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The characterization of developmental phenotypes often relies on the accurate linear measurement of structures that are small and require laborious preparation. This is tedious and prone to errors, especially when repeated for the multiple replicates that are required for statistical analysis, or when multiple distinct structures have to be analysed. To address this issue, we have developed a pipeline for characterization of long-bone length and inter-vertebral distance using X-ray microtomography (XMT) scans. The pipeline involves semi-automated algorithms for automatic thresholding and fast interactive isolation and 3D-model generation of the main limb bones, using either the open-source ImageJ plugin BoneJ or the commercial Mimics Innovation Suite package. The tests showed the appropriate combination of scanning conditions and analysis parameters yields fast and comparable length results, highly correlated with the measurements obtained via ex vivo skeletal preparations. Moreover, since XMT is not destructive, the samples can be used afterwards for histology or other applications. Our new pipelines will help developmental biologists and evolutionary researchers to achieve fast, reproducible and non-destructive length measurement of bone samples from multiple animal species.Summary statementBeltran Diaz et al. present a semi-automated pipeline for fast and versatile characterization of bone length from micro-CT images of mouse developmental samples.

A transcription factor gradient conveys positional information in the vertebrate limb bud., The positional information theory proposes that a coordinate system provides information to embryonic cells about their position and orientation along a patterning axis. Cells interpret this information to produce the appropriate pattern. During development, morphogens and interpreter transcription factors provide this information. We report a gradient of Meis homeodomain transcription factors along the mouse limb bud proximo-distal (PD) axis antiparallel to and shaped by the inhibitory action of distal fibroblast growth factor (FGF). Elimination of Meis results in premature limb distalization and HoxA expression, proximalization of PD segmental borders, and phocomelia. Our results show that Meis transcription factors interpret FGF signaling to convey positional information along the limb bud PD axis. These findings establish a new model for the generation of PD identities in the vertebrate limb and provide a molecular basis for the interpretation of FGF signal gradients during axial patterning.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

The study of the mechanisms controlling organ size during development and regeneration is critical to understanding how complex life arises from cooperating single cells. Long bones are powerful models in this regard, as their size depends on a scaffold made from another tissue (cartilage, composed of chondrocytes), and both tissues interact during the growth period. Investigating long bone growth offers a valuable window into the processes that integrate internal and external cues to yield finely controlled size of organs. Within the cellular and molecular pathways that control bone growth, the regulation of stem-cell renewal, along with amplification and differentiation of their progeny, are key to understanding normal and perturbed long-bone development. The phenomenon of "catch-up" growth-where cellular hyperproliferation occurs following injury to restore a normal growth trajectory-reveals key aspects of this regulation, such as the fact that bone growth is target-seeking. The control mechanisms that lead to this behavior are either bottom-up or top-down, and the interaction between these modes is likely critical to achieve a highly nuanced, yet flexible, degree of control. The role of cartilage-intrinsic mechanisms has been well studied, establishing a very solid groundwork for this field. However, addressing the unanswered questions of bone growth arguably requires new hypotheses and approaches. Future research could for example address to what extent extrinsic signals and cells, as well as communication with other tissues, modulate intra-limb and inter-organ growth coordination. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Vertebrate Organogenesis > Musculoskeletal and Vascular.

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches utilizing two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach utilizes available lines expressing tTA/rTA to control timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf) and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.Summary statementAhmadzadeh et al. present a collection of four mouse lines and genetic tools for misexpression-mediated manipulation of cellular activity with high spatiotemporal control, in a reversible manner.

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

The regulation of organ size is essential to human health and has fascinated biologists for centuries. Key to the growth process is the ability of most organs to integrate organ-extrinsic cues (eg, nutritional status, inflammatory processes) with organ-intrinsic information (eg, genetic programs, local signals) into a growth response that adapts to changing environmental conditions and ensures that the size of an organ is coordinated with the rest of the body. Paired organs such as the vertebrate limbs and the long bones within them are excellent models for studying this type of regulation because it is possible to manipulate one member of the pair and leave the other as an internal control. During development, growth plates at the end of each long bone produce a transient cartilage model that is progressively replaced by bone. Here, we review how proliferation and differentiation of cells within each growth plate are tightly controlled mainly by growth plate-intrinsic mechanisms that are additionally modulated by extrinsic signals. We also discuss the involvement of several signaling hubs in the integration and modulation of growth-related signals and how they could confer remarkable plasticity to the growth plate. Indeed, long bones have a significant ability for “catch-up growth” to attain normal size after a transient growth delay. We propose that the characterization of catch-up growth, in light of recent advances in physiology and cell biology, will provide long sought clues into the molecular mechanisms that underlie organ growth regulation. Importantly, catch-up growth early in life is commonly associated with metabolic disorders in adulthood, and this association is not completely understood. Further elucidation of the molecules and cellular interactions that influence organ size coordination should allow development of novel therapies for human growth disorders that are noninvasive and have minimal side effects.

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions. DOI: http://dx.doi.org/10.7554/eLife.27210.001, eLife digest As bones grow, their size is carefully controlled and coordinated with the growth of the other organs in the body. The mechanisms that control organ size also help the body to recover from injury, and play a key role in controlling body size and proportions. Over the course of evolution, these mechanisms have likely changed to produce the distinct body sizes and proportions seen in humans and other animals. Despite their importance, it is not well understood how signals from both inside and outside an organ work together to regulate its size. In growth disorders this signaling goes wrong, which can lead to a person having unusual proportions such as a very short stature or having one leg shorter than the other. Currently, most growth disorders that affect leg proportions are treated with painful surgical procedures. Researchers would like to know how bone growth is affected by signals from the surrounding tissues because this could help them to develop new non-invasive treatments for these conditions. Long bones, for example those in the leg, grow from structures near their ends called growth plates. Roselló-Díez et al. have now engineered mice in which an injury shortly after birth caused cells in the knee in the rear left leg to die off. At the same time, the rear right leg of the mice developed as normal, allowing the growth of the two legs to be compared. Roselló-Díez et al. found that the left leg of these mice grew more slowly than the right leg, even though none of the cells in the growth plate of the left leg bone had been damaged. Further investigation revealed that this was because the injury caused an imbalance between the growth-promoting and growth-restricting signals that are produced by the fat pad and articular cartilage in the knee joint. Restoring the lost balance allowed the left leg bone to grow to a more normal length. In the future, boosting bone growth signals might provide a way to treat conditions like dwarfism or leg-length discrepancies. Understanding how different tissues influence body proportions could also help researchers to investigate how different animals evolved different body proportions. DOI: http://dx.doi.org/10.7554/eLife.27210.002

Summary Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor, and therefore, plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood, and vasculature. In addition, Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and non-homeodomain transcription factors. During post-natal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with pre-disposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1-expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1-expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and disease. genesis 52:967–975, 2014. © 2014 Wiley Periodicals, Inc.

Developing vertebrate limbs initiate proximo-distal patterning by interpreting opposing gradients of diffusible signaling molecules. We report two thresholds of proximo-distal signals in the limb bud: a higher threshold that establishes the upper-arm to forearm transition; and a lower one that positions a later transition from forearm to hand. For this last transition to happen, however, the signal environment seems to be insufficient, and we show that a timing mechanism dependent on histone acetylation status is also necessary. Therefore, as a consequence of the time dependence, the lower signaling threshold remains cryptic until the timing mechanism reveals it. We propose that this timing mechanism prevents the distal transition from happening too early, so that the prospective forearm has enough time to expand and form a properly sized segment. Importantly, the gene expression changes provoked by the first transition further regulate proximo-distal signal distribution, thereby coordinating the positioning of the two thresholds, which ensures robustness. This model is compatible with the most recent genetic analyses and underscores the importance of growth during the time-dependent patterning phase, providing a new mechanistic framework for understanding congenital limb defects.

Background The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies. Methods and Results In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1−/+;En2−/−), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice. Conclusions Our results reveal that gc production is differentially regulated in each zone of the cerebellar vermis, and our mutant analysis indicates that the dynamics of gc production plays a role in determining the 3D structure of the Cb. Electronic supplementary material The online version of this article (doi:10.1186/s13064-016-0072-z) contains supplementary material, which is available to authorized users.

We have used the chick limb as a model to gain insight into the longstanding question of regulative vs. mosaic development. To test the influence of signals on limb proximodistal development, distal limb bud tips of several stages were grafted to regions of the embryo known to provide different signaling environments. Of interest, thin grafts (100-micron thick) formed elements more proximal in character when grafted to the proximal limb region than when grafted to other regions. The extra elements were derived from host tissue, presumably distalized and recruited by the graft's apical ectodermal ridge signals. The results of classic and recent experiments have been reinterpreted in light of our conclusions.

Report, Vertebrate limbs develop three main proximodistal (PD) segments (upper arm, forearm, and hand) in a proximal-to-distal sequence. Despite extensive research into limb development, whether PD specification occurs through nonautonomous or autonomous mechanisms is not resolved. Heterotopic transplantation of intact and recombinant chicken limb buds identifies signals in the embryo trunk that proximalize distal limb cells to generate a complete PD axis. In these transplants, retinoic acid induces proximalization, which is counteracted by fibroblast growth factors from the distal limb bud; these related actions suggest that the first limb-bud PD regionalization results from the balance between proximal and distal signals. The plasticity of limb progenitor cell identity in response to diffusible signals provides a unifying view of PD patterning during vertebrate limb development and regeneration., Supported by grants RD06/0010/0008, BFU2009-08331/BMC (M.T.) and BFU2008-00397 (M.A.R.), from the Spanish Ministry of Science and Innovation (MICINN) and fellowship CPI/0051/2007 (A. R.-D.) from Madrid’s Regional Government (CAM). The CNIC is supported by the MICINN and the Pro-CNIC Foundation.

Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor, and therefore, plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood, and vasculature. In addition, Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and non-homeodomain transcription factors. During post-natal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with pre-disposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1-expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1-expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and disease.

Recently a label-free FLIM imaging method has been proposed to distinguish metabolic states of germ cells (Stringari et al, 2011). This method, based on the phasor analysis of autofluorescence lifetime (AF-phasorFLIM), avoids some problems of state-of-art approaches due to multi-exponential fitting. The capability of phasorFLIM analysis to separate the different molecular species in a pixel lies on the rule of phasor addition. PhasorFLIM identifies molecules by their position in the phasor plot because every molecular species has a specific phasor that can be calibrated in reference samples.We applied phasorFLIM to detect retinoids autofluorescence in live P19 embryonal carcinoma cells that undergo differentiation when treated with retinoic acid (RA).RA and retinol are essential for cell-cell signaling during vertebrate organogenesis. RA is involved in stem cell differentiation and it is a morphogen. It is a diffusible signal that generates patterns in embryo because it induces different cellular responses depending on its local concentration. The presence of RA in tissues has been inferred through gene activation, and the RA average tissular concentration measured by HPLC. However, the spatial distribution in tissues and cells of the active molecule has never been observed or quantified directly.We built a database of phasorFLIM standards of the major molecules that contribute to autofluorescence. The database allows assigning the contribution of fluorescent molecules to P19 autofluorescence.P19 cells were treated with retinol to follow in time the effect of the molecule on cellular metabolism. PhasorFLIM analysis was performed using SimFCS and showed an increase of retinoids fluorescence after treatment. In cells treated with retinol, we observed retinol in plasma and nuclear membrane and an increase of RA in the nucleus.AF-phasorFLIM allowed determining, distinguishing and localizing the contribution of retinoids to autofluorescence in live P19 cells.

A full list of authors and affiliations appears on page 5 of this article., The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10-14), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10-11; ncases = 98,742 and ncontrols = 409,511). Using an En1cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10-11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population., peer-reviewed