H. Scott Stadler

Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia

In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, we show that Hoxa13 is essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13GFP-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restored proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified.

HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis.

In humans and mice, loss of HOXA13 function causes defects in the growth and patterning of the digits and interdigital tissues. Analysis of Hoxa13 expression reveals a pattern of localization overlapping with sites of reduced Bmp2 and Bmp7 expression in Hoxa13 mutant limbs. Biochemical analyses identified a novel series of Bmp2 and Bmp7 enhancer regions that directly interact with the HOXA13 DNA-binding domain and activate gene expression in the presence of HOXA13. Immunoprecipitation of HOXA13-Bmp2 and HOXA13-Bmp7 enhancer complexes from the developing autopod confirm that endogenous HOXA13 associates with these regions. Exogenous application of BMP2 or BMP7 partially rescues the Hoxa13 mutant limb phenotype, suggesting that decreased BMP signaling contributes to the malformations present in these tissues. Together, these results provide conclusive evidence that HOXA13 regulates Bmp2 and Bmp7 expression, providing a mechanistic link between HOXA13, its target genes and the specific developmental processes affected by loss of HOXA13 function.

HOXA13 Is Essential for Placental Vascular Patterning and Labyrinth Endothelial Specification

In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placentas function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13–DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (Kd = 27–42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia.

Hypospadias, a common defect affecting the growth and closure of the external genitalia, is often accompanied by gross enlargements of the genital tubercle (GT) vasculature. Because Hoxa13 homozygous mutant mice also exhibit hypospadias and GT vessel expansion, we examined whether genes playing a role in angiogenesis exhibit reduced expression in the GT. From this analysis, reductions in EphA6 and EphA7 were detected. Characterization of EphA6 and EphA7 expression in the GT confirmed colocalization with HOXA13 in the GT vascular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a series of highly conserved cis‐regulatory elements bound by HOXA13 with high affinity. GT chromatin immunoprecipitation confirmed that HOXA13 binds these gene‐regulatory elements in vivo. In vitro, HOXA13 activates gene expression through the EphA6 and EphA7 gene‐regulatory elements. Together these findings indicate that HOXA13 directly regulates EphA6 and EphA7 in the developing GT and identifies the GT vascular endothelia as a novel site for HOXA13‐dependent expression of EphA6 and EphA7. Developmental Dynamics 236:951–960, 2007.

Transcriptome analysis of the murine forelimb and hindlimb autopod

To gain insight into the coordination of gene expression profiles during forelimb and hindlimb differentiation, a transcriptome analysis of mouse embryonic autopod tissues was performed using Affymetrix Murine Gene Chips (MOE‐430). Forty‐four transcripts with expression differences higher than 2‐fold (T test, P ≤0.05) were detected between forelimb and hindlimb tissues including 38 new transcripts such as Rdh10, Frzb, Tbx18, and Hip that exhibit differential limb expression. A comparison of gene expression profiles in the forelimb, hindlimb, and brain revealed 24 limb‐signature genes whose expression was significantly enriched in limb autopod versus brain tissue (fold change >2, P ≤ 0.05). Interestingly, the genes exhibiting enrichment in the developing autopod also segregated into significant fore‐ and hindlimb‐specific clusters (P ≤ 0.05) suggesting that by E 12.5, unique gene combinations are being used during the differentiation of each autopod type. Developmental Dynamics 234:74–89, 2005.

Musculoskeletal integration at the wrist underlies the modular development of limb tendons

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes. Highlighted article: The analysis of tendon development in mice that have defective muscle or cartilage developmental programmes reveals a novel integrated model for limb tendon development.

Elucidation, Quantitative Refinement, and in Vivo Utilization of the HOXA13 DNA Binding Site

Mutations in Hoxa13 cause malformations of the appendicular skeleton and genitourinary tract, including digit loss, syndactyly, and hypospadias. To determine the molecular basis for these defects, the DNA sequences bound by HOXA13 were empirically determined, revealing a novel high affinity binding site. Correlating the utilization of this high affinity binding site with genes exhibiting perturbed expression in Hoxa13 mutant limbs, we identified that HOXA13 suppresses the expression of the BMP antagonist, Sostdc1. In the absence of HOXA13 function, Sostdc1 is ectopically expressed in the distal limb, causing reduced expression of BMP-activated genes and decreased SMAD phosphorylation. Limb chromatin immunoprecipitation revealed HOXA13 binding at its high affinity site in two conserved Sostdc1 regulatory sites in vivo. In vitro, HOXA13 represses gene expression through the Sostdc1 high affinity binding sites in a dosage-dependent manner. Together, these findings confirm that the high affinity HOXA13 binding site deduced by quantitative analyses is used in vivo to facilitate HOXA13 target gene regulation, providing a critical advance toward understanding the molecular basis for defects associated with the loss of HOXA13 function.

Modelling genitourinary defects in mice: an emerging genetic and developmental system.

The rising incidence of genitourinary (GU) defects among newborns establishes the need and opportunity to focus research efforts on the amelioration of this growing public-health concern. Sadly, our inability to explain the causes of GU defects can be directly attributed to our lack of understanding of GU gene function. Recently, mouse models have been used to provide new insights into the mechanisms that underlie congenital GU malformations.

Survival of Hoxa13 homozygous mutants reveals a novel role in digit patterning and appendicular skeletal development

The loss of HOXA13 function severely disrupts embryonic limb development. However, because embryos lacking HOXA13 die by mid‐gestation, the defects present in the mutant limb could arise as a secondary consequence of failing embryonic health. In our analysis of the mutant Hoxa13GFP allele, we identified a surviving cohort of homozygous mutants exhibiting severe limb defects including: missing phalanx elements, fusions of the carpal/tarsal elements, and significant reductions in metacarpal/metatarsal length. Characterization of prochondrogenic genes in the affected carpal/tarsal regions revealed significant reduction in Gdf5 expression, whereas Bmp2 expression was significantly elevated. Analysis of Gdf5 mRNA localization also revealed diffuse expression in the carpal/tarsal anlagen, suggesting a role for HOXA13 in the organization of the cells necessary to delineate individual carpal/tarsal elements. Together these results identify Gdf5 as a potential target gene of HOXA13 target gene and confirm a specific role for HOXA13 during appendicular skeletal development. Developmental Dynamics 239:446–457, 2010.

Structural basis for sequence specific DNA binding and protein dimerization of HOXA13.

The homeobox gene (HOXA13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of genes during embryonic morphogenesis. Here we present the NMR structure of HOXA13 homeodomain (A13DBD) bound to an 11-mer DNA duplex. A13DBD forms a dimer that binds to DNA with a dissociation constant of 7.5 nM. The A13DBD/DNA complex has a molar mass of 35 kDa consistent with two molecules of DNA bound at both ends of the A13DBD dimer. A13DBD contains an N-terminal arm (residues 324 – 329) that binds in the DNA minor groove, and a C-terminal helix (residues 362 – 382) that contacts the ATAA nucleotide sequence in the major groove. The N370 side-chain forms hydrogen bonds with the purine base of A5* (base paired with T5). Side-chain methyl groups of V373 form hydrophobic contacts with the pyrimidine methyl groups of T5, T6* and T7*, responsible for recognition of TAA in the DNA core. I366 makes similar methyl contacts with T3* and T4*. Mutants (I366A, N370A and V373G) all have decreased DNA binding and transcriptional activity. Exposed protein residues (R337, K343, and F344) make intermolecular contacts at the protein dimer interface. The mutation F344A weakens protein dimerization and lowers transcriptional activity by 76%. We conclude that the non-conserved residue, V373 is critical for structurally recognizing TAA in the major groove, and that HOXA13 dimerization is required to activate transcription of target genes.