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"Strategic ablation of Id compensation triggers the emergence of a constellation of novel phenotypes in the adult"

by
Corey Chang
MD/Ph.D. Program
B.S. 2007, Johns Hopkins University



Thesis Advisor: Diego Fraidenraich, Ph.D., FAHA
Assistant Professor
Department of Cell Biology and Molecular Medicine

Tuesday, May 13, 2014
2:00 P.M., MSB G Level Conference Room


Abstract

The Id family of transcriptional regulatory proteins (Id1-Id4) are highly expressed in embryonic development and are involved in a diverse array of biological processes including cell proliferation, differentiation, growth, cell cycle regulation and specification. While Id1, Id2 and Id3 exhibit overlapping patterns of expression along more undifferentiated, actively cycling cells along multiple cell lineages in the developing embryo, Id4 expression exhibits a unique pattern limited specifically to nervous tissue and the ventral portion of the epithelium of the developing foregut in early stages of development. Of particular interest is the striking degree of similarity in pattern of expression between Id1 and Id3 in various embryonic tissues like the bone, brain, gut, heart and vasculature, suggesting a degree of redundancy or compensation between these two Id members.
Attempts to study compensation between Id1 and Id3 have been impeded by the embryonic lethality of Id1/Id3 double knockout (Id DKO) mice at midgestation. Id DKO mice exhibit a number of gross abnormalities including small size, brain hemorrhage and multiple cardiac defects, reminiscent of the ‘thin myocardial wall syndrome’. Meanwhile single Id knockout (Id1 KO or Id3 KO) mice are viable and exhibit postnatal phenotypes. In an attempt to study compensation between Id1 and Id3 in vivo, we employed a conditional knockout strategy in which we ablated Id3 globally and conditionally ablated Id1 using Tie2Cre recombination to target important Id1 expressing tissues including the endothelium, endocardial cushion and hematopoietic cells. We found that approximately 70% of these conditional double knockout mice (Id cDKO) survived into adulthood manifesting a number of novel postnatal phenotypes.
Surviving Id cDKO mice are small in stature compared to littermates and most die by 1 year of age. Upon examination, we found that Id cDKOs exhibit pale bones with a 50% decrease in bone marrow cellularity and splenomegaly to levels not observed in single Id knockouts. Analysis of the peripheral blood revealed that these mice exhibit marked anemia with marked reticulocytosis, a novel finding unreported in previous single knockout models of Id ablation. Immunophenotypic analysis unveiled multi-lineage (erythroid, myeloid, lymphoid) hematopoietic abnormalities with a significant degree of hematopoietic apoptosis. Molecular studies demonstrated marked disruptions in the hematopoietic transcriptional network. Bone marrow transplantation studies highlighted important intrinsic and extrinsic components to the development of anemia in Id cDKO mice. Chromatin immunoprecipitation (ChIP) assays from Id cDKO bone marrow cells unveiled for the first time upregulation of E2A (a canonical ubiquitous E protein binding partner of Id) in the absence of Id compensation as well as increased occupancy of E47 (an E2A gene product) at the promoter region of E2A, suggesting the presence of an important autoregulatory loop.
Tie2Cre recombination also results in global loss of Id compensation in endothelial cells lining the vasculature of Id cDKO mice. Would healing studies reveal that Id cDKO mice exhibit marked delays in wound closure with evidence of decreased granulation tissue formation and dilated neovessels at the wound periphery. Bone marrow transplantation studies reveal a significant bone marrow cellular contribution to the wound healing process. WT recipients of Id cDKO bone marrow exhibit significant delays in wound closure compared to WT recipients of WT bone marrow while Id cDKO recipients of WT bone marrow showed improvements in wound closure time. These findings help elucidate the impact of Id loss in the wound healing process and provide a framework for understanding how Id loss impacts the ability of the bone marrow to contribute to the wound healing process.
In addition to hematopoietic defects, Id cDKO also develop dilated cardiomyopathy in adulthood with a unique pattern of marked perivascular and endocardial fibrosis. Ejection fraction and fractional shortening (measures of cardiac function) were both reduced. Microarray analysis of Id cDKO hearts revealed upregulation of hypertrophic, fibrotic and angiostatic genes and downregulation of angiogenic genes. Immuohistochemical and qPCR analysis reveal increased expression of thrombospondin-1 (TSP-1) as well as several downstream genes including connective tissue growth factor (CTGF) and collagens I & III (Col1a1 & Col3a1), which have previously been shown to increase in the context of heart failure. Immunohistochemical analysis revealed that TSP1 expression in Id cDKO hearts was endothelial and was more abundant near regions of disruption within the endocardium. TUNEL analysis revealed increased percentage of apoptotic cells in Id cDKO hearts. CD31 staining of Id cDKO hearts revealed a distended and diffuse capillary network within the endocardial region. Western blot analysis of Id cDKO hearts showed no changes in phosphorylation of Smad2, a downstream mediator of the TSP1/TGFâ/Smad profibrotic pathway. Furthermore, ex vivo cardiac explant experiments revealed downregulation of CTGF and Col3a1 in the presence of LSKL, an antagonistic peptide that blocks TSP1 mediated activation of TGFâ. These findings suggest that loss of Id compensation might impact the TSP1/TGFâ/CTGF axis through noncanonical (non-Smad) pathways. Furthermore, bone marrow transplantation studies reveal that recipients of Id cDKO bone marrow exhibit early signs of cardiac disease reminiscent of the Id cDKO cardiac phenotype with similar changes in gene expression and a corresponding decrease in cardiac function, suggesting that there is an important hematopoietic component to this cardiac pathology.
Collectively, these findings provide a novel paradigm for understanding the impact of lost Id compensation in different tissues and organ systems in the adult.


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