Genitourinary functions of Hoxa13 and Hoxd13

In the United States, Japan, United Kingdom, and Sweden, birth defects affecting the growth and development of the genitourinary (GU) regions are becoming increasingly prevalent, with incidences ranging as high as 1 in 125 live births. To understand the basis for these malformations, scientists have begun to examine the function of developmental genes in GU tissues. At the forefront of these investigations are studies examining the role of the 5' HOX proteins during the formation of the GU region. In this report we discuss what is known about HOXA13 and HOXD13 function during GU development, highlighting some of the cellular and molecular mechanisms controlled by these proteins during the GU formation. Finally, the translational benefits of identifying HOX target genes are discussed; first to explain the prevalence of some GU defects as well as a mechanism to facilitate their prevention in the birth population.     

Hoxd13 expression in the developing limbs of the short-tailed fruit bat, Carollia perspicillata

Bat forelimbs are highly specialized for sustained flight, providing a unique model to explore the genetic programs that regulate vertebrate limb diversity. Hoxd9-13 genes are important regulators of stylopodium, zeugopodium, and autopodium development and thus evolutionary changes in their expression profiles and biochemical activities may contribute to divergent limb morphologies in vertebrates. We have isolated the genomic region that includes Hoxd12 and Hoxd13 from Carollia perspicillata, the short-tailed fruit bat. The bat Hoxd13 gene encodes a protein that shares 95% identity with human and mouse HOXD13. The expression pattern of bat Hoxd13 mRNA during limb development was compared with that of mouse. In bat and mouse hindlimbs, the expression patterns of Hoxd13 are relatively similar. However, although the forelimb Hoxd13 expression patterns in both organisms during early limb bud stages are similar, at later stages they diverge; the anterior expression boundary of bat Hoxd13 is posterior-shifted relative to the mouse. These findings, compared with the Hoxd13 expression profiles of other vertebrates, suggest that divergent Hoxd13 expression patterns may contribute to limb morphological variation.     

Hoxa11 and Hoxd11 regulate branching morphogenesis of the ureteric bud in the developing kidney

Hoxa11 and Hoxd11 are functionally redundant during kidney development. Mice with homozygous null mutation of either gene have normal kidneys, but double mutants have rudimentary, or in extreme cases, absent kidneys. We have examined the mechanism for renal growth failure in this mouse model and find defects in ureteric bud branching morphogenesis. The ureteric buds are either unbranched or have an atypical pattern characterized by lack of terminal branches in the midventral renal cortex. The mutant embryos show that Hoxa11 and Hoxd11 control development of a dorsoventral renal axis. By immunohistochemical analysis, Hoxa11 expression is restricted to the early metanephric mesenchyme, which induces ureteric bud formation and branching. It is not found in the ureteric bud. This suggests that the branching defect had been caused by failure of mesenchyme to epithelium signaling. In situ hybridizations with Wnt7b, a marker of the metanephric kidney, show that the branching defect was not simply the result of homeotic transformation of metanephros to mesonephros. Absent Bf2 and Gdnf expression in the midventral mesenchyme, findings that could by themselves account for branching defects, shows that Hoxa11 and Hoxd11 are necessary for normal gene expression in the ventral mesenchyme. Attenuation of normal gene expression along with the absence of a detectable proliferative or apoptotic change in the mutants show that one function of Hoxa11 and Hoxd11 in the developing renal mesenchyme is to regulate differentiation necessary for mesenchymal-epithelial reciprocal inductive interactions.     

Identification and functional analysis of phosphorylated tyrosine residues within EphA2 receptor tyrosine kinase

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr(587) and Tyr(593) bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)(734) binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.     

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.     

Estrogen and Myc negatively regulate expression of the EphA2 tyrosine kinase

Estrogen receptor and c-Myc are frequently overexpressed during breast cancer progression but are downregulated in many aggressive forms of the disease. High levels of the EphA2 tyrosine kinase are consistently found in the most aggressive breast cancer cells, and EphA2 overexpression can increase metastatic potential. We demonstrate, herein, that estrogen and Myc negatively regulate EphA2 expression in mammary epithelial cells. These data reveal EphA2 as a downstream target of estrogen and Myc and suggest a mechanism by which estrogen and Myc may regulate breast cancer.     

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.     

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.     

A critical role for the EphA3 receptor tyrosine kinase in heart development

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.     

Analysis of EphA4 receptor tyrosine kinase substrate specificity using peptide-based arrays

Eph receptor tyrosine kinases regulate many important biological processes. In the present study, we explored the substrate specificity of the EphA4 receptor tyrosine kinase using peptide arrays. We define a consensus substrate motif for EphA4 and go on to identify and test a number of potential EphA4 substrates and map their putative site(s) of phosphorylation. Cotransfection studies validate two of the predicted substrates: Nck2 and Dok1. Our findings identify several potential EphA4 substrates and demonstrate the general utility of using peptide arrays to rapidly identify and map protein kinase phosphorylation sites.     

Cancer somatic mutations disrupt functions of the EphA3 receptor tyrosine kinase through multiple mechanisms

The Eph receptor tyrosine kinases make up an important family of signal transduction molecules that control many cellular processes, including cell adhesion and movement, cell shape, and cell growth. All of these are important aspects of cancer progression, but the relationship between Eph receptors and cancer is complex and not fully understood. Genetic screens of tumor specimens from cancer patients have revealed somatic mutations in many Eph receptors. The most highly mutated Eph receptor is EphA3, but its functional role in cancer is currently not well established. Here we show that many EphA3 mutations identified in lung, colorectal, and hepatocellular cancers, melanoma, and glioblastoma impair kinase activity or ephrin ligand binding and/or decrease the level of receptor cell surface localization. These results suggest that EphA3 has ephrin- and kinase-dependent tumor suppressing activities, which are disrupted by somatic cancer mutations.     

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis

In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.     

Blockade of EphA receptor tyrosine kinase activation inhibits vascular endothelial cell growth factor-induced angiogenesis

Angiogenesis is a multistep process involving a diverse array of molecular signals. Ligands for receptor tyrosine kinases (RTKs) have emerged as critical mediators of angiogenesis. Three families of ligands, vascular endothelial cell growth factors (VEGFs), angiopoietins, and ephrins, act via RTKs expressed in endothelial cells. Recent evidence indicates that VEGF cooperates with angiopoietins to regulate vascular remodeling and angiogenesis in both embryogenesis and tumor neovascularization. However, the relationship between VEGF and ephrins remains unclear. Here we show that interaction between EphA RTKs and ephrinA ligands is necessary for induction of maximal neovascularization by VEGF. EphA2 RTK is activated by VEGF through induction of ephrinA1 ligand. A soluble EphA2-Fc receptor inhibits VEGF-, but not basic fibroblast growth factor-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis. As an independent, but complementary approach, EphA2 antisense oligonucleotides inhibited endothelial expression of EphA2 receptor and suppressed ephrinA1- and VEGF-induced cell migration. Taken together, these data indicate an essential role for EphA receptor activation in VEGF-dependent angiogenesis and suggest a potential new target for therapeutic intervention in pathogenic angiogenesis.     

Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.     

Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.     

Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.     

Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs.

Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, we generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation.     

Developmental expression and distinctive tyrosine phosphorylation of the Eph-related receptor tyrosine kinase Cek9

Cek9 is a receptor tyrosine kinase of the Eph subfamily for which only a partial cDNA sequence was known (Sajjadi, F.G., and E.B. Pasquale. 1993. Oncogene. 8:1807-1813). We have obtained the entire cDNA sequence and identified a variant form of Cek9 that lacks a signal peptide. We subsequently examined the spatio-temporal expression and tyrosine phosphorylation of Cek9 in the chicken embryo by using specific antibodies. At embryonic day 2, Cek9 immunoreactivity is concentrated in the eye, the brain, the posterior region of the neural tube, and the most recently formed somites. Later in development, Cek9 expression is widespread but particularly prominent in neural tissues. In the developing visual system, Cek9 is highly concentrated in areas containing retinal ganglion cell axons, suggesting a role in regulating their outgrowth to the optic tectum. Unlike other Eph-related receptors, Cek9 is substantially phosphorylated on tyrosine in many tissues at various developmental stages. Since autophosphorylation of receptor protein-tyrosine kinases typically correlates with increased enzymatic activity, this suggests that Cek9 plays an active role in embryonic signal transduction pathways.     

Hoxa4 expression in developing mouse hair follicles and skin

We have examined the expression of the Hoxa4 gene in embryonic vibrissae and developing and cycling postnatal pelage hair follicles by digoxigenin-based in situ hybridization. Hoxa4 expression is first seen in E13.5 vibrissae throughout the follicle placode. From E15.5 to E18.5 its expression is restricted to Henle's layer of the inner root sheath. Postnatally, Hoxa4 expression is observed at all stages of developing pelage follicles, from P0 to P4. Sites of expression include both inner and outer root sheaths, matrix cells, and the interfollicular epidermis. Hoxa4 is not expressed in hair follicles after P4. Hoxb4, however, is expressed both in developing follicles at P2 and in catagen at P19, suggesting differential expression of these two paralogous genes in the hair follicle cycle.