Developing Limb Bud Research Articles

Single cell RNA-sequencing reveals cellular heterogeneity and trajectories of lineage specification during murine embryonic limb development

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9’s functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9’s regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.

Comprehensive clinical and molecular studies in split-hand/foot malformation: identification of two plausible candidate genes (LRP6 and UBA2).

Split-hand/foot malformation (SHFM) is a clinically and genetically heterogeneous condition. We sequentially performed screening of the previously identified Japanese founder 17p13.3 duplication/triplication involving BHLHA9, array comparative genomic hybridization, and whole exome sequencing (WES) in newly recruited 41 Japanese families with non-syndromic and syndromic SHFM. We also carried out WES in seven families with nonsyndromic and syndromic SHFM in which underlying genetic causes including pathogenic copy-number variants (CNVs) remained undetected in our previous studies of 56 families. Consequently, we identified not only known pathogenic CNVs (17p13.3 duplications/triplications [n = 21], 2q31 deletion [n = 1], and 10q24 duplications [n = 3]) and rare variants in known causative genes (TP63 [n = 3], DLX5 [n = 1], IGF2 [n = 1], WNT10B [n = 3], WNT10B/PORCN [n = 1], and PORCN [n = 1]), but also a de novo 19q13.11 deletion disrupting UBA2 (n = 1) and variants that probably affect function in LRP6 (n = 1) and UBA2 (n = 1). Thus, together with our previous data based on testing of 56 families, molecular studies for a total of 97 families with SHFM revealed underlying genetic causes in 75 families, and clinical studies for the 75 families indicated a certain degree of correlation between genetic causes and phenotypes. The results imply that SHFM primarily occurs as a genetic disorder with genotype-phenotype correlations. Furthermore, the results together with previous data such as the development of SHFM in Lrp6 knockout mice, the presence of SHFM in two subjects with 19q13 deletions involving UBA2, and strong mouse Uba2 expression in the developing limb buds, imply that LRP6 and UBA2 represent plausible candidate genes for SHFM.

Multiple Variations in Third Segment of Axillary Artery

Variations in the subscapular artery (SA) have been well documented and acknowledged as the most common variation in the axillary artery (AA). Although this is true, a variation of the third portion of the AA was found during dissection of the right upper limb of a female cadaver. This portion of the AA was found to give rise to a proximal SA, followed by a common trunk with 4 ramifications and distally, to the anterior circumflex humeral a. The common trunk gave rise to a second SA, posterior circumflex humeral a., scapular circumflex a., and the thoracodorsal a. Both SA branches were found to be irrigating the subscapularis m., implicating a possible dual and collateral supply. The posterior circumflex humeral a. was identified as it traversed posteriorly to reach the surgical neck of the humerus along with the axillary n. through the quadrangular space. The scapular circumflex a. was identified as it passed backward around the lower border of the scapula into the triangular space. Finally, the thoracodorsal a. gave off a branch to the latissimus dorsi m. and continued its course along the lateral thoracic wall to give off multiple branches to the serratus anterior m. It appears that the thoracodorsal branch reaching the serratus anterior m. is its main source of blood supply since the lateral thoracic a. was not found arising from the AA at any point. These findings are different from what is commonly documented. Usually, the AA is divided into three segments based on its relationship with the pectoralis minor m. The second portion of the AA gives rise to the thoracoacromial a., just posterior to the medial superior margin of the pectoralis m., and to the lateral thoracic a., passing downward behind the pectoralis minor m. along its inferolateral border. The third portion of the AA, which lies on the subscapularis m. and teres major m., gives rise to the anterior circumflex humeral a., the posterior circumflex humeral a., and the SA. The SA is commonly the largest branch of the AA, it runs downward on the costal surface of the subscapularis m. before it divides into the scapular circumflex a. and the thoracodorsal a. These arteries provide the major blood supply to the posterior wall of the axilla. Acknowledging the possibility of a variation from normal anatomy is relevant when interpreting imaging studies such as angiographs or during axillary surgical preparation due to possible pressure on the brachial plexus and vessels of the axilla when there is downward displacement of the head of the humerus. The suitability of the SA system as a donor for arterial grafts during upper limb circulation restoration is of great clinical importance. Their angle of rotation and their large, stable diameter render these arteries appropriate for a variety of flaps in plastic surgery. These clinical correlations can be further studied by exploring the embryology of these arteries. Normally, the subclavian a. arises from the 4th aortic arch and the 7th intersegmental a. It continues to develop as the limb bud grows; therefore, since the variations observed occur past the subclavian a., it seems that the vascular plexus was affected during development of the upper limb bud. Knowing this will help guide surgeons when exposed to such variations if a malformation during fetal limb development is suspected.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synchronization of Hes1 oscillations coordinates and refines condensation formation and patterning of the avian limb skeleton

The tetrapod appendicular skeleton is initiated as spatially patterned mesenchymal condensations. The size and spacing of these condensations in avian limb buds are mediated by a reaction-diffusion-adhesion network consisting of galectins Gal-1A, Gal-8 and their cell surface receptors. In cell cultures, the appearance of condensations is synchronized across distances greater than the characteristic wavelength of their spatial pattern. We explored the possible role of observed oscillations of the transcriptional co-regulator Hes1 in this phenomenon. Treatment of micromass cultures with DAPT, a γ-secretase inhibitor, damped Hes1 oscillations, elevated Gal-1A and -8 mRNA levels, and led to irregularly-sized proto-condensations that subsequently fused. In developing limb buds, DAPT led to spatially non-uniform Hes1 expression and fused, truncated and misshapen digits. Periodicity in adhesive response to Gal-1A, a plausible Hes1-dependent function, was added to a previously tested mathematical model for condensation patterning by the two-galectin network. The enhanced model predicted regularization of patterning due to synchronization of Hes1 oscillations and resulting spatiotemporal coordination of its expression. The model also predicted changes in galectin expression and patterning in response to suppression of Hes1 expression, which were confirmed in in vitro experiments. Our results indicate that the two-galectin patterning network is regulated by Hes1 dynamics, the synchronization of which refines and regularizes limb skeletogenesis.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-β1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-β may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia’s influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-β3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia’s effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. Statement of SignificanceCartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia’s effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia’s chondrogenic effects may be enhanced on soft materials.

Rabbit Whole Embryo Culture.

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

ABSTRACTThe key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

A point mutation in the pre-ZRS disrupts sonic hedgehog expression in the limb bud and results in triphalangeal thumb–polysyndactyly syndrome

PurposeThe zone of polarizing activity regulatory sequence (ZRS) is an enhancer that regulates sonic hedgehog during embryonic limb development. Recently, mutations in a noncoding evolutionary conserved sequence 500 bp upstream of the ZRS, termed the pre-ZRS (pZRS), have been associated with polydactyly in dogs and humans. Here, we report the first case of triphalangeal thumb–polysyndactyly syndrome (TPT-PS) to be associated with mutations in this region and show via mouse enhancer assays how this mutation leads to ectopic expression throughout the developing limb bud. MethodsWe used linkage analysis, whole-exome sequencing, Sanger sequencing, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, single-nucleotide polymorphism array, and a mouse transgenic enhancer assay. ResultsTen members of a TPT-PS family were included in this study. The mutation was linked to chromosome 7q36 (LOD score 3.0). No aberrations in the ZRS could be identified. A point mutation in the pZRS (chr7:156585476G>C; GRCh37/hg19) was detected in all affected family members. Functional characterization using a mouse transgenic enhancer essay showed extended ectopic expression dispersed throughout the entire limb bud (E11.5). ConclusionOur work describes the first mutation in the pZRS to be associated with TPT-PS and provides functional evidence that this mutation leads to ectopic expression of this enhancer within the developing limb.

1, 25-Dihydroxyvitamin D3 suppresses cell cycle progression and thus growth of prostate cancer cells by inducing expression of limb bud and heart development (LBH)

Purpose: To investigate the function of limb bud and heart development (LBH) in 1α, 25-dihydroxyvitamin D3 (1,25D)-mediated inhibitory effect on proliferation of prostate cancer cells. Methods: The inhibitory effect of 1,25D on growth and cell cycle progression of lymph node carcinoma of the prostate (LNCaP) cells was determined using cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry, while the expression levels of LBH in response to treatment with 1,25D were determined by quantitative reverse-transcription PCR (qRT-PCR) and western blottingting. The expression levels of LBH in cells were down/up regulated by transfection with siRNA or overexpression plasmids, and then cell growth and cell cycles were measured using the CCK-8 assay, EdU assay, and flow cytometry. Finally, the growth inhibitory effect of 1,25D on LBH knockdown cells were determined using CCK-8 and EdU assays. Results: Treatment with 1,25D arrested LNCaP cells in G0/G1 phase of cell cycle, suppressed the growth of the cells and induced the expression of LBH. Overexpression/knockdown of LBH in LNCaP cells suppressed/promoted cell growth and accumulated/decreased cells in the G0/G1 phase. Moreover, knockdown of LBH reversed the inhibitory effect of 1,25D on cell proliferation of LNCaP cells. Conclusion: Inhibitory effect of 1,25D on cell cycle progression and cell proliferation might be via LBH. Keywords: Cell proliferation, Limb bud and heart development, Prostate cancer, 1α, 25-Dihydroxyvitamin D3

A quantitative method for staging mouse embryos based on limb morphometry.

To determine the developmental stage of embryonic mice, we apply a geometric morphometric approach to the changing shape of the mouse limb bud as it grows from embryonic day 10 to embryonic day 15 post-conception. As the ontogenetic sequence results in the de novo emergence of shape features not present in the early stages, we have created a standard ontogenetic trajectory for limb bud development - a quantitative characterization of shape change during limb morphogenesis. This trajectory of form as a function of time also gives us the reverse function: the ability to infer developmental stage from form, with a typical uncertainty of 2 h. We introduce eMOSS (embryonic mouse ontogenetic staging system) as a fast, reliable, convenient and freely available online tool for staging embryos from two-dimensional images of their limb buds, and illustrate its use in phenotyping early limb abnormalities.

Methotrexate inhibits effects of platelet-derived growth factor and interleukin-1\u03b2 on rheumatoid arthritis fibroblast-like synoviocytes

BackgroundA key feature of joints in rheumatoid arthritis (RA) is the formation of hyperplastic destructive pannus tissue, which is orchestrated by activated fibroblast-like synoviocytes (FLS). We have demonstrated that the RA risk gene and tumor suppressor Limb bud and heart development (LBH) regulates cell cycle progression in FLS. Methotrexate (MTX) is the first-line treatment for RA, but its mechanisms of action remain incompletely understood. Here, we studied the effects of MTX on mitogen-induced FLS proliferation and expression of cell cycle regulators in vitro.MethodsPrimary FLS from patients with RA or osteoarthritis were stimulated with the mitogen platelet-derived growth factor (PDGF) and the cytokine interleukin-1β (IL-1β) in the presence or absence of MTX. Cells were then subjected to qPCR for gene expression and cell cycle analysis by flow cytometry.ResultsStimulation with PDGF and IL-1β increased the percentage of FLS in the G2/M phase and shifted the cell morphology to a dendritic shape. These effects were inhibited by MTX. Furthermore, PDGF + IL-1β reduced LBH mRNA expression. However, MTX treatment yielded significantly higher transcript levels of LBH, and of CDKN1A (p21) and TP53 (p53), compared to untreated samples upon mitogen stimulation. The expression of DNA methyltransferase-1 (DNMT1) was also higher in the presence of MTX and there was strong correlation between DNMT1 and LBH expression.ConclusionsTherapeutic concentrations of MTX abolish the effects of PDGF and IL-1β on tumor suppressor expression and inhibit mitogen-promoted FLS proliferation. These data demonstrate novel and important effects of MTX on pathogenic effector cells in the joint, which might involve epigenetic mechanisms.

Comparative Genomics Reveals Accelerated Evolution in Conserved Pathways during the Diversification of Anole Lizards.

Squamates include all lizards and snakes, and display some of the most diverse and extreme morphological adaptations among vertebrates. However, compared with birds and mammals, relatively few resources exist for comparative genomic analyses of squamates, hampering efforts to understand the molecular bases of phenotypic diversification in such a speciose clade. In particular, the ∼400 species of anole lizard represent an extensive squamate radiation. Here, we sequence and assemble the draft genomes of three anole species—Anolis frenatus, Anolis auratus, and Anolis apletophallus—for comparison with the available reference genome of Anolis carolinensis. Comparative analyses reveal a rapid background rate of molecular evolution consistent with a model of punctuated equilibrium, and strong purifying selection on functional genomic elements in anoles. We find evidence for accelerated evolution in genes involved in behavior, sensory perception, and reproduction, as well as in genes regulating limb bud development and hindlimb specification. Morphometric analyses of anole fore and hindlimbs corroborated these findings. We detect signatures of positive selection across several genes related to the development and regulation of the forebrain, hormones, and the iguanian lizard dewlap, suggesting molecular changes underlying behavioral adaptations known to reinforce species boundaries were a key component in the diversification of anole lizards.

Polarizing Region Tissue Grafting in the Chick Embryo Limb Bud.

The polarizing region of the developing limb bud is an important organizing center that is involved in anteroposterior (thumb to little finger) patterning and has three main functions that are now considered to depend on the secreted protein Sonic hedgehog (Shh). These are (1) specifying anteroposterior positional values by autocrine and graded paracrine signaling; (2) promoting growth in adjacent mesenchyme; (3) maintaining the distal epithelium that is essential for limb outgrowth by induction of a factor in adjacent mesenchyme. The polarizing region was identified using classical tissue grafting techniques in chicken embryos. Here we describe this procedure using tissue from transgenic Green Fluorescent Protein-expressing chicken embryos that allows the long-term fate of the polarizing region to be determined. This technique provides a highly useful and effective method to understand how the polarizing region patterns the limb and has implications for other organizing centers.

Reactivation of larval keratin gene (krt62.L) in blastema epithelium during Xenopus froglet limb regeneration

Limb regeneration is considered a form of limb redevelopment because of the molecular and morphological similarities. Forming a regeneration blastema is, in essence, creating a developing limb bud in an adult body. This reactivation of a developmental process in a mature body is worth studying. Xenopus laevis has a biphasic life cycle that involves distinct larval and adult stages. These distinct developmental stages are useful for investigating the reactivation of developmental processes in post-metamorphic frogs (froglets). In this study, we focused on the re-expression of a larval gene (krt62.L) during Xenopus froglet limb regeneration. Recently renamed krt62.L, this gene was known as the larval keratin (xlk) gene, which is specific to larval-tadpole stages. During limb regeneration in a froglet, krt62.L was re-expressed in a basal layer of blastema epithelium, where adult-specific keratin (Krt12.6.S) expression was also observable. Nerves produce important regulatory factors for amphibian limb regeneration, and also play a role in blastema formation and maintenance. The effect of nerve function on krt62.L expression could be seen in the maintenance of krt62.L expression, but not in its induction. When an epidermis-stripped limb bud was grafted in a froglet blastema, the grafted limb bud could reach the digit-forming stage. This suggests that krt62.L-positive froglet blastema epithelium is able to support the limb development process. These findings imply that the developmental process is locally reactivated in an postmetamorphic body during limb regeneration.

Effects of insulin like growth factors on early embryonic chick limb myogenesis.

Limb muscles derive from pax3 expressing precursor cells that migrate from the hypaxial somite into the developing limb bud. Once there they begin to differentiate and express muscle determination genes such as MyoD. This process is regulated by a combination of inductive or inhibitory signals including Fgf18, retinoic acid, HGF, Notch and IGFs. IGFs are well known to affect late stages of muscle development and to promote both proliferation and differentiation. We examined their roles in early stage limb bud myogenesis using chicken embryos as an experimental model. Grafting beads soaked in purified recombinant IGF-I, IGF-II or small molecule inhibitors of specific signaling pathways into developing chick embryo limbs showed that both IGF-I and IGF-II induce expression of the early stage myogenic markers pax3 and MyoD as well as myogenin. Their effects on pax3 and MyoD expression were blocked by inhibitors of both the IGF type I receptor (picropodophyllotoxin, PPP) and MEK (U0126). The PI3K inhibitor LY294002 blocked IGF-II, but not IGF-I, induction of pax3 mRNA as well as the IGF-I, but not IGF-II, induction of MyoD mRNA. In addition SU5402, an FGFR/ VEGFR inhibitor, blocked the induction of MyoD by both IGFs but had no effect on pax3 induction, suggesting a role for FGF or VEGF signaling in their induction of MyoD. This was confirmed by in situ hybridization showing that FGF18, a known regulator of MyoD in limb myoblasts, was induced by IGF-I. In addition to their well-known effects on later stages of myogenesis via their induction of myogenin expression, both IGF-I and IGF-II induced pax3 and MyoD expression in developing chick embryos, indicating that they also regulate early stages of myogenesis. The data suggests that the IGFs may have slightly different effects on IGF1R signal transduction via PI3K and that their stimulatory effects on MyoD expression may be indirect, possibly via induction of FGF18 expression.

Regulation of the Cell Cycle and Inflammatory Arthritis by the Transcription Cofactor LBH Gene.

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display unique aggressive behavior, invading the articular cartilage and promoting inflammation. Using an integrative analysis of RA risk alleles, the transcriptome and methylome in RA FLS, we recently identified the limb bud and heart development (LBH) gene as a key dysregulated gene in RA and other autoimmune diseases. Although some evidence suggests that LBH could modulate the cell cycle, the precise mechanism is unknown and its impact on inflammation in vivo has not been defined. Our cell cycle analysis studies show that LBH deficiency in FLS leads to S-phase arrest and failure to progress through the cell cycle. LBH-deficient FLS had increased DNA damage and reduced expression of the catalytic subunit of DNA polymerase α. Decreased DNA polymerase α was followed by checkpoint arrest due to phosphorylation of checkpoint kinase 1. Because DNA fragments can increase arthritis severity in preclinical models, we then explored the effect of LBH deficiency in the K/BxN serum transfer model. Lbh knockout exacerbated disease severity, which is associated with elevated levels of IL-1β and checkpoint kinase 1 phosphorylation. These studies indicate that LBH deficiency induces S-phase arrest that, in turn, exacerbates inflammation. Because LBH gene variants are associated with type I diabetes mellitus, systemic lupus erythematosus, RA, and celiac disease, these results suggest a general mechanism that could contribute to immune-mediated diseases.

Mechanical transformations of living cavitary bodies

All living organisms and many of their parts are organised in an essentially similar manner: they are closed cavitary bodies consisting of 1) the inner mass and 2) a relatively thin sheath enclosing it. This organisation allows living bodies to change shape by employing hydrostatic forces. These forces are generally recognised to govern changes of shape in walled cells. To explain transformations of other organisms, other factors are usually sought. In this paper, the hydrostatic mechanism is represented as a universal mode of shape formation. It acts in all kinds of organisms, determining the course of diverse processes such as development of cell outgrowths, limb buds, gut derivatives and sense organs; endocytosis; cell division; branching of capillaries; gastrulation; cell locomotion; muscle contraction etc.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Pitx1 determines characteristic hindlimb morphologies in cartilage micromass culture.

The shapes of homologous skeletal elements in the vertebrate forelimb and hindlimb are distinct, with each element exquisitely adapted to their divergent functions. Many of the signals and signalling pathways responsible for patterning the developing limb bud are common to both forelimb and hindlimb. How disparate morphologies are generated from common signalling inputs during limb development remains poorly understood. We show that, similar to what has been shown in the chick, characteristic differences in mouse forelimb and hindlimb cartilage morphology are maintained when chondrogenesis proceeds in vitro away from the endogenous limb bud environment. Chondrogenic nodules that form in high-density micromass cultures derived from forelimb and hindlimb buds are consistently different in size and shape. We described analytical tools we have developed to quantify these differences in nodule morphology and demonstrate that characteristic hindlimb nodule morphology is lost in the absence of the hindlimb-restricted limb modifier gene Pitx1. Furthermore, we show that ectopic expression of Pitx1 in the forelimb is sufficient to generate nodule patterns characteristic of the hindlimb. We also demonstrate that hindlimb cells are less adhesive to the tissue culture substrate and, within the limb environment, to the extracellular matrix and to each other. These results reveal autonomously programmed differences in forelimb and hindlimb cartilage precursors of the limb skeleton are controlled, at least in part, by Pitx1 and suggest this has an important role in generating distinct limb-type morphologies. Our results demonstrate that the micromass culture system is ideally suited to study cues governing morphogenesis of limb skeletal elements in a simple and experimentally tractable in vitro system that reflects in vivo potential.