Mouse Forelimb Buds Research Articles

Conserved and species-specific chromatin remodeling and regulatory dynamics during mouse and chicken limb bud development

Chromatin remodeling and genomic alterations impact spatio-temporal regulation of gene expression, which is central to embryonic development. The analysis of mouse and chicken limb development provides important insights into the morphoregulatory mechanisms, however little is known about the regulatory differences underlying their morphological divergence. Here, we identify the underlying shared and species-specific epigenomic and genomic variations. In mouse forelimb buds, we observe striking synchrony between the temporal dynamics of chromatin accessibility and gene expression, while their divergence in chicken wing buds uncovers species-specific regulatory heterochrony. In silico mapping of transcription factor binding sites and computational footprinting establishes the developmental time-restricted transcription factor-DNA interactions. Finally, the construction of target gene networks for HAND2 and GLI3 transcriptional regulators reveals both conserved and species-specific interactions. Our analysis reveals the impact of genome evolution on the regulatory interactions orchestrating vertebrate limb bud morphogenesis and provides a molecular framework for comparative Evo-Devo studies.

Developmental hourglass and heterochronic shifts in fin and limb development.

How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark (Chiloscyllium punctatum). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls.

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.

RING1 proteins contribute to early proximal-distal specification of the forelimb bud by restricting Meis2 expression.

Polycomb group (PcG) proteins play a pivotal role in silencing developmental genes and help to maintain various stem and precursor cells and regulate their differentiation. PcG factors also regulate dynamic and complex regional specification, particularly in mammals, but this activity is mechanistically not well understood. In this study, we focused on proximal-distal (PD) patterning of the mouse forelimb bud to elucidate how PcG factors contribute to a regional specification process that depends on developmental signals. Depletion of the RING1 proteins RING1A (RING1) and RING1B (RNF2), which are essential components of Polycomb repressive complex 1 (PRC1), led to severe defects in forelimb formation along the PD axis. We show that preferential defects in early distal specification in Ring1A/B-deficient forelimb buds accompany failures in the repression of proximal signal circuitry bound by RING1B, including Meis1/2, and the activation of distal signal circuitry in the prospective distal region. Additional deletion of Meis2 induced partial restoration of the distal gene expression and limb formation seen in the Ring1A/B-deficient mice, suggesting a crucial role for RING1-dependent repression of Meis2 and likely also Meis1 for distal specification. We suggest that the RING1-MEIS1/2 axis is regulated by early PD signals and contributes to the initiation or maintenance of the distal signal circuitry.

Regionalized Twist1 activity in the forelimb bud drives the morphogenesis of the proximal and preaxial skeleton

Development of the mouse forelimb bud depends on normal Twist1 activity. Global loss of Twist1 function before limb bud formation stops limb development and loss of Twist1 throughout the mesenchyme after limb bud initiation leads to polydactyly, the ulnarization or loss of the radius and malformations and reductions of the shoulder girdle. Here we show that conditional deletion of Twist1 by Mesp1-Cre in the mesoderm that migrates into the anterior-proximal part of the forelimb bud results in the development of supernumerary digits and carpals, the acquisition of ulna-like characteristics by the radius and malformations of the humerus and scapula. The mirror-like duplications and posteriorization of pre-axial tissues are preceded by disruptions to anterior–posterior Shh, Bmp and Fgf signaling gradients and dysregulation of transcription factors that regulate anterior–posterior limb patterning.

Distinct Roles of Hand2 in Initiating Polarity and Posterior Shh Expression during the Onset of Mouse Limb Bud Development

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

Cholesterol Metabolism: the Main Pathway Acting Downstream of Cytochrome P450 Oxidoreductase in Skeletal Development of the Limb

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

Proteomic analysis to characterize differential mouse strain sensitivity to cadmium‐induced forelimb teratogenesis

Cadmium ion (Cd2+) is a ubiquitous environmental contaminant, and it is a potent teratogen in mice. An intraperitoneal dose of 4 mg/kg of CdCl2 at gestational day 9 causes forelimb ectrodactyly in the C57BL/6N mouse strain, but the SWV/Fnn strain is resistant. The objective of this study was to identify differentially displayed proteins in two target tissues for cadmium teratogenesis, and to derive hypotheses regarding the mechanisms involved in the murine strain difference in Cd-induced forelimb ectrodactyly. The global proteomics strategy used two-dimensional polyacrylamide gel electrophoresis for protein separation, and MALDI-TOF-MS and LC-MS/MS for protein identification, to compare and identify proteins in forelimb buds and yolk sacs from the two mouse strains following Cd administration. More than 1,000 protein spots were detected by two-dimensional polyacrylamide gel electrophoresis in day 10.0 mouse forelimb buds and yolk sacs. Thirty-eight proteins had identifiable differences in abundance levels in Cd-treated forelimb buds between the two strains. Of those 38 proteins, 14 could be associated with the unfolded protein response process and seven are associated with actin polymerization. The proteins that were found to be differentially abundant between the strains in yolk sacs that were exposed to CdCl2 were predominantly different than the proteins detected differentially in the limb buds of the two strains with an overlap of approximately 20%. These patterns of differentially displayed proteins rationalize a hypothesis that the differential murine strain response to cadmium-induced forelimb ectrodactyly is due to differences in their pathways for the unfolded protein response and/or actin polymerization.

A teratoproteomics analysis: Heat shock protein 70 is upregulated in mouse forelimb bud by methoxyacetic acid treatment

Methoxyacetic acid (MAA) causes fetal limb abnormalities when the substance is administrated on gestation day (GD) 11 in mice. Limb abnormalities are caused mainly by extensive cell death in the mesoderm of the limb plate. This investigation focused on identifying a protein that is linked with mouse limb teratogenicity. A single dose of MAA at 10 mmol/kg body weight was administered by gavage on GD 11; controls were administered vehicle only. Dams were killed by cervical dislocation 4 hr after treatment and forelimb buds were isolated from both the control and treated embryos. Proteins in forelimb buds GD 11 + 4 hr were precipitated out using 40-60% ammonium sulfate and were then analyzed by 2D SDS-PAGE. Excised protein spots were identified by mass spectrometry and amino acid internal sequence analysis. Identified protein was further confirmed by Western blotting. Two-dimensional gel analysis indicated that 1 protein spot of 81.7 kDa/pI 7.3 was overexpressed, and the protein matched heat shock protein 70 (HSP70; accession no. P08109, SwissProt). The results suggest that MAA, when administered to pregnant mice, upregulates HSP70 in the forelimb buds.

Effect of platelet-derived growth factor isoforms on the migration of mouse embryo limb myogenic cells.

The effect of platelet-derived growth factor (PDGF) isoforms on limb myoblast migration was examined in vitro. Using Blindwell Chemotaxis chambers, the ability of PDGF-AA, -AB and -BB to stimulate the migration of myoblasts, obtained from the proximal region of 11.5 day mouse forelimb buds, was examined. Immunocytochemistry, with the anti-sarcomeric myosin antibody MF-20, was used to identify the myogenic cells in the heterogeneous cell population. Myoblasts, suspended in PDGF-free medium in the upper chamber, migrated across the polycarbonate filter of the Blindwell chamber to 1-10 ng/ml PDGF-AB and 1-100 ng/ml PDGF-BB situated in the lower well. At 1-10 ng/ml of either PDGF-AB or -BB migration increased in a dose-dependent manner. PDGF-AA, however, was unable to elicit a significant locomotory response in forelimb myoblasts. A Checkerboard assay, with various concentrations of PDGF-AA, -AB or -BB in the upper and lower wells of the chamber, indicated that -AB and -BB but not -AA stimulated the random migration of limb myoblasts. The differential effect of PDGF isoforms on myoblast migration was compared with other aspects of skeletal muscle development. At 0.1-10 ng/ml all three isoforms were able to stimulate an increase in the number of differentiated myoblasts, indicated by the expression of sarcomeric myosin, on examination after 48 h when cultured at low density. In high density cultures, however, these isoforms inhibited myoblast fusion when compared to the spontaneous fusion observed in untreated cultures. Immunohistochemical studies of both cultured limb cells and cryosections of 11.5 day whole limbs revealed that myoblasts expressed both PDGF alpha- and beta-receptors which suggests that the action of PDGF isoforms on limb myoblasts is receptor-mediated. Finally, having demonstrated that the PDGF-B monomer stimulates migration in limb myoblasts, by immunohistochemistry, the presence of PDGF-B was confirmed and its distribution examined in the 11.5 day forelimb.

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: Effects of serum and albumin

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups ( P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups ( P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater ( P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be succesfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.

Regenerative capacity of forelimb buds after amputation in mouse embryos at the early‐organogenesis stage

The ability of mouse forelimb buds at stage 1 (Wanek et al., '89a) of development to regenerate after amputation was investigated. The findings were as follows: 1. Outgrowths in the form of hillocks were found at the sites of amputation in 116 (95%) out of 122 embryos examined 24 hours after amputation. Examination of the amputated region after various intervals of time revealed that the outgrowths were established from flank tissues at the anterior and posterior borders of the wound. 2. Ectodermal thickening was found on the distal margin of the outgrowths in 21 (66%) out of 32 specimens examined. These thickenings were histologically similar to the apical ectodermal ridge (AER) present on the control limb buds. 3. Alkaline phosphatase activity was detected on the ectodermal thickening in 11 (79%) out of 14 experimental limb buds examined. The pattern of expression of alkaline phosphatase activity was similar to that observed in control limb buds. 4. There was no correlation between the size of the outgrowths and the presence of the ectodermal thickening or the enzymatic activity. The outgrowths developed despite the absence of ectodermal thickening and enzymatic activity, suggesting that the thickening and the presence of alkaline phosphatase are not crucial for the initiation and formation of the outgrowths. 5. Explants of the outgrowths, when grafted beneath adult kidney capsules, differentiated extensively into various tissues, which included bones, epiphyseal plates, skeletal muscles, and skin derivatives. Control explants also gave rise to the same spectrum of tissues. Hence, the flank tissues surrounding the site of amputation in E10 mouse embryos can regenerate to form a structure that is morphologically and histochemically similar to a limb bud and the mesenchyme within the structure is histogenetically competent to produce the variety of tissues that is normally found in the adult limb.

A study on the regenerative potential of partially excised mouse embryonic fore-limb bud

The ability of day E10 mouse fore-limb bud to regulate following the removal of a portion of limb tissue was investigated. A longitudinal strip of tissue, two to three somites in width and extending from the base of the limb bud to its distal tip, was excised. The embryos were then maintained in a roller culture system for periods of 6 h, 12 h or 24 h post-operatively prior to fixation and subsequent examination. The embryos were examined with scanning electron microscopy (SEM) and light microscopy. SEM revealed that about two thirds of the operated limbs grossly restored their overall morphology. The sequence of morphological changes involved in the restoration process is described. The ability of the restored limb bud to develop an apical ectodermal ridge (AER) is shown in histological sections.

Specific effects of retinoic acid on the skeletal morphogenesis of the 11-day mouse embryo forelimb bud in vitro

Using our improved method for culturing 11-day mouse forelimb buds in vitro, we have investigated the effects of a local application of all- trans-retinoic acid (RA) on growth, cartilaginous differentiation and skeletal patterning in the mammalian limb bud. Carrier implants of catgut impregnated with DMSO or various doses of RA in DMSO were inserted at the apex of the buds in the proximo-distal axis just beneath the apical ectodermal ridge. After 6 days of culture, cartilaginous skeletons were stained and explants were processed for morphological analysis and quantitative study using computerized optical image analysis. Buds treated with low doses of RA exhibited stimulated growth and chondrogenesis. Moreover, hypertrophied and fused metacarpals were seen within explants treated with the lowest dose. High doses strongly inhibited growth and skeletal morphogenesis. An intermediate dose sustained cartilaginous differentiation at the same level as low doses, but concomitantly disturbed the skeletal pattern. These results are discussed considering reported RA effects on other experimental systems including avian limb bud as an in vivo model or cell cultures as an in vitro simplified model.

New in vitro procedures for experimental studies on the development of 11-day mouse embryo forelimb buds.

A new method has been developed for culturing 11-day mouse forelimb buds in vitro. In cultures performed with conventional procedures, skeletal pieces frequently appeared distorted and reduced in size. Moreover, forelimb buds explanted from embryos younger than a stage corresponding to 50 pairs of somites developed narrow hand plates devoid of radiated autopods. By contrast, in the new procedure using media supplemented with fetal calf serum and growth factors and enhancing distal feeding with carrier implants of catgut, enlarged pads were obtained that exhibited at least 4 digital rays in buds explanted from embryos with 40-44 pairs of somites. Compared with conventional procedures, the mean value of DNA content per limb bud was twice as great with use of our improved method. The ability of limb bud cells to proliferate and differentiate when cultured either in classical or in modified conditions, and the importance of the technical procedures, are discussed in the new prospect of in vitro developmental studies.

Developmental Changes of Cell Process Meshwork and Extracellular Space in Subepidermal Mesenchyme in Mouse Forelimb Buds: (apical ectodermal ridge/mesenchymal cell process/subepidermal extracellular space/ limb bud/mouse embryo).

This study was designed to reveal morphological changes in epithelial-mesenchymal interface during limb development. An electron microscopic and morphometric analysis was done on the cell process meshwork (CPM) and subepidermal extracellular space (SEECS) in mesenchyme in mouse forelimb buds at embryonic ages from day 9.5 to 12.5 (vaginal plug=day 0). At days 9.5 and 10.0, when the apical ectodermal ridge (AER) had not yet appeared, no spatial differences were noted in the CPM density and SEECS width. However, at days 10.5, 11.0 and 11.5, when the AER was distinct, the SEECS was wide and the number of mesenchymal cell processes was small beneath the AER as compared with those at days 9.5 and 10.0; they differed significantly from those beneath the dorsal or ventral non-ridge epidermis. Between days 10.0 and 10.5, the SEECS width decreased and the number of cell processes increased in the dorsal region. At days 12.0 and 12.5, when the AER was regressing, the spatial differences in the CPM density and the SEECS width became less obvious. These findings indicate that spatial and temporal differences of the SEECS width and CPM density exist in mouse limb buds, and these differences are closely associated with the AER development.

Accelerated maturation of limb mesenchyme by the Brachypod H mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism H ( bp H ) mutation are shown to behave similarly to ‘normal’ proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than ‘normal’ distal or whole limb bud cells. These results support the hypothesis that the bp H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.

In vitro analysis of susceptible stages of mouse forelimb buds to retinol.

In vitro analysis of susceptible stages of mouse forelimb buds to retinol.