Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.     

Deciphering skeletal patterning: clues from the limb

Even young children can distinguish a Tyrannosaurus rex from a Brontosaurus by observing differences in bone size, shape, number and arrangement, that is, skeletal pattern. But despite our extensive knowledge about cartilage and bone formation per se, it is still largely a mystery how skeletal pattern is established. Much of what we do know has been learned from studying limb development in chicken and mouse embryos. Based on the data from such studies, models for how limb skeletal pattern is established have been proposed and continue to be hotly debated.     

Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud

There is increasing evidence that retinoic acid is a morphogen involved in vertebrate development. This evidence comes in part from studies of the chick wing bud, in which local application of all-trans-retinoic acid results in a duplication of the digit pattern along the anteroposterior axis. Retinoic acid may be only one of several morphogenetic signalling compounds required for limb pattern formation. To identify novel morphogenetically active compounds, fractionated extracts of whole chick embryos were tested for their ability to induce digit pattern duplications. We describe here the isolation of a new activity present in the limb bud, which we have identified as all-trans-3,4-didehydroretinoic acid. The 3,4-didehydroretinoic acid is generated in situ from retinol through a 3,4-didehydroretinol intermediate. We show that 3,4-didehydroretinoic acid and retinoic acid are equipotent in evoking digit duplications. These findings suggest that there are at least two endogenous retinoids with morphogenetic properties in the chick limb.     

Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud.

Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.     

Polarizing activity and retinoid synthesis in the floor plate of the neural tube

In many developing organisms the establishment of axial polarity and the patterning of cells depend on local signals that derive from restricted regions of the embryo. In vertebrate embryos, the origins of tissue polarity have been examined extensively in the developing limb. The anteroposterior pattern of the chick limb seems to be controlled by a morphogen, possibly retinoic acid, that is enriched in a region of the limb known as the zone of polarizing activity (ZPA). Certain tissues other than the ZPA have also shown polarizing activity experimentally in the chick limb, raising the possibility that signalling molecules involved in pattern formation in different embryonic tissues are conserved. Here we provide evidence that a similar polarizing activity is also present in a restricted region of the developing central nervous system (CNS). We show that a specialized group of neural cells termed the floor plate, but not other regions of the CNS, mimics the ZPA in respecifying the digit pattern in the developing chick limb. In addition, using an in vitro biochemical assay, we show that the floor plate can synthesize retinoic acid and 3,4-didehydroretinol, the precursor of a second morphogenetically active retinoid, 3,4-didehydroretinoic acid. These results show that the floor plate is a local source of a ZPA-like polarizing signal, possibly a retinoid, which may regulate the pattern of cell differentiation in the developing CNS.     

Mox2 is a component of the genetic hierarchy controlling limb muscle development.

The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate into the limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4) and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.     

Distal-less encodes a homoeodomain protein required for limb development in Drosophila

The spatial organization of the Drosophila embryo depends on the activity of three axial pattern-forming systems. In addition to the anterior-posterior and dorsal-ventral systems that organize the segmented body plan, a proximal-distal pattern-forming system is required to provide positional information for the developing limbs. The development of both the larval and adult limbs depends directly on the activity of the Distal-less gene. Genetic analysis has shown that Distal-less functions as a developmental switch that is required to promote the development of limb structures above the evolutionary ground-state of body wall. Here we provide genetic evidence that indicates a graded requirement for Distal-less activity during limb development. Reduction of this activity has a global effect on pattern formation in the limb. The molecular structure of the Distal-less locus indicates that the gene encodes a homoeodomain-containing protein which is therefore likely to specify limb development through differential regulation of subordinate genes.     

Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud

In recent years there has been considerable interest in the role of retinoic acid (RA) in vertebrate-limb pattern formation. When RA is applied to the anterior of the chick wing bud, a mirror-image duplication of the limb pattern develops that is identical to the pattern resulting from grafts of posterior tissue (zone of polarizing activity, or ZPA). It has been proposed that position along the anterior-posterior axis in the chick limb is specified by a gradient of a diffusible factor produced by the ZPA. The ZPA-mimicking action of RA has led to the hypothesis that exogenously applied RA acts by providing graded spatial information across the anterior-posterior limb axis. An alternative interpretation is that RA changes anterior cells into ZPA cells, which in turn provide the actual pattern-duplicating stimulus; there is already some preliminary evidence that this occurs. A hybrid interpretation has also been suggested whereby ZPA cells are formed in response to RA exposure and then begin to release retinoids that act as graded spatial cues. We have used a functional assay to test anterior chick wing-bud cells for ZPA activity after exposure to RA. The results of our studies indicate that the action of RA is to change anterior cells into ZPA cells. Further, our results indicate that it is unlikely that RA-treated anterior cells then begin producing RA in such a way as to provide a graded positional signal.     

Spatial and temporal expression of the retinoic acid receptor in the regenerating amphibian limb

Retinoic acid is known to have dramatic effects on vertebrate limb pattern in development and regeneration, supporting a model in which a gradient of retinoic acid serves as a morphogen to differentially supply positional information to a developing limb. The discovery of a retinoic acid receptor (RAR) and its homology to the steroid and thyroid hormone receptors provided a potential molecular mechanism for limb morphogenesis. One prediction of this model is that the receptor must be expressed in the developing and regenerating limb anlage. We investigated the expression of the RAR in the adult newt, Notophthalmus viridescens, whose amputated limbs are capable of regenerating and upon which retinoic acid can act to alter pattern. We report the cloning of cDNAs encoding a functional newt RAR and the localization of high and uniform levels of RAR mRNA specifically in the regenerating cells that control limb pattern. These results indicate that the morphogenic field is established through differential activation of pre-existing retinoic acid receptors rather than differential expression of the RAR gene.     

Identification and spatial distribution of retinoids in the developing chick limb bud

All-trans-retinoic acid (RA) induces striking digit pattern duplications when locally applied to the developing chick limb bud. Instead of the normal digit pattern (234) a mirror-symmetrical 432234 pattern can be specified. Hence, RA closely mimics posterior limb bud tissue (the zone of polarizing activity, ZPA) that causes very similar duplications when grafted to an anterior site of a host limb bud. This resemblance raises an intriguing possibility: that RA is related to the as yet unidentified inducer substance thought to be released by the ZPA. Here we report that chick limb buds contain endogenous RA and we show that RA, but not its biosynthetic precursor retinol, forms a concentration gradient across the limb anlage with a high-point in the posterior domain of the limb bud, the part that also contains the ZPA.     

Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning

Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal-distal (P-D) axis, but how these signals influence limb patterning is still much debated. Fibroblast growth factor (FGF) gene family members are key AER-derived signals, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER. Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P-D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P-D patterning can be integrated with the concept of early specification to explain the genetic data presented here.     

一种基于卷积神经网络的下肢动作模式识别方法

针对目前下肢动作模式识别技术存在的数据量少、识别率低的问题,提出了一种新的基于卷积神经网络的下肢动作模式识别方法。以下肢步态动作识别为对象,采集无负重平地行走,无负重上/下楼及负重上/下楼5种步态的表面肌电信号（surface electromyography,sEMG）,对sEMG进行特征提取,构建了一种以特征集作为输入的卷积神经网络,并比较了其与另外几种传统分类识别方法的识别准确率和工作特征。实验结果表明,新方法对于5种步态的平均识别准确率大于95%,错误率都低于8%,具有较高的准确性。因此所提方法的输入特征集更能代表预测模型特征,模式识别率更高,可为康复医疗机器人、助力机器人等设备改善下肢运动功能提供参考。     

Homeobox gene expression correlated with the bifurcation process of limb cartilage development.

The complex architecture of the limb cartilage pattern probably develops by the sequential segmentation and branching process of precartilaginous cell condensation under the control of positional signalling provided by the zone of polarizing activity (anteroposterior) and the apical ectodermal ridge (proximodistal). This signalling is monitored and interpreted in the mesenchymal cells and induces the position-specific response of subsets of genes. Homeobox genes may be responsible for the interpretation of signalling. A correlation between limb pattern and expression domains of the homeobox genes in the upstream region of Hox/Chox-4 has been proposed. We have analysed the spatial expression pattern of the Chox-1 genes during development of chick limb buds. In contrast to genes in Hox/Chox-4 expressed coordinately along the anteroposterior axis, homeobox genes in Chox-1 have unique and mutually exclusive expression domains along the proximodistal axis. We report here that the expression domains of the Chox-1 genes are closely related to the segmental structure of cartilage along the proximodistal axis, whereas the expression domains of the Chox-4 genes are related to the cartilage branching pattern.     

前交叉韧带重建术后步行过程中的生物力学特征

 为明确前交叉韧带（anterior cruciate ligament,ACL）重建术后动作模式的改变情况,应用三维运动捕捉系统和表面肌电系统同步采集ACL重建术后患者在步行过程中的运动学、动力学和肌电数据。结果发现,与健侧相比,术侧膝关节屈曲角度峰值和屈伸活动范围均显著减小,但承重反应期的外旋幅度显著增加;术侧承重反应期膝关节伸直力矩峰值、支撑相末期膝关节屈曲力矩峰值和外旋力矩峰值均小于健侧;术侧股直肌在承重反应期的激活程度、股二头肌和半腱肌在摆动前期的激活程度均大于健侧。结果表明：ACL重建术后6~12个月（7.4±1.3月）步态呈僵硬特征,大腿肌群激活程度增加。建议ACL重建术后针对肌肉功能特征及步态特征的改变进行精准康复干预,改善术后效果。