sertoli cells into epithelial tubules enclosing germ cells and partitioning mesenchymal cells and vasculature to the interstitial space of the testis. Recent experiments investigating this dynamic process in four dimensions have begun to shed new light on the collective interactions of multiple cell types during morphogenesis of testis cords.

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.