Chick Limb Bud Research Articles

Research Note: SOCS2 contributes to reduction of the third digit during development of the chicken forelimb

The development of the avian wing pattern has been the subject of heated debate due to its special shape. The Suppressor of cytokine signaling 2 (SOCS2) gene encodes a negative regulator of growth hormone (GH) signaling and bone growth and is known to be strongly expressed in the third digit of chicken forelimbs. These observations suggest that SOCS2 might regulate the morphology of the avian wing, however, the function of SOCS2 in avian limb development remains unknown. Here, we reexamined SOCS2 expression in successive developmental stages of chicken limb development by in situ hybridization (ISH) and describe extended expression from the posterior of the stypolod to the third digit of the forelimbs. We used the RCAS avian retrovirus to overexpress SOCS2 in the developing chicken limb buds, which resulted in reduced or malformed chicken wings while hindlimbs developed normally. Transcriptome sequencing (mRNA-Seq) revealed changes in expression of genes known to be associated with growth and development in forelimbs with overexpressed SOCS2. This study highlights a pivotal role for SOCS2 during the development of the wing in the chicken and provides new insight into molecular mechanisms regulating avian limb development.

Commitment of human mesenchymal stromal cells to skeletal lineages is independent of their morphogenetic capacity.

Mesenchymal stromal cells (MSCs) are multipotent cell populations obtained from fetal and adult tissues. They share some characteristics with limb bud mesodermal cells such as differentiation potential into osteogenic, chondrogenic, and tenogenic lineages and an embryonic mesodermal origin. Although MSCs differentiate into skeletal-related lineages in vitro, they have not been shown to self-organize into complex skeletal structures or connective tissues, as in the limb. In this work, we demonstrate that the expression of molecular markers to commit MSCs to skeletal lineages is not sufficient to generate skeletal elements in vivo. To evaluate the potential of MSCs to differentiate into skeletal lineages and generate complex skeletal structures using the recombinant limb (RL) system. We used the experimental system of RLs from dissociated-reaggregated human placenta (PL) and umbilical cord blood (UCB) MSCs. After being harvested and reaggregated in a pellet, cultured cells were introduced into an ectodermal cover obtained from an early chicken limb bud. Next, this filled ectoderm was grafted into the back of a donor chick embryo. Under these conditions, the cells received and responded to the ectoderm's embryonic signals in a spatiotemporal manner to differentiate and pattern into skeletal elements. Their response to differentiation and morphogenetic signals was evaluated by quantitative polymerase chain reaction, histology, immunofluorescence, scanning electron microscopy, and in situ hybridization. We found that human PL-MSCs and UCB-MSCs constituting the RLs expressed chondrogenic, osteogenic, and tenogenic molecular markers while differentially committing into limb lineages but could not generate complex structures in vivo. MSCs-RL from PL or UCB were committed early to chondrogenic lineage. Nevertheless, the UCB-RL osteogenic commitment was favored, although preferentially to a tenogenic cell fate. These findings suggest that the commitment of MSCs to differentiate into skeletal lineages differs according to the source and is independent of their capacity to generate skeletal elements or connective tissue in vivo. Our results suggest that the failure to form skeletal structures may be due to the intrinsic characteristics of MSCs. Thus, it is necessary to thoroughly evaluate the biological aspects of MSCs and how they respond to morphogenetic signals in an in vivo context. PL-MSCs and UCB-MSCs express molecular markers of differentiation into skeletal lineages, but they are not sufficient to generate complex skeletal structures in vivo.

Mathematical modeling for developmental processes.

We review several mathematical models and concepts in developmental biology that have been established over the last decade. (1) Feedback vertex set: Ascidian embryos contain cells of seven types, and cell fate is controlled by ~100 interacting genes. The "feedback vertex set" of the directed graph of the gene regulatory network consists of a small number of genes. By experimentally manipulating them, we can differentiate cells into any cell type. (2) Tissue deformation: Describing morphological changes in tissues and relating them to gene expression and other cellular processes is key in understanding morphogenesis. Expansion and anisotropy of the tissue are described by a "deformation tensor" at each location. A study on chick limb bud formation revealed that both the volume growth rate and anisotropy in deformation differed significantly between locations and stages. (3) Mechanobiology: Forces operating on each cell may alter cell shape and gene expression, which may subsequently exert forces on their surroundings. Measurements of force, tissue shape, and gene expression help us understand autonomous tissue deformation. (4) Adaptive design of development: An optimal growth schedule in fluctuating environments explains the growth response to starvation in Drosophila larvae. Adaptive placement of morphogen sources makes development robust to noises.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Regulation of LIM homeodomain 2 (Lhx2) in the distal limb bud during patterning and development

During limb morphogenesis, fibroblast growth factors (Fgfs) secreted from the apical ectodermal ridge (AER) direct proximodistal outgrowth of the limb, while sonic hedgehog (Shh) released from the zone of polarizing activity modulate anteroposterior expansion of the limb. Fgfs and Shh maintain each other’s expression through an autoregulatory loop. The transcription factor LIM homeodomain 2 (Lhx2) is expressed in the distal sub‐AER mesoderm and is important for Shh expression. We have identified a chicken enhancer upstream of the LHX2 promoter that is active in the distal sub‐AER LHX2 expression domain that contains putative transcription factor binding sites for known downstream targets of FGF signaling that are critical for activity. Therefore, we hypothesized that LASARM1 interacts with the LHX2 promoter and that FGF works through LASARM1 to regulate LHX2 expression. To determine this, we performed chromosome conformation capture (3C) on cells isolated from distal chicken limb buds and compared them to non‐LHX2 expressing tail cells. Promoter and enhancer specific primers were used to isolate ligated promoter‐enhancer fragments by end‐point PCR. Amplified sequences were then sequence validated. A 3C ligation product library was used as a control. We also evaluated the role of FGF to regulate LASARM1 activity in subAER mesoderm by analyzing LASARM1 activity following inhibition of FGF signaling by an FGF‐receptor inhibitor. End‐point PCR revealed promoter‐enhancer ligated amplicons in distal limb bud cells, but not in tail cells. DNA sequencing of enhancer‐ligated fragments confirmed LHX2‐LASARM1 fusion fragments for distal limb bud cells. Interestingly, disruption of FGF signaling via a specific FGF receptor inhibitor did not alter LASARM1 activity, although LHX2 expression was markedly reduced. These findings suggest that LASARM1 interacts with the LHX2 promoter during limb patterning, but additional enhancers are required to mediate the FGF‐mediated up‐regulation of LHX2. Evaluation of additional LHX2 enhancers is underway. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying LHX2 regulation and the FGF‐to‐SHH reciprocal loop.

Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior–posterior axis in the limb bud

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 ​h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 ​h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

Expression and function of Ebf1 gene during chondrogenesis in chick embryo limb buds

The expression profile of early B-cell factor (Ebf) genes and loss of function experiments denote a crucial role for these genes during the late stage of skeletogenesis. However, little is known regarding the expression and function of these genes during the early stage of skeletogenesis. Therefore, this study aimed to detail the spatiotemporal expression pattern of cEbf1, in comparison to cEbf2 and cEbf3, in chick limb buds and investigate its function during chondrogenesis. cEbf1-3 were co-expressed in the distal mesenchyme from a very early stage and later in the outer perichondrium and the surrounding noncartilaginous mesenchymal cells. Ebf1 loss of function through injection of RCASBP virus-carrying Ebf1 dominant-negative form (ΔEbf1) into the wing buds resulted in shortened skeletal elements with a clear defect in the chondrocyte differentiation program. In RCASBP-ΔEbf1 injected wing, the chondrogenesis was initiated normally but hindered at the maturation stage. Subsequently, the chondrocytes failed to become mature or hypertrophic and the long bone diaphysis was not properly developed. The final phenotype included shorter, thicker, and fused long bones. These phenotypic changes were associated with downregulation of the early [Sox9 and collagen type II (Col2a1)] and the late [alkaline phosphatase (AP)] chondrocytes differentiation markers in the limb buds. These results conclude that cEbf1 could be involved in a molecular cascade that promotes the terminal stages of chondrogenesis in the long bone anlagen.

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.

In ovo electroporation of chicken limb bud ectoderm: Electroporation to chick limb ectoderm: Electroporation to chick limb ectoderm.

Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

ETS Binding Sites are Critical for Activity of the LHX2‐Associated Sub‐AER Regulatory Module 1 (LASARM1)

Fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) coordinate proximal-distal patterning, while sonic hedgehog (SHH) from the zone of polarizing activity (ZPA) directs anterior-posterior growth of the limb. These signaling centers maintain each other's expression in a reciprocal feedback loop; however, the molecular intermediates involved are only partially characterized. LIM homeobox 2 (LHX2) has been identified as an intermediate regulator downstream of FGF in the reciprocal loop. We have identified a cis-regulatory module (CRM) located upstream of the LHX2 promoter that is active in the sub-AER LHX2 expression domain. Overexpression of FGF in Hamburger-Hamilton stage 24 (HH24) chicken limb buds, resulted in increased expression of several FGF-associated transcription factors, including E26 transformation-specific proteins (ETS). Members of the ETS family have been known to regulate the expression of SHH via an enhancer known as the Zone of Polarizing Activity Regulatory Sequence (ZRS). We wondered whether the regulation of LHX2 was mediated through this LHX2-associated sub-AER Regulatory Module 1 (LASARM1) via ETS transcription factors. In silico analysis of LASARM1 revealed three ETS binding sites within LASARM1 and hypothesized that mutation of all three binding sites would disrupt FGF-mediated enhancer activity. To determine whether these ETS transcription factor binding sites contribute to LASARM1 activity, we performed site directed mutagenesis on all three ETS binding sites (triple∆ETS) within a LASARM1-ptk-eGFP reporter construct, followed by targeted regional electroporation (TREP) into the limb buds of HH24 chick embryos. Transfection efficiency was determined by co-transfection with a β-actin promoter-driven RFP construct. After 24 hours of incubation, LASARM1 activity was determined by fluorescence microscopy. We found that mutation of all three ETS binding sites within LASARM1 resulted in fluorescence that was undetectable, suggesting reduced LASARM1 activity. Our findings suggest that FGF signaling utilizes ETS transcription factors to regulate LHX2 expression through the LASARM1 enhancer. Further work is needed to confirm this connection. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF-SHH regulatory loop.

Knockdown of slit signaling during limb development leads to a reduction in humerus length.

Slits (1-3) and their Robo (1-3) receptors play multiple non-neuronal roles in development, including in development of muscle, heart and mammary gland. Previous work has demonstrated expression of Slit and Robo family members during limb development, where their functions are unclear. In situ hybridisation confirmed strong expression of Slit2, Slit3, Robo1, and Robo2 throughout mouse limb and joint development. No expression of Slit1 or Robo3 was detected. Analysis of Slit1/2 or Slit3 knockout mice revealed normal limb development. In contrast, locally blocking Slit signaling though grafting of cells expressing a dominant-negative Robo2 construct in the proximo-central region of developing chicken limb buds caused significant shortening of the humerus. These findings demonstrate an essential role for Slit/Robo signaling in regulating bone length during chicken limb development.

CNS Activity of an LHX2‐Associated Cis‐Regulatory Module

Limb development can be described along three axes – proximal‐distal, anterior‐posterior, and dorsal‐ventral. Fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) coordinate growth along the proximal‐distal axis, while sonic hedgehog (SHH), secreted from the zone of polarizing activity, directs anterior‐posterior patterning and expansion. AER‐related FGFs and SHH regulate each other in a positive feedback loop. This reciprocal loop is critical for proper limb patterning, however the molecular intermediates involved in this loop are only partially characterized. Previously, we identified LIM homeobox 2 (LHX2) as a downstream target of FGF in the FGF to SHH loop and determined that FGF upregulates LHX2 in the absence of de novo protein synthesis. This suggests that LHX2, expressed in the mesoderm subjacent to the AER, is a primary response target of FGF signaling.Cis‐regulatory modules (CRMs) within noncoding DNA can convey temporal and spatial regulation of an associated gene and are good candidates to convey the FGF‐mediated regulation of LHX2. We identified several potential CRMs (PCRMs) upstream of the LHX2 locus based on sequence conservation, active chromatin marks, and presence of transcription factor binding sites related to FGF signaling. Based on the number of active chromatin marks, we chose four of these PCRMs to investigate. We suspect that FGF regulates LHX2 through one or more of these PCRMs. Thus, we hypothesized that one or more of these PCRMs is active in the LHX2 expression domain during limb development.To test for activity of LHX2 PCRMs we used targeted regional electroporation (TREP) to introduce PCRM‐ptk‐GFP reporter constructs into the distal mesoderm of Hamburger‐Hamilton stage 23 (HH23) chicken limb buds. Transfection efficiency was determined by co‐transfection with a b‐actin promoter‐driven RFP construct. We determined PCRM activity by fluorescence microscopy, following 24 hours of incubation after TREP.None of the four PCRMs tested exhibited activity in the limb. However, subsequent TREP of the reporter construct containing PCRM ‐7 (~ 800 bp conserved region located 215 kb upstream of the LHX2 promoter) into the neural tube of HH10 embryos revealed green fluorescence at the midbrain‐forebrain boundary 24 hours post‐TREP. This activity suggests PCRM ‐7 may play a role in regulating LHX2 expression in the developing forebrain. Further studies are underway to correlate the activity of this CRM with expression of LHX2 or other genes in the topologically associated domain.

An ELK4 Binding Site is Critical for Activity of the LHX2‐Associated Sub‐AER Regulatory Module 1 (LASARM1)

Fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) coordinate limb growth and proximal‐distal patterning, while sonic hedgehog (SHH) from the zone of polarizing activity directs anterior‐posterior growth and patterning. Moreover, these signaling centers maintain each other’s expression in a reciprocal feedback loop. This regulatory loop coordinates limb patterning during outgrowth. However, the molecular intermediates involved in in this loop are only partially characterized. Previously, we identified LIM homeobox 2 (LHX2), expressed in the mesoderm subjacent to the AER, as a downstream target of FGF in the FGF to SHH arm of the feedback loop.LHX2‐associated Cis‐regulatory modules (CRMs) are reasonable candidates to convey the FGF‐mediated regulation of LHX2. We have identified a 1745 bp CRM located 483 kb upstream of the LHX2 promoter that is active in the sub‐AER region within the expression domain of LHX2. In silico analysis of this LHX2‐associated sub‐AER regulatory module 1 (LASARM1) revealed several FGF‐related transcription factor binding sites, including a site for ETS like‐4 (ELK4). We performed in situ hybridization for ELK4 mRNA and found a pattern coincident with LHX2 expression. Thus, we hypothesized that mutation of the ELK4 binding site within LASARM1 would disrupt FGF‐mediated enhancer activity.To identify whether the putative ELK4 transcription factor binding site contributes to LASARM1 activity, we performed site‐directed mutagenesis on the ELK4 binding site in the LASARM1‐ptk‐EGFP reporter construct. We used targeted regional electroporation (TREP) to introduce control and mutated reporter constructs into the distal mesoderm of Hamburger‐Hamilton stage 23 chicken limb buds. Transfection efficiency was determined by co‐transfection with a β‐actin promoter‐driven RFP construct. After 24 hours of incubation LASARM1 activity was determined by fluorescence microscopy.The fluorescence of LASARM1mutELK4 was markedly reduced relative to the LASARM1control. Our findings suggest that FGF signaling utilizes the ELK4 transcription factor to regulate LHX2 expression through the LASARM1 enhancer. Further work is needed to confirm this connection.

RA Signaling in Limb Development and Regeneration in Different Species.

This chapter brings together data on the role of retinoic acid (RA) in the embryonic development of fins in zebrafish , limbs in amphibians , chicks , and mice, and regeneration of the amphibian limb . The intention is to determine whether there is a common set of principles by which we can understand the mode of action of RA in both embryos and adults. What emerges from this synthesis is that there are indeed commonalities in the involvement of RA in processes that ventralize, posteriorize, and proximalize the developing and regenerating limb . Different axes of the limb have historically been studied independently; as for example, the embryonic development of the anteroposterior (AP) axis of the chick limb bud versus the regeneration of the limb bud proximodistal (PD) axis . But when we take a broader view, a unifying principle emerges that explains why RA administration to embryos and regenerating limbs results in the development of multiple limbs in both cases. As might be expected, different molecular pathways govern the development of different systems and model organisms, but despite these differences, the pathways involve similar RA signaling genes, such as tbx5, meis, shh, fgfs and hox genes. Studies of developing and regenerating systems have highlighted that RA acts by being synthesized in one embryonic location while acting in another one, exactly as embryonic morphogens do, although there is no evidence for the presence of an RA gradient within the limb . What also emerges is that there is a paucity of information on the involvement of RA in development of the dorsoventral (DV) axis . A molecular explanation as to how RA establishes and alters positional information in all three axes is the most important area of study for the future.

Regulation of a GDF5 Associated Enhancer During Limb Development

Growth differentiation factor 5 (GDF5) is an anabolic growth factor that plays a critical role in the formation, maintenance and repair of bones and joints. Reduced levels of GDF5 are associated with accelerated degradation of joint cartilage, a disease called osteoarthritis (OA). Identification of factors involved in GDF5 regulation may offer therapeutic targets for treating or preventing OA. A GDF5 Associated Regulatory Region (GARR) has been previously identified as a 900 bp enhancer located 78 kb downstream of the GDF5 locus. GARR contains a number of potential transcription factor binding sites, including sites for odd‐skipped related (Osr) zinc‐finger and Sry box (Sox) transcription factors. As Osr1/2 and Sox11 are important for normal Gdf5 expression, and their expression patterns overlap that of Gdf5, we hypothesized that mutation of OSR and SOX binding sites within GARR would disrupt enhancer activity.To identify whether putative OSR and SOX transcription factor binding sites contribute to GARR activity, we performed site‐directed mutagenesis on both potential OSR binding sites (GARRmutOSR), or on both potential SOX binding sites (GARRmutSOX) in the GARR‐tk‐EGFP reporter construct (GARRcontrol). We used targeted regional electroporation (TREP) to introduce control and mutated reporter constructs into Hamburger‐Hamilton stage 23 chicken limb buds near the developing elbow joint. Transfection efficiency was determined by co‐transfection with a β‐actin promoter‐driven RFP construct. We determined GARR activity by fluorescence microscopy, following 48 hours of incubation after TREP.Comparison of GARRcontrol and GARRmutOSR demonstrated similar GFP fluorescence intensity localized to the elbow joint. In contrast, fluorescence of GARRmutSOX was markedly reduced relative to GARRcontrol, although similarly restricted to the elbow. Though Osr transcription factors are associated with Gdf5 expression, mutation of the OSR binding sites in GARR was insufficient to substantially alter the level of GARR activity. Mutation of SOX binding sites, however, dramatically reduced GARR activity, implicating SOX transcription factors as critical regulators of GDF5 expression.Support or Funding InformationThe research was funded in part by a grant from the LLU Pathology Research Endowment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds

BackgroundIntercalary pattern formation is an important regulatory step in amphibian limb regeneration. Amphibian limb regeneration is composed of multiple steps, including wounding, blastema formation, and intercalary pattern formation. Attempts have been made to transfer insights from regeneration-competent animals to regeneration-incompetent animalsat each step in the regeneration process. In the present study, we focused on the intercalary mechanism in chick limb buds. In amphibian limb regeneration, a proximodistal axis is organized as soon as a regenerating blastema is induced. Intermediate structures are subsequently induced (intercalated) between the established proximal and distal identities. Intercalary tissues are derived from proximal tissues. Fgf signaling mediates the intercalary response in amphibian limb regeneration.ResultsWe attempted to transfer insights into intercalary regeneration from amphibian models to the chick limb bud. The zeugopodial part was dissected out, and the distal and proximal parts were conjunct at st. 24. Delivering ectopic Fgf2 + Fgf8 between the distal and proximal parts resulted in induction of zeugopodial elements. Examination of HoxA11 expression, apoptosis, and cell proliferation provides insights to compare with those in the intercalary mechanism of amphibian limb regeneration. Furthermore, the cellular contribution was investigated in both the chicken intercalary response and that of axolotl limb regeneration.ConclusionsWe developed new insights into cellular contribution in amphibian intercalary regeneration, and found consistency between axolotl and chicken intercalary responses. Our findings demonstrate that the same principal of limb regeneration functions between regeneration-competent and -incompetent animals. In this context, we propose the feasibility of the induction of the regeneration response in amniotes.

Syndromic mutations in WNT5A disrupt cellular organization in the chicken limb bud

Syndromic mutations in WNT5A disrupt cellular organization in the chicken limb bud

Upstream regulation for initiation of restricted Shh expression in the chick limb bud.

The organizing center, which serves as a morphogen source, has crucial functions in morphogenesis in animal development. The center is necessarily located in a certain restricted area in the morphogenetic field, and there are several ways in which an organizing center can be restricted. The organizing center for limb morphogenesis, the ZPA (zone of polarizing activity), specifically expresses the Shh gene and is restricted to the posterior region of the developing limb bud. The pre-pattern along the limb anteroposterior axis, provided by anterior Gli3 expression and posterior Hand2 expression, seems insufficient for the initiation of Shh expression restricted to a narrow, small spot in the posterior limb field. Comparison of the spatiotemporal patterns of gene expression between Shh and some candidate genes (Fgf8, Hoxd10, Hoxd11, Tbx2, and Alx4) upstream of Shh expression suggested that a combination of these genes' expression provides the restricted initiation of Shh expression. Taken together with results of functional assays, we propose a model in which positive and negative transcriptional regulatory networks accumulate their functions in the intersection area of their expression regions to provide a restricted spot for the ZPA, the source of morphogen, Shh. Developmental Dynamics 246:417-430, 2017. © 2017 Wiley Periodicals, Inc.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

Transgene Introduction into the Chick Limb Bud by Electroporation.

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.