Mapping the <i>Shh</i> long-range regulatory domain

Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains

Development of five digits is controlled by a bipartite long-range <i>cis</i>-regulator

Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs

Impaired Spleen Formation Perturbs Morphogenesis of the Gastric Lobe of the Pancreas

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/−, Bapx1−/− and Sox11−/−, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas

The dynamics of spleen morphogenesis

AbstractThe mammalian spleen has important functions in immunity and haematopoiesis but little is known about the events that occur during its early embryonic development. Here we analyse the origin of the cells that gives rise to the splenic mesenchyme and the process by which the precursors assume their position along the left lateral side of the stomach. We report a highly conserved regulatory element that regulates the Nkx2-5 gene throughout early spleen development. A transgenic mouse line carrying this element driving a reporter gene was used to show that morphogenesis of the spleen initiates bilaterally and posterior to the stomach, before the splenic precursors grow preferentially leftward. In addition the transgenic line was used in an organ culture system to track spleen precursor cells during development. Spleen cells were shown to move from the posterior mesenchyme and track along the left side of the stomach. Removal of tissue from the anterior stomach resulted in splenic cells randomly scattering suggesting a guidance role for the anterior stomach. Using a mouse line carrying a conditional Cre recombinase to mark early precursor cell populations, the spleen was found to derive from posterior mesenchyme distinct from the closely adjacent stomach mesenchyme

The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis

Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE‐class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme‐specific inactivation of Pbx1 (SKO) on a Pbx2‐ or Pbx3‐deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub‐lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine‐encoding genes, including KitL/SCF, Cxcl12/SDF‐1,IL‐3,IL‐4,GM‐CSF/Csf2 IL‐10, and Igf‐1, compared with controls. KitL/SCF and Cxcl12/SDF‐1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non‐cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF‐1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury

YAC transgenic analysis reveals Wilms' Tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb

Wilms' Tumour 1 gene (WT1) is required for the correct development of the urogenital system. To examine its regulation and expression, we created several transgenic mouse lines containing a beta-galactosidase reporter driven by the human WT1 promoter. A 5 kb promoter weakly recapitulated a subset of the endogenous Wt1 expression pattern. In contrast, 470 and 280 kb YAC transgenes reproduced the correct pattern with high activity and highlighted new expression sites. Wt1 is expressed in the septum transversum revealing how its mutation causes diaphragmatic defects. Wt1 expression in the limb demarcates a zone between chondrogenic and apoptotic domains. Finally, Wt1 is expressed in mesenchymal cells derived from the coelomic epithelium. Based upon these and further data we discuss a Wt1 role in epithelialmesenchymal transitions

The Transcriptional Activity of Neurog3 Affects Migration and Differentiation of Ectopic Endocrine Cells in Chicken Endoderm

Neurog3 is expressed transiently in pancreatic endocrine progenitors where it is responsible for activating a transcription factor cascade which eventually defines the mature endocrine cells. However, the mechanism by which Neurog3 regulates different aspects of the endocrine differentiation program is less clear. In this report we used in ovo electroporation to investigate how manipulation of Neurog3 protein activity affected migration, differentiation and fate determination. We found that changes in the onset of Neurog3 expression only had minor effect on differentiation. However increasing the transcriptional activity of Neurog3 by fusing it to VP16 or co-electroporating with Ep300 caused the electroporated cells to migrate rather than differentiate. In contrast, reducing the transcriptional activity of Neurog3 by deleting parts of the activation domain, by fusing Neurog3 to the engrailed repressor domain, or co-electroporating with Hdac1 greatly increased the proportion of glucagon expressing cells. Developmental Dynamics 239:1950-1966, 2010. (C) 2010 Wiley-Liss, Inc