In vertebrates, Hox genes play major roles in the formation of most vital organs. It has been proposed that the exquisite DNA-binding specificities that allow different Hox proteins to regulate specific target genes, thus instructing the identity of distinct body structures, depend on interactions with other homeoproteins, which act as Hox cofactors. For the last fifteen years, based on molecular and biochemical analyses, the prevailing view has been that TALE homeodomain proteins, which comprise the products encoded by the Pbx gene family, act as ancillary cofactors for Hox. Pbx1 is a homolog of Drosophila extradenticle (exd), which has critical roles in patterning of the fly body. While exd is the sole Pbx-encoding gene in the fly, the mouse has four such genes (Pbx1-4). Despite their paramount roles in organogenesis and patterning of the body and limb axes, the molecular mechanisms of Hox regulation remain elusive. Our objectives are to use the mouse limb as the most tractable and established system to delineate whether regulation of Hox "collinear" expression, a basic and mysterious biological phenomenon, is governed by Pbx. We have established that different Pbx genes, similarly to Hox genes, share overlapping roles in limb patterning and outgrowth. Accordingly, Pbx1/Pbx2 double homozygous (Pbx1-/-;Pbx2-/-) embryos lack limbs altogether, while Pbx1-/-;Pbx2 mutants exhibit limb truncations similar to those of HoxA/D mutants. Additionally, we have found that Pbx1/Pbx2 control the onset and spatial distribution of 5'HoxA/D expression in limb mesenchyme. These findings establish that Pbx proteins hierarchically govern 5'HoxA/D gene expression in the limb. In view of these new findings, our hypothesis proposes a novel mechanism for Hox gene regulation, whereby 5'Hox expression is directly controlled at the transcriptional level by Pbx in the bud mesenchyme for limb morphogenesis and digit formation. We will test our hypothesis using embryologic, genetic and molecular approaches in the mouse. First, by molecular methods, we will determine whether Pbx1/2 regulate 5'HoxD transcription by direct control of the HoxD GCR, a genomic region that governs HoxD collinear expression in the autopod. We will then test whether Pbx binding to the HoxD GCR has functional bearings on transcription by both transient transfections in cell culture and transient transgenesis experiments in the mouse. Moreover, by tissue-specific and inducible genetic ablation, using our new Pbx1 conditional allele (on a Pbx2-deficient background), and available Cre lines (one of which inducible in the mesenchyme), we will dissect Pbx1/Pbx2 spatial and temporal requirements in the limb field and bud mesenchyme. By this approach, we will determine when Hox expression is first affected by Pbx loss in the limb bud. Completion of these studies will define novel regulatory networks that govern transcription of Hox genes and will directly contribute to the understanding of human congenital limb malformations. Broadly, given the involvement of human HOX genes in leukemias and solid tumors, our studies will inform general comprehension of HOX regulation also in human neoplasia.

Public Health Relevance

Hox proteins play essential roles in the formation of many critical organs in mammals, including the limb;however, the molecular mechanisms underlying Hox gene regulation remain elusive. The proposed studies will use mouse models to provide novel insights into the control of Hox regulation by Pbx proteins in the limb. Accordingly, a broad impact of this work will be the generation of new knowledge on the pathogenesis of human congenital malformations, including those that affect limb skeletal development and function, occurring in approximately 1 of 500 human live births.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD043997-07
Application #
8238822
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Javois, Lorette Claire
Project Start
2003-04-01
Project End
2016-12-31
Budget Start
2012-01-07
Budget End
2012-12-31
Support Year
7
Fiscal Year
2012
Total Cost
$350,675
Indirect Cost
$143,175
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
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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
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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
Arora, Nitin; Novak, Zdenek; Fowler, Karen B et al. (2010) Cytomegalovirus viruria and DNAemia in healthy seropositive women. J Infect Dis 202:1800-3

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