Expression patterns of Hox10 paralogous genes during lumbar spinal cord development

We have examined the expression of three paralogous Hox genes from E11.5 through E15.5 in the mouse spinal cord. These ages coincide with major phases of spinal cord neurogenesis, neuronal differentiation, cell migration, gliogenesis, and motor neuron cell death. The three genes, Hoxa10, Hoxc10, and Hoxd10, are all expressed in the lumbar spinal cord and have distinct expression patterns. Mutations in these three genes are known to affect motor neuron patterning. All three genes show lower levels of expression at the rostral limits of their domains, with selective regions of higher expression more caudally. Hoxa10 and Hoxd10 expression appears confined to postmitotic cell populations in the intermediate and ventral gray, while Hoxc10 is also expressed in proliferating cells in the dorsal ventricular zone. Hoxc10 and Hoxd10 expression is clearly excluded from the lateral motor columns at rostral lumbar levels but is present in this region more caudally. Double labeling demonstrates that Hoxc10 expression is correlated with ventrolateral LIM gene expression in the caudal part of the lumbar spinal cord.     

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.     

Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons

A central question in neural development is how the broad diversity of neurons is generated in the vertebrate CNS. We have investigated the function of Hoxc10 and Hoxd10 in mouse lumbar motoneuron development. We show that Hoxc10 and Hoxd10 are initially expressed in most newly generated lumbar motoneurons, but subsequently become restricted to the lateral division of the lateral motor column (lLMC). Disruption of Hoxc10 and Hoxd10 caused severe hindlimb locomotor defects. Motoneurons in rostral lumbar segments were found to adopt the phenotype of thoracic motoneurons. More caudally the lLMC and dorsal-projecting axons were missing, yet most hindlimb muscles were innervated. The loss of the lLMC was not due to decreased production of motoneuron precursors or increased apoptosis. Instead, presumptive lLMC neurons failed to migrate to their normal position, and did not differentiate into other motoneurons or interneurons. Together, these results show that Hoxc10 and Hoxd10 play key roles in establishing lumbar motoneuron columnar, divisional and motor pool identity.     

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.     

Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes

We present a detailed study of the genetic basis of mesodermal axial patterning by paralogous group 8 Hox genes in the mouse. The phenotype of Hoxd8 loss-of-function mutants is presented, and compared with that of Hoxb8- and Hoxc8-null mice. Our analysis of single mutants reveals common features for the Hoxc8 and Hoxd8 genes in patterning lower thoracic and lumbar vertebrae. In the Hoxb8 mutant, more anterior axial regions are affected. The three paralogous Hox genes are expressed up to similar rostral boundaries in the mesoderm, but at levels that strongly vary with the axial position. We find that the axial region affected in each of the single mutants mostly corresponds to the area with the highest level of gene expression. However, analysis of double and triple mutants reveals that lower expression of the other two paralogous genes also plays a patterning role when the mainly expressed gene is defective. We therefore conclude that paralogous group 8 Hox genes are involved in patterning quite an extensive anteroposterior (AP) axial region. Phenotypes of double and triple mutants reveal that Hoxb8, Hoxc8 and Hoxd8 have redundant functions at upper thoracic and sacral levels, including positioning of the hindlimbs. Interestingly, loss of functional Hoxb8 alleles partially rescues the phenotype of Hoxc8- and Hoxc8/Hoxd8-null mutants at lower thoracic and lumbar levels. This suggests that Hoxb8 affects patterning at these axial positions differently from the other paralogous gene products. We conclude that paralogous Hox genes can have a unique role in patterning specific axial regions in addition to their redundant function at other AP levels.     

Retinoid receptors in chronic degeneration of the spinal cord: observations in a rat model of amyotrophic lateral sclerosis

Changes in distribution and expression of retinoid receptors may be part of a spinal cord protective response to acute injury and to chronic degeneration. In this study, we have combined RNA and protein expression analysis to characterize the expression profile of retinoid receptors in the lumbar spinal cord of the superoxide dismutase 1 G93A mutant rat model of amyotrophic lateral sclerosis, a fatal neurodegenerative disorder causing extensive motor neuron loss. We also report a nonsignificant change in RNA expression of binding proteins and metabolizing enzymes for retinol and retinoic acid in the mutant rat spinal cord at end-stage disease. Only retinoid X receptor beta (RXRbeta), and to a lesser extent retinoic acid receptor beta and alpha (RARbeta/alpha) were reliably detected in lumbar spinal cord at an early pre-symptomatic phase and throughout the disease progression. The expression of RXRbeta in lamina II neurons in the dorsal horn of transgenic and wild type (WT) animals was associated with extensive astrocyte staining in end-stage lumbar spinal cord from transgenic rats. RARbeta and RARalpha diffuse staining of large motor neurons in the pre-symptomatic transgenic and in the WT lumbar cord appear to decline in end-stage disease, when a selective and strong gamma motor neuron RARalpha staining becomes evident. As gliosis and motor neuron loss are key pathogenic features in amyotrophic lateral sclerosis, the selective expression of retinoid receptors in astrocytes and motor neurons may provide further clues to the role of retinoid signalling in neurodegeneration and suggest new treatment strategies based on retinoid-modulating agents.     

Hoxd10 induction and regionalization in the developing lumbosacral spinal cord

We have used Hoxd10 expression as a primary marker of the lumbosacral region to examine the early programming of regional characteristics within the posterior spinal cord of the chick embryo. Hoxd10 is uniquely expressed at a high level in the lumbosacral cord, from the earliest stages of motor column formation through stages of motoneuron axon outgrowth. To define the time period when this gene pattern is determined, we assessed Hoxd10 expression after transposition of lumbosacral and thoracic segments at early neural tube stages. We present evidence that there is an early prepattern for Hoxd10 expression in the lumbosacral neural tube; a prepattern that is established at or before stages of neural tube closure. Cells within more posterior lumbosacral segments have a greater ability to develop high level Hoxd10 expression than the most anterior lumbosacral segments or thoracic segments. During subsequent neural tube stages, this prepattern is amplified and stabilized by environmental signals such that all lumbosacral segments acquire the ability to develop high levels of Hoxd10, independent of their axial environment. Results from experiments in which posterior neural segments and/or paraxial mesoderm segments were placed at different axial levels suggest that signals setting Hoxd10 expression form a decreasing posterior-to-anterior gradient. Our experiments do not, however, implicate adjacent paraxial mesoderm as the only source of graded signals. We suggest, instead, that signals from more posterior embryonic regions influence Hoxd10 expression after the early establishment of a regional prepattern. Concurrent analyses of patterns of LIM proteins and motor column organization after experimental surgeries suggest that the programming of these characteristics follows similar rules.     

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.     

ABRA在不同年龄大鼠腰段脊髓中的表达变化

目的检测Actin binding Rho activator（ABRA）在不同年龄大鼠腰段脊髓中的表达变化。方法采用Western blot定量检测不同年龄大鼠腰段脊髓中ABRA蛋白水平表达变化,采用免疫荧光染色显示不同年龄大鼠腰髓中ABRA细胞定位。结果Western blot显示ABRA在新生鼠腰段脊髓中表达显著高于成年鼠及老年鼠。免疫荧光染色显示ABRA广泛表达于神经元的胞核、胞浆和突起,在腰髓前角,与前角运动神经元存在共定位,在腰髓后角,与小的NeuN阳性感觉神经元存在共定位。腰髓前角、后角的阳性细胞计数均显示新生鼠ABRA＋NeuN双阳性细胞占总ABRA阳性细胞百分比显著低于成年鼠及老年鼠。结论ABRA广泛表达于腰髓中的神经元,ABRA在新生鼠腰髓中表达最强,随年龄的增长呈现明显的时相变化,提示ABRA可能参与了腰髓中神经元的发育和成熟。     

Hoxa11 and hoxd11 regulate chondrocyte differentiation upstream of runx2 and shox2 in mice

During limb development, posterior Hox genes of the Hoxa- and Hoxd cluster provide positional information along the limb axis. Here we report a new function for Hoxa11 and Hoxd11 in regulating the early steps of chondrocyte differentiation. We analyzed forelimbs of Hoxa11(-/-);d11(-/-) and Ulnaless mice, which are characterized by specifically shortened zeugopods. By detailed morphological and molecular analyses, we show that loss of Hoxa11 and Hoxd11 in the ulna of both mutants leads to an arrest of chondrocyte differentiation at a step before the separation into round and columnar cells takes place. Furthermore, we demonstrate that Hoxa11 and Hoxd11 act upstream of Runx2 and Shox2, two key regulators of chondrocyte differentiation. We hypothesize that Runx2 activates Shox2 in early chondrocytes, which at later stages induces Runx2 expression to regulate hypertrophic differentiation. These results give insight into mechanisms by which positional information might be translated into a specific bone pattern.     

FLD interacts with CO to affect both flowering time and floral initiation in Arabidopsis thaliana.

fld and co, both with significantly delayed flowering, are characterized as late-flowering mutations in Arabidopsis thaliana. Double mutants between fld-2 and co-3 were generated and the phenotypes characterized. Double mutants flower later than both single mutant parents, suggesting that there is an additive effect. In addition, the formation of flowers in double mutants was altered and showed a novel phenotype. Double mutant flowers contained a much longer stalk (pedicel). Sepals and petals were absent. Several leaf-like structures were produced in the position normally occupied by sepals and the organ numbers were reduced. The carpels were morphologically normal. The stamens produced were usually aborted in the early stage, thus, the flowers were sterile. The additive phenotype observed in double mutants provides evidence to support that these two genes, FLD and CO, are not only involved in rosette-to-inflorescence transition but also involved in the flower formation. This result also indicates that FLD and CO promote the reproductive program through two different pathways.     

Hoxa3 regulates integration of glossopharyngeal nerve precursor cells.

In vertebrates, certain Hox genes are known to control cellular identities along the anterior-posterior (A-P) axis in the developing hindbrain. In mouse Hoxa3 mutants, truncation of the glossopharyngeal (IXth) nerve or the fusion of the IXth and vagus (Xth) nerves was reported, although its underlying mechanism is largely unknown. To elucidate the mechanism of the IXth nerve defects, we reexamined the phenotype of Hoxa3 mutant embryos. In Hoxa3 mutants, we observed an abnormal caudal stream of the migrating Hoxa3-expressing neural crest cells at the prospective IXth nerve-forming area. Dorsomedial migration of the placode-derived neuronal precursor cells of the IXth nerve was also affected. Motor neurons at rhombomere 6 (r6), where those of the IXth nerve were positioned, often projected axons to the Xth nerve. In summary, the Hoxa3 gene has crucial roles in ensuring the correct axon projection pattern of all three components of the IXth nerve, i.e., motor neurons and sensory neurons of the proximal and distal ganglia.     

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.     

An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.