Organogenesis begins with the specification, positioning and assembly of the cell types specific to an organ into the organ primordium (anlage). Active cell proliferation also takes place to build a critical mass for organ morphogenesis and expansion to occur. In this proposal, we will use the vertebrate spleen as a model to investigate these fundamental steps. The complex architecture and functions of the spleen result from intimate interactions among different cell types: mesenchymal cells ("basic parenchyma"), invading endothelial cells and colonizing hematopoietic cells. In humans, the spleen has critical roles in early hematopoiesis, immunity and blood filtering and its absence (as in congenital asplenia, an under-diagnosed disorder often recognized only at autopsy) results in a high risk for life-threatening bacterial infections in newborns and children. Our long-term objective is to identify genetic pathways that control the successive stages of spleen development: i.e. morphogenesis, expansion, and influx of hematopoietic and endothelial cells, since these interrelated organogenetic processes are of utmost importance to spleen function and yet mostly unknown. Using genetic approaches and asplenic mouse strains, we defined key steps in the genetic pathways that govern early spleen development. We reported that the homeobox gene Pbx1 is required for spleen cell fates and is a hierarchical co-regulator of Nkx2.5 and Hox11 (which are also essential for spleen formation). We also found that Pbx1 expression commences earlier than that of both Nkx2.5 and Hox11 in the Lateral Plate Mesoderm (LPM). Additionally, we uncovered that Pbx1 is expressed in the endothelium of the developing spleen anlage. In view of these findings, our hypothesis is that a distinct sub-population of Pbx1-positive progenitor cells within the LPM is required for spleen parenchyma specification, morphogenesis, and expansion and that Pbx expression in the endothelium also contributes to its function in spleen morphogenesis and expansion. In addition, we hypothesize that both an intact mesenchymal anlage and endothelium are essential for normal spleen hematopoietic colonization and function. Using available lines of gene-targeted and transgenic mice, we will test our hypothesis through embryologic, genetic, and molecular approaches. First, we will establish genetic and molecular pathways that control spleen morphogenesis and expansion. To this end, we will characterize the spleen morphogenesis and cellular proliferation defects in a mouse line with conditional inactivation of Pbx1 in the spleen mesenchymal parenchyma, but not in the endothelium. We will further utilize immortalized cell cultures generated from these embryonic spleens to determine the roles of Pbx in cell cycle regulation. Second, we will assess whether an intact endothelium is essential for spleen morphogenesis and expansion by characterizing a mouse line in which only the endothelium is altered by genetic inactivation of Pbx1. Also, by Pbx1 inducible inactivation, we will establish Pbx temporal requirements in the spleen endothelium. Third, we will genetically dissect the role of the mesenchyme and endothelium, respectively, in spleen hematopoietic colonization, development, and function. Our studies will shed light on novel genetic and molecular networks that underlie the development of the spleen, a neglected organ in regard to its ontogeny. In light of the intimate interactions among the mesenchymal spleen anlage, invading endothelial cells and hematopoietic cells, the new knowledge generated from this work will have a deep impact on the understanding of spleen function. Lastly, our studies aspire to provide a better comprehension of the pathogenesis of congenital asplenia, as we put forth the prerequisite basic genetic background towards prenatal molecular diagnosis of this condition.

Public Health Relevance

The complex architecture and functions of the spleen result from intimate interactions among different cell types: mesenchymal cells ("basic parenchyma"), invading endothelial cells and colonizing hematopoietic cells. In humans, the spleen has critical roles in early hematopoiesis, immunity and blood filtering and its absence (as in congenital asplenia, an under-diagnosed disorder often recognized only at autopsy) results in a high risk for life-threatening bacterial infections and fatal sepsis in newborns and children. Our long-term objective is to identify genetic pathways that control the successive stages of spleen development: morphogenesis, expansion, and influx of endothelial and hematopoietic cells, since these interrelated organogenetic processes are of utmost importance to spleen function and, as of yet, mostly unknown. We anticipate that the new knowledge generated from these studies will have a deep impact on the understanding of spleen function, including hematopoiesis and immune response. In summary, the proposed work will shed light on novel genetic and molecular networks that underlie the ontogeny of the spleen, a neglected organ in regard to its development. Lastly, our studies aspire to provide a better comprehension of the pathogenesis of congenital asplenia, as we put forth the prerequisite basic genetic background towards prenatal molecular diagnosis of this condition, which results in heavily impaired immune response to deadly bacterial infections.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD061403-03
Application #
8278674
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Coulombe, James N
Project Start
2010-09-15
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$336,648
Indirect Cost
$137,448
Name
Weill Medical College of Cornell University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Bolze, Alexandre; Mahlaoui, Nizar; Byun, Minji et al. (2013) Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia. Science 340:976-8
Vitobello, Antonio; Ferretti, Elisabetta; Lampe, Xavier et al. (2011) Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation. Dev Cell 20:469-82
Capellini, Terence D; Handschuh, Karen; Quintana, Laura et al. (2011) Control of pelvic girdle development by genes of the Pbx family and Emx2. Dev Dyn 240:1173-89
Ferretti, Elisabetta; Li, Bingsi; Zewdu, Rediet et al. (2011) A conserved Pbx-Wnt-p63-Irf6 regulatory module controls face morphogenesis by promoting epithelial apoptosis. Dev Cell 21:627-41
Capellini, Terence D; Zappavigna, Vincenzo; Selleri, Licia (2011) Pbx homeodomain proteins: TALEnted regulators of limb patterning and outgrowth. Dev Dyn 240:1063-86
Capellini, Terence D; Vaccari, Giulia; Ferretti, Elisabetta et al. (2010) Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1. Development 137:2559-69