Hoxa10 and Hoxd10 coordinately regulate lumbar motor neuron patterning

The paralogous Hox genes Hoxa10 and Hoxd10 are expressed in overlapping domains in the developing lumbar spinal cord and surrounding mesoderm. Independent inactivation of these two genes alters the trajectory of spinal nerves and decreases the complement of motor neurons present in the lumbar spinal cord, whereas dual inactivation of these two genes has been shown to alter peripheral nerve growth and development in the mouse hindlimb. We have examined the organization and distribution of lumbar motor neurons in the spinal cords of Hoxa10/Hoxd10 double mutant animals. Double mutant animals have decreased numbers of lumbar motor neurons in both the medial and lateral motor columns. The anteroposterior position of the lumbar motor column is shifted caudally in double mutant animals, and the distribution of motor neurons is altered across individual spinal segments. Distinctions between classes of motor neurons based on positional specificity appear disrupted in double mutants. Double mutants also demonstrate abnormal spinal cord vasculature and altered kidney placement and size. Our observations suggest that Hoxa10 and Hoxd10 activity is required to specify the position of the lumbar motor column and to provide segmental specification and identity for the lumbar motor neurons.     

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.     

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.     

Functional specificity of the Hoxa13 homeobox.

To better define Abd-B type homeodomain function, to test models that predict functional equivalence of all Hox genes and to initiate a search for the downstream targets of Hoxa13, we have performed a homeobox swap by replacing the homeobox of the Hoxa11 gene with that of the Hoxa13 gene. The Hoxa11 and Hoxa13 genes are contiguous Abd-B type genes located at the 5' end of the HoxA cluster. The modified Hoxa11 allele (A11(13hd)) showed near wild-type function in the development of the kidneys, axial skeleton and male reproductive tract, consistent with functional equivalence models. In the limbs and female reproductive tract, however, the A11(13hd) allele appeared to assume dominant Hoxa13 function. The uterus, in particular, showed a striking homeotic transformation towards cervix/vagina, where Hoxa13 is normally expressed. Gene chips were used to create a molecular portrait of this tissue conversion and revealed over 100 diagnostic gene expression changes. This work identifies candidate downstream targets of the Hoxa13 gene and demonstrates that even contiguous Abd-B homeoboxes have functional specificity.     

The paralogous Hox genes Hoxa10 and Hoxd10 interact to pattern the mouse hindlimb peripheral nervous system and skeleton

The most 5' mouse Hoxa and Hoxd genes, which occupy positions 9-13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.     

Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeoboxes

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5' cluster positions were generally dominant over more 3' homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development.     

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 Hoxa13(GFP)-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.     

Hmgn5 functions downstream of Hoxa10 to regulate uterine decidualization in mice

Although Hmgn5 is involved in the regulation of cellular proliferation and differentiation, its physiological function during decidualization is still unknown. Here we showed that Hmgn5 was highly expressed in the decidual cells. Silencing of Hmgn5 expression by specific siRNA reduced the proliferation of uterine stromal cells and expression of Ccnd3 and Cdk4 in the absence or presence of estrogen and progesterone, whereas overexpression of Hmgn5 exhibited the opposite effects. Simultaneously, Hmgn5 might induce the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP and progesterone could up-regulate the expression of Hmgn5, but the up-regulation was impeded by H89 and RU486, respectively. Attenuation of Hmgn5 expression could block the differentiation of uterine stromal cells in response to cAMP and progesterone. Further studies found that regulation of cAMP and progesterone on Hmgn5 expression was mediated by Hoxa10. During in vitro decidualization, knockdown of Hmgn5 could abrogate Hoxa10-induced upregulation of Prl8a2 and Prl3c1, while overexpression of Hmgn5 reversed the inhibitory effects of Hoxa10 siRNA on the expression of Prl8a2 and Prl3c1. In the stromal cells undergoing decidualization, Hmgn5 might act downstream of Hoxa10 to regulate the expression of Cox-2, Vegf and Mmp2. Collectively, Hmgn5 may play an important role during mouse decidualization.     

Altered Hox expression and increased cell death distinguish Hypodactyly from Hoxa13 null mice.

Hypodactyly (Hoxa13Hd) mice have a small deletion within the coding sequence of Hoxa13 and a limb phenotype that is more severe than that of mice with an engineered null allele of Hoxa13. We used whole-mount in situ hybridization, Nile blue sulfate staining and genetic crosses to determine the basis for the phenotypic differences between these two mutants. Expression of Hoxd13 was unaffected in Hoxa13-/- mice, but its domain was reduced at the anterior and posterior margins of the autopod in Hoxa13Hd/Hd limb buds. The maturation of Hoxd11 expression was delayed and expression of Hoxa11 failed to become restricted to the autopod/zeugopod junction in both Hoxa13Hd/Hd and Hoxa13-/- limb buds compared to wild-type mice. Fgf8 expression was normal in both Hoxa13Hd/Hd and Hoxa13-/- mice throughout limb development. A dramatic increase in cell death was observed in limb bud mesenchyme of Hoxa13Hd/Hd mice as early as E11.5 but not in mice homozygous for the null allele. Genetic background was excluded as the basisforthe phenotypic differences. Compound heterozygotes (Hoxa13-/Hd) displayed an intermediate phenotype relative to both homozygotes suggesting that Hoxa13Hd has an effect on the development of the autopod beyond that which may result from a loss of HOXA13 protein. These results showthat Hoxa13Hd has a negative effect on the survival of the mesenchyme in the autopod, unlike the Hoxa13 null mutation, that cannot be explained by a failure of the AER to express Fgfs. In addition, at least one target of HOXA13 may be Hoxa11.     

Roles of Hoxa1 and Hoxa2 in patterning the early hindbrain of the mouse

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, we generated mice simultaneously mutant at both loci. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres.     

Targeted disruption of Hoxd9 and Hoxd10 alters locomotor behavior, vertebral identity, and peripheral nervous system development

The five most 5' HoxD genes, which are related to the Drosophila Abd-B gene, play an important role in patterning axial and appendicular skeletal elements and the nervous system of developing vertebrate embryos. Three of these genes, Hoxd11, Hoxd12, and Hoxd13, act synergistically to pattern the hindlimb autopod. In this study, we examine the combined effects of two additional 5' HoxD genes, Hoxd9 and Hoxd10. Both of these genes are expressed posteriorly in overlapping domains in the developing neural tube and axial mesoderm as well as in developing limbs. Locomotor behavior in animals carrying a double mutation in these two genes was altered; these alterations included changes in gait, mobility, and adduction. Morphological analysis showed alterations in axial and appendicular skeletal structure, hindlimb peripheral nerve organization and projection, and distal hindlimb musculature. These morphological alterations are likely to provide the substrate for the observed alterations in locomotor behavior. The alterations observed in double-mutant mice are distinct from the phenotypes observed in mice carrying single mutations in either gene, but exhibit most of the features of both individual phenotypes. This suggests that the combined activity of two adjacent Hox genes provides more patterning information than activity of each gene alone. These observations support the idea that adjacent Hox genes with overlapping expression patterns may interact functionally to provide patterning information to the same regions of developing mouse embryos.     

Comparative genomics of chondrichthyan Hoxa clusters

Background  

The chondrichthyan or cartilaginous fish (chimeras, sharks, skates and rays) occupy an important phylogenetic position as the sister group to all other jawed vertebrates and as an early lineage to diverge from the vertebrate lineage following two whole genome duplication events in vertebrate evolution. There have been few comparative genomic analyses incorporating data from chondrichthyan fish and none comparing genomic information from within the group. We have sequenced the complete Hoxa cluster of the Little Skate (Leucoraja erinacea) and compared to the published Hoxa cluster of the Horn Shark (Heterodontus francisci) and to available data from the Elephant Shark (Callorhinchus milii) genome project.     

Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.