Impact of neuropathic pain on participation: perspectives from the Canadian SCI community survey.

STUDY DESIGN: Secondary analysis of an observational cohort study. OBJECTIVES: To determine if the perceived overall limitation of neuropathic pain on activities (NP limitation) covaries in 26 specific life activities in people with SCI, taking into account the severity of injury. SETTING: Community-based in Canada. METHODS: Secondary analysis of a cohort (N = 1481) was performed using questionnaires to rate NP frequency and limitation on activities as well as participation in 26 life activities. Relative risks (RR) analyses using Poisson regression were used to examine the data. RESULTS: Most participants (N = 1158; 78%) reported living with NP (from once a year to every day). When NP limitation was described as "not at all" or "very little" (N = 394; 34%), there was no statistically significant RR (p > 0.0019), suggesting no additional risk of not participating "as much as wanted" in any of activities compared to participants with no NP. When NP limitation was described as "to some extent" (N = 411; 35%), a significantly higher risk was observed for 5 of the 26 activities (1.34 < RR < 1.62), and for 23 activities with a large range (1.24 < RR < 3.20) when NP limitation was rated as "to a great extent or more" (N = 353; 31%). CONCLUSION: The variation of RR observed across the 26 activities suggests that the NP limitation may not be general but rather related to specific activity characteristics, which should be taken into account when evaluating NP limitations with a specific focus on the life activities being affected.

Animal Models for Understanding Human Skeletal Defects.

Skeletal defects, such as cleft palate, scoliosis, and shortening of the limb bones are common in the human population. Animal models have been essential for characterizing the molecular and cellular mechanisms that underlie these and other skeletal disorders. This chapter will explore the cellular origins of the vertebrate skeleton and introduce a selection of animal models for human disorders of the skull and facial bones, spinal column, and limbs. The common genetic pathways that build the skeleton of various vertebrate species and how these similarities facilitate the study of human developmental processes in laboratory animals will be a focus of discussion. This chapter will also highlight how current genome editing technologies can be applied to model various perturbations of human chromatin structure in laboratory animals.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis.

Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

Mice lacking the transcription factor SHOX2 display impaired cerebellar development and deficits in motor coordination.

Purkinje cells of the developing cerebellum secrete the morphogen sonic hedgehog (SHH), which is required to maintain the proliferative state of granule cell precursors (GCPs) prior to their differentiation and migration to form the internal granule layer (IGL). Despite a wealth of knowledge regarding the function of SHH during cerebellar development, the upstream regulators of Shh expression during this process remain largely unknown. Here we report that the murine short stature homeobox 2 (Shox2) gene is required for normal Shh expression in dorsal-residing Purkinje cells. Using two different Cre drivers, we show that elimination of Shox2 in the brain results in developmental defects in the inferior colliculus and cerebellum. Specifically, loss of Shox2 in the cerebellum results in precocious differentiation and migration of GCPs from the external granule layer (EGL) to the IGL. This correlates with premature bone morphogenetic protein 4 (Bmp4) expression in granule cells of the dorsal cerebellum. The size of the neonatal cerebellum is reduced in Shox2-mutant animals, which is consistent with a reduction in the number of GCPs present in the EGL, and could account for the smaller vermis and thinner IGL present in adult Shox2mutants. Shox2-mutant mice also display reduced exploratory activity, altered gait and impaired motor coordination. Our findings are the first to show a role for Shox2 in brain development. We provide evidence that Shox2 plays an important role during cerebellar development, perhaps to maintain the proper balance of Shh and Bmp expression levels in the dorsal vermis, and demonstrate that in the absence of Shox2, mice display both cerebellar impairments and deficits in motor coordination, ultimately highlighting the importance of Shox2 in the cerebellum.

Shox2 is a molecular determinant of depot-specific adipocyte function.

Visceral and s.c. fat exhibit different intrinsic properties, including rates of lipolysis, and are associated with differential risk for the development of type 2 diabetes. These effects are in part related to cell autonomous differences in gene expression. In the present study, we show that expression of Shox2 (Short stature homeobox 2) is higher in s.c. than visceral fat in both rodents and humans and that levels are further increased in humans with visceral obesity. Fat-specific disruption of Shox2 in male mice results in protection from high fat diet-induced obesity, with a preferential loss of s.c. fat. The reduced adipocyte size is secondary to a twofold increase in the expression of ß3 adrenergic receptor (Adrb3) at both the mRNA and protein level and a parallel increase in lipolytic rate. These effects are mimicked by knockdown of Shox2 in C3H10T1/2 cells. Conversely, overexpression of Shox2 leads to a repression of Adrb3 expression and decrease lipolytic rate. Shox2 does not affect differentiation but directly interacts with CCAAT/enhancer binding protein alpha and attenuates its transcriptional activity of the Adrb3 promoter. Thus, Shox2 can regulate the expression of Adrb3 and control the rate of lipolysis and, in this way, exerts control of the phenotypic differences between visceral and s.c. adipocytes.

Comparative transgenic analysis of enhancers from the human SHOX and mouse Shox2 genomic regions.

Disruption of presumptive enhancers downstream of the human SHOX gene (hSHOX) is a frequent cause of the zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse Shox2 gene (mShox2) is also required for limb development, but in the more proximal stylopodium. In this study, we used transgenic mice in a comparative approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. Among conserved noncoding elements (CNEs) that function as enhancers in vertebrate genomes, those that are maintained near paralogous genes are of particular interest given their ancient origins. Therefore, we first analyzed the regulatory potential of a genomic region containing one such duplicated CNE (dCNE) downstream of mShox2 and hSHOX. We identified a strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene. Interestingly, this enhancer requires sequences only conserved in the mammalian lineage in order to drive strong limb expression, whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. Similarly, we found that a conserved element downstream of hSHOX (CNE9) also functions as a neural enhancer in transgenic mice. However, when the CNE9 transgenic construct was enlarged to include adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the limbs. Therefore, both hSHOX and mShox2 limb enhancers are coupled to distinct neural enhancers. This is the first report demonstrating the activity of cis-regulatory elements from the hSHOX and mShox2 genomic regions in mammalian embryos.

Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves.

BACKGROUND: Axons from the visceral motor neurons (vMNs) project from nuclei in the hindbrain to innervate autonomic ganglia and branchial arch-derived muscles. Although much is known about the events that govern specification of somatic motor neurons, the genetic pathways responsible for the development of vMNs are less well characterized. We know that vMNs, like all motor neurons, depend on sonic hedgehog signaling for their generation. Similarly, the paired-like homeobox 2b (Phox2b) gene, which is expressed in both proliferating progenitors and post-mitotic motor neurons, is essential for the development of vMNs. Given that our previous study identified a novel role for the short stature homeobox 2 (Shox2) gene in the hindbrain, and since SHOX2 has been shown to regulate transcription of islet 1 (Isl1), an important regulator of vMN development, we sought to determine whether Shox2 is required for the proper development of the facial motor nucleus. RESULTS: Using a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs. We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants. Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants. CONCLUSIONS: Combined, our data show that Shox2 is required for development of the facial motor nucleus and its associated facial (VII) nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region.

Shox2 regulates progression through chondrogenesis in the mouse proximal limb.

In humans, loss of SHOX gene function is responsible for the mesomelic short stature characteristic of Turner syndrome, Leri-Weill dyschondrosteosis, and Langer dysplasia. In a mouse model of SHOX deficiency, Prrx1-Cre-driven limb-specific deletion of the paralogous gene Shox2 results in severe rhizomelia. In this study, we show that Col2a1-Cre-driven deletion of Shox2 in developing chondrocytes also results in shortening of the stylopodial skeleton (i.e. humerus, femur) and that this rhizomelia is due to precocious chondrocyte maturation and hypertrophy. We demonstrate, using the micromass culture model system, that increased BMP activity triggers accelerated maturation and hypertrophy in Col2a1-Cre Shox2 mutant chondrocytes and we confirm in vivo that elevated transcript levels and expanded expression domains of Bmp2 and 4 are associated with premature formation of the hypertrophic zone in mutant humeri. In micromass cultures of Prrx1-Cre Shox2 mutant limb cells, we find that Shox2 deletion in undifferentiated mesenchymal cells results in increased BMP activity that enhances early chondrogenesis, but is insufficient to provoke chondrocyte maturation and hypertrophy. Similarly, shRNA-mediated Shox2 knockdown in multipotent C3H10T1/2 cells and primary mouse bone marrow mesenchymal stem cells results in spontaneous chondrogenesis in the absence of chondrostimulation, but again fails to induce progression through the later stages of chondrogenic differentiation. Importantly, exogenous BMP supplementation can overcome the block to maturation and hypertrophy caused by Shox2 depletion prior to overt chondrogenesis. Thus, we provide evidence that Shox2 regulates progression through chondrogenesis at two distinct stages--the onset of early differentiation and the transition to maturation and hypertrophy.

mRNA fluorescence in situ hybridization to determine overlapping gene expression in whole-mount mouse embryos.

BACKGROUND: Whole-mount in situ hybridization (ISH) is a prevalent tool to examine the spatial distribution of gene transcripts in intact embryos. Chromogenic-based methods of signal development are commonly used in mouse embryos because of their high sensitivity. Fluorescence techniques, however, offer several advantages over chromogenic methods including the ability to visualize multiple signals in a specimen at once. RESULTS: We describe a procedure for fluorescence in situ hybridization (FISH) for whole mouse embryos up to embryonic day 13.5. We show that this approach successfully produces a bright expression signal for several genes, validating the procedure in multiple tissues. Further, we show that double FISH can be used to visualize the expression of two genes in a single embryo by determining that Hoxd13 and Shh are co-expressed in both the limb bud and the hindgut. Finally, we demonstrate that FISH can be paired with confocal microscopy to take optical sections of interior regions of the embryo. CONCLUSIONS: FISH is a valid alternative to chromogenic-based ISH for visualizing gene expression in whole mouse embryos. This work provides a framework to add additional fluorescence signals in the mouse such as visualizing both mRNA and protein by pairing the procedure with immunofluorescence.

Spinal cord injury community survey: a national, comprehensive study to portray the lives of canadians with spinal cord injury.

BACKGROUND: To better understand service-related needs and the current situation of persons with spinal cord injury (SCI) living in the community, a more comprehensive approach for studying their interrelationships (needs vs community living outcomes) is greatly needed. OBJECTIVE: To describe the development, design, and findings of a Canadian survey portraying the life situation of people with SCI. METHOD: The SCI Community Survey covers demographics, health, SCI-specific needs, community participation, employment, quality of life, health care utilization, and overall health rating. A total of 1,549 persons with SCI completed the survey (Web or phone) between May 2011 and August 2012. RESULTS: Some major expressed needs for services to support community living are met to a great extent for a substantial proportion of people with SCI. Complications remain highly prevalent for some health issues, including pain, sexual dysfunction, and musculoskeletal disorders. The extent of community participation based on values and preferences varies tremendously among daily activities and social roles. Some dimensions of quality of life are rated positively (eg, family life) while others are greatly disrupted (eg, sex life and physical health). Most of these findings vary significantly between people with traumatic and nontraumatic lesions. CONCLUSION: This survey is the first in Canada and among the first worldwide to draw a comprehensive picture of major aspects of the lives of people with SCI including service needs. The results will help to determine the links between various aspects of community living and guide service providers and policy makers in focusing on major issues to enhance quality of life after SCI.

Spinal Cord Injury Community Survey: Understanding the Needs of Canadians with SCI.

BACKGROUND: There is a lack of literature regarding service needs of people with SCI living in the community. Better assessment of expressed and met and unmet needs would help in the development of effective service delivery. OBJECTIVE: From a national SCI Community Survey in Canada, the aim was to identify the most critical service needs of people living in the community at least 1 year post discharge from rehabilitation and the support they received to meet their needs. METHOD: Data were collected mainly through a secure Web site and encompassed demographics, personal and household income, an SCI severity measure, and an SCI community needs measure containing information on 13 SCI-related needs. RESULTS: A total of 1,549 persons with SCI (traumatic lesion, n = 1,137; nontraumatic lesion, n = 412) across Canada completed the survey. Most critical needs for community integration were expressed by a substantial proportion of survey participants, but significantly more expressed and met needs were reported by persons with a traumatic than a nontraumatic lesion. Personal and environmental characteristics influenced the probability of expressing and meeting needs (eg, severity of injury and household income). Help and support to meet expressed needs were received from government agencies, community organizations, and friends or family. CONCLUSION: Better assessment of expressed and met or unmet needs for services remains a challenge but will serve as a tool to optimize service delivery in the community. Environmental barriers to services, particularly the process of getting needs met and associated costs, remain an issue that requires a reconsideration of some aspects of access to services.

An Exploratory Analysis of the Potential Association Between SCI Secondary Health Conditions and Daily Activities.

BACKGROUND: Secondary health conditions (SHCs) are common following traumatic spinal cord injury (tSCI) and are believed to influence a person's ability to participate in daily activities (DAs). This association should be understood so that health care providers may target interventions with clarity and purpose to manage SHCs and facilitate DAs to maximal effect. OBJECTIVE: To explore the association between SHCs and DAs expressed as the increased chance of not participating as much as wanted in a DA when an SHC is present. METHODS: Community-dwelling persons with tSCI (n = 1,137) responded to the SCI Community Survey. The occurrence and frequency of 21 SHCs were determined. The extent of participation in 26 DAs was measured. The relative risk (RR) of not participating as much as wanted in a DA when a SHC is present was calculated. RESULTS: When some SHC were present, the RR of not participating as much as wanted increased significantly (range, 15%-153%; P < .001). Certain SHCs (light-headedness/dizziness, fatigue, weight problems, constipation, shoulder problems) were associated with a greater chance of not participating in many DAs. No single SHC was associated with every DA and conversely not every DA was associated with an SHC. CONCLUSIONS: Maximizing participation in DAs requires minimizing SHCs in every instance. Understanding the association between SHCs and DAs may facilitate targeted care resulting in less severe SHCs, greater participation in DAs, and benefits to both the individual and society.

Health care utilization in persons with traumatic spinal cord injury: the importance of multimorbidity and the impact on patient outcomes.

BACKGROUND: Persons with spinal cord injury (SCI) living in the community have high health care utilization (HCU). To date, the interrelationships among multiple secondary health conditions (multimorbidity due to comorbidities and complications) that drive HCU and their impact on patient outcomes are unknown. OBJECTIVE: To determine the association among multimorbidity, HCU, health status, and quality of life. METHODS: Community-dwelling persons with traumatic SCI participated in an online/phone SCI Community Survey. Participants were grouped using the 7-item HCU questionnaire (group 1 did not receive needed care and/or rehospitalized; group 2 received needed care but rehospitalized; group 3 received needed care and not rehospitalized). Personal, injury, and environmental factors; multimorbidity (presence/absence of 30 comorbidities/ complications); health status (Short Form-12); and quality of life measures (Life Satisfaction-11 first question and single-item quality of life measure) were collected. Associations among these variables were assessed using multivariate analysis. RESULTS: The 1,137 survey participants were divided into 3 groups: group 1 (n = 292), group 2 (n = 194), and group 3 (n = 650). Group 1 had the greatest number of secondary health conditions (15.14 ± 3.86) followed by group 2 (13.60 ± 4.00) and group 3 (12.00 ± 4.16) (P < .05). Multimorbidity and HCU were significant risk factors for having a lower SF-12 Mental (P < .001) and Physical Component Score (P < .001). They in turn were associated with participants reporting a lower quality of life (P < .001, for both questions). CONCLUSIONS: Multimorbidity and HCU are interrelated and associated with lower health status, which in turn is associated with lower quality of life. Future work will include the development of a screening tool to identify persons with SCI at risk of inappropriate HCU (eg, rehospitalization, not able to access care), which should lead to better patient outcomes and cost savings.

Development and assessment of a community follow-up questionnaire for the Rick Hansen spinal cord injury registry.

OBJECTIVE: To develop a comprehensive community follow-up questionnaire for participants enrolled in the Rick Hansen Spinal Cord Injury Registry (RHSCIR). DESIGN: Development and preliminary assessment of measurement properties (reliability and validity) of instruments used during a community follow-up and aligned with the International Classification of Functioning, Disability and Health (ICF). SETTING: General community setting. PARTICIPANTS: People with spinal cord injury (N=50) living in the community. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: A comprehensive follow-up questionnaire, referred to as the RHSCIR Community Follow-up Questionnaire Version 2.0 (CFQ-V2.0), includes 8 instruments. Four new instruments were developed, 2 existing instruments were modified, and 2 previously published instruments were included. RESULTS: Intra- and interrater reliability statistics (Gwet's AC1) support the measurement properties of the new and modified instruments. Correlations between new and existing instruments and between groups based on the severity of injury support the construct validity of the secondary complications and person-perceived participation instruments. CONCLUSIONS: The RHSCIR CFQ-V2.0 is a comprehensive community follow-up questionnaire that aligns to the ICF. Initial study results suggest that it covers all relevant aspects of community living, and the measurement properties are promising.

Novel Candidate Regulators and Developmental Trajectory of Pituitary Thyrotropes.

The pituitary gland regulates growth, metabolism, reproduction, the stress response, uterine contractions, lactation, and water retention. It secretes hormones in response to hypothalamic input, end organ feedback, and diurnal cues. The mechanisms by which pituitary stem cells are recruited to proliferate, maintain quiescence, or differentiate into specific cell types, especially thyrotropes, are not well understood. We used single-cell RNA sequencing in juvenile P7 mouse pituitary cells to identify novel factors in pituitary cell populations, with a focus on thyrotropes and rare subtypes. We first observed cells coexpressing markers of both thyrotropes and gonadotropes, such as Pou1f1 and Nr5a1. This was validated in vivo by both immunohistochemistry and lineage tracing of thyrotropes derived from Nr5a1-Cre; mTmG mice and demonstrates that Nr5a1-progenitors give rise to a proportion of thyrotropes during development. Our data set also identifies novel factors expressed in pars distalis and pars tuberalis thyrotropes, including the Shox2b isoform in all thyrotropes and Sox14 specifically in Pou1f1-negative pars tuberalis thyrotropes. We have therefore used single-cell transcriptomics to determine a novel developmental trajectory for thyrotropes and potential novel regulators of thyrotrope populations.

A conditional allele of Rspo3 reveals redundant function of R-spondins during mouse limb development.

R-spondins are secreted ligands that bind cell surface receptors and activate Wnt/ß-catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1-4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R-spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R-spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2-4 of Rspo3, allowing tissue-specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes.

Transcription factor short stature homeobox 2 is required for proper development of tropomyosin-related kinase B-expressing mechanosensory neurons.

Dorsal root ganglia (DRG) contain somatosensory neurons of diverse sensory modalities. Among these different types of sensory neurons, the molecular mechanisms that regulate the development and specification of touch neurons are the least well understood. We took a candidate approach and searched for transcription factors that are expressed in subsets of DRG neurons, and found that the transcription factor Shox2 (short stature homeobox 2) is expressed in subpopulations of TrkB (tropomyosin-related kinase B)- and Ret-expressing neurons at neonatal stages. Since TrkB is a known marker that is selectively expressed in touch sensory neurons, we decided to examine the function of Shox2 in specifying TrkB-positive DRG neurons. Conditional deletion of Shox2 in neural crest cells (which give rise to all DRG neurons) caused a 60 ∼ 65% reduction in the number of TrkB-expressing neurons. It also resulted in an increase in coexpression of TrkC in Ret-positive sensory neurons. Deletion of Shox2 in differentiating DRG neurons at later time points caused only a moderate reduction in TrkB expression. Overexpression of Shox2 in all neural crest cells resulted in a small increase in the number of TrkB-expressing neurons. Finally, Shox2 deletion also caused reduced touch sensory axonal innervation to layers III/IV of the spinal cord. Together, our findings identify Shox2 as an essential but not sufficient component of the transcription programs required in neural progenitor cells for the proper specification of subsets of TrkB-expressing touch/mechanosensory neurons.

Shox2 function couples neural, muscular and skeletal development in the proximal forelimb.

The mouse Shox2 gene codes for a homeodomain transcription factor that is required to form the proximal bones of the limbs, the humerus and femur. Shox2 is the only gene known to be essential for the specific development of these skeletal elements. Shox2 is also of special interest because it is closely related to the human SHOX gene, deficiencies of which cause the short stature in Turner, Langer and Léri-Weill syndromes. In order to understand in more detail the development of the proximal limb, we searched for Shox2-dependent gene expression patterns using Affymetrix microarrays. We compared the mRNA of Shox2-mutant and wild-type forelimb buds at 10.5 and 11.5 days of embryonic development (E10.5 and E11.5) and successfully identified a set of genes whose wild-type expression pattern requires Shox2 function, as confirmed by in situ hybridization for eleven of the candidates. Strikingly, several of the identified genes were predicted to have functions in tissues other than the skeleton, including nerves and muscle precursors, prompting us to analyze neural and muscular patterning in Shox2 mutants. We report here an axonal migration defect in Shox2 mutants resulting in a profound innervation deficiency of the dorsal forelimb, including the complete absence of the radial and axillary nerves. Muscular development was also altered as early as E11.5. Specifically, the triceps muscles that develop along the posterior face of the humerus had severe abnormalities. These data demonstrate that Shox2 is required for normal skeletal, neural and muscular development in the forelimb at a similar early developmental stage in each tissue.

Interspecies transcriptomics identify genes that underlie disproportionate foot growth in jerboas.

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has "mouse-like" arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as "nodes" for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

R-Spondin 3 Regulates Mammalian Dental and Craniofacial Development.

Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.