Chick Limb 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.

Unveiling the evolutionary significance: Atavisms transition from ancestral traits to a fundamental biological phenomenon

This essay delves into the nuanced concept of atavism, tracing its evolution from the abrupt manifestation of ancestral traits in wild populations to a pivotal element in contemporary biology. Explored through recent studies such as the Atavism Theory of Cancer, Single-Cell Atavism of Cnidocytes, and Atavism in the Developmental Polarity of Chicken Limb, this research reveals atavisms journey from speculative fiction to empirical reality. By scrutinizing cancer as a series of atavistic changes, experimental atavism at the single-cell level, and the atavism observed in avian limb development, the essay proposes that atavism offers fresh perspectives on evolution, adaptation, and the dynamic reuse of gene reservoirs. This study underscores atavisms transition into a tangible and crucial component of ecological and developmental biology, providing insights that traverse disciplinary boundaries and deepen our comprehension of the natural world.

A two-galectin network establishes mesenchymal condensation phenotype in limb development

Previous work showed that Gal-1A and Gal-8, two proteins belonging to the galactoside-binding galectin family, are the earliest determinants of the patterning of the skeletal elements of embryonic chicken limbs, and further, that their experimentally determined interactions in the embryonic limb bud can be interpreted via a reaction–diffusion–adhesion (2GL: two galectin plus ligands) model. Here, we use an ordinary differential equation-based approach to analyze the intrinsic switching modality of the 2GL network and characterize the network behavior independent of the diffusive and adhesive arms of the patterning mechanism. We identify two states: where the concentrations of both the galectins are respectively, negligible, and very high. This bistable switch-like system arises via a saddle–node bifurcation from a monostable state. For the case of mass-action production terms, we provide an explicit Lyapunov function for the system, which shows that it has no periodic solutions. Our model therefore predicts that the galectin network may exist in low expression and high expression states separated in space or time, without any intermediate states. We test these predictions in experiments performed with high density cultures of chick limb mesenchymal cells and observe that cells inside precartilage protocondensations express Gal-1A at a much higher rate than those outside, for which it was negligible. The Gal-1A and -8-based patterning network is therefore sufficient to partition the mesenchymal cell population into two discrete cell states with different developmental (chondrogenic vs. non-chondrogenic) fates. When incorporated into an adhesion and diffusion-enabled framework this system can generate a spatially patterned limb skeleton.

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.

Sox, Fox, and Lmx1b binding sites differentially regulate a Gdf5-Associated regulatory region during elbow development.

Introduction: The articulating ends of limb bones have precise morphology and asymmetry that ensures proper joint function. Growth differentiation factor 5 (Gdf5) is a secreted morphogen involved in cartilage and bone development that contributes to the architecture of developing joints. Dysregulation of Gdf5 results in joint dysmorphogenesis often leading to progressive joint degeneration or osteoarthritis (OA). The transcription factors and cis-regulatory modules (CRMs) that regulate Gdf5 expression are not well characterized. We previously identified a Gdf5-associated regulatory region (GARR) that contains predicted binding sites for Lmx1b, Osr2, Fox, and the Sox transcription factors. These transcription factors are recognized factors involved in joint morphogenesis and skeletal development. Methods: We used in situ hybridization to Gdf5, Col2A1, and the transcription factors of interest in developing chicken limbs to determine potential overlap in expression. We further analyzed scRNA-seq data derived from limbs and knees in published mouse and chicken datasets, identifying cells with coexpression of Gdf5 and the transcription factors of interest. We also performed site-directed mutatgenesis of the predicted transcription factor binding sites in a GARR-reporter construct and determined any change in activity using targeted regional electroporation (TREP) in micromass and embryonic chicken wing bioassays. Results: Gdf5 expression overlapped the expression of these transcription factors during joint development both by in situ hybridization (ISH) and scRNA-seq analyses. Within the GARR CRM, mutation of two binding sites common to Fox and Sox transcripstion factors reduced enhancer activity to background levels in micromass cultures and in ovo embryonic chicken wing bioassays, whereas mutation of two Sox-only binding sites caused a significant increase in activity. These results indicate that the Fox/Sox binding sites are required for activity, while the Sox-only sites are involved in repression of activity. Mutation of Lmx1b binding sites in GARR caused an overall reduction in enhancer activity in vitro and a dorsal reduction in ovo. Despite a recognized role for Osr2 in joint development, disruption of the predicted Osr2 site did not alter GARR activity. Conclusion: Taken together, our data indicates that GARR integrates positive, repressive, and asymmetrical inputs to fine-tune the expression of Gdf5 during elbow joint development.

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.

Differential Regulation of Lhx9 Expression in Mouse and Chicken Limbs

The distal tip of the developing vertebrate limb is covered by a rim of thickened ectoderm called the apical ectodermal ridge (AER). The AER secretes fibroblast growth factors (Fgfs) into the underlying mesoderm to control outgrowth and proximal‐distal patterning. Patterning during outgrowth proceeds from proximal to distal, with the proximal segment or stylopod being laid down first, then the zeugopod, and the distal segment or autopod forming last. Secreted Fgfs from the AER maintain a sub‐AER population of undifferentiated mesoderm, called the progress zone, that provides cells for this progressive differentiation.In mice, Lhx9and its homologue Lhx2are expressed in the sub‐AER mesoderm and appear to play redundant roles in maintaining the expression of other patterning molecules, such as Shh. Interestingly in the chicken, the expression of LHX9 is restricted to the anterior aspect of the sub‐AER mesoderm, while LHX2 retains the full distal expression seen in mice. Recent clinical reports of an LHX9 missense mutation in a patient with hypoplastic/missing thumbs and great toes suggest that LHX9 can play a critical role in normal limb development. We hypothesized that the chicken cis‐regulatory modules (CRMs) regulating Lhx9 might have subtle species‐specific sequence variations compared to the murine CRMs that correlate with this differential expression pattern.To test this hypothesis, potential CRMs were identified near the Lhx9 locus by in silicoanalysis, using sequence conservation and regulatory chromatin markers from published ChIP‐seq data. The potential CRMs were isolated and inserted into CRM‐reporter constructs. The limb‐related activity of a potential CRM was then determined by targeted regional electroporation into the distal chicken limb mesoderm.We identified a highly conserved, transcription‐factor‐rich 623 bp sequence within intron 12 of DENDD1B 132 kbp upstream of the chicken LHX9 transcription start site. The potential CRM was associated with H3k4me2, H3k27ac, p300 and H3k27me3. The associated CRM‐reporter construct displayed activity in both anterior and posterior sub‐AER limb mesoderm overlapping LHX9 and LHX2 expression. These findings are consistent with a distal sub‐AER LHX9enhancer, but do not account for the lack of posterior expression. This suggests that additional CRMs contribute to the final expression pattern of LHX9 in the limb. Twenty‐two additional potential CRMs were identified and will be further characterized to determine their role in restricting LHX9expression from the posterior mesoderm in chickens. Characterizing the differential regulation of Lhx9 during limb development may provide insight into the species‐specific variation of limb patterning.

The Role of Sox9 in Regulating Joint Development through a Gdf5 Enhancer

Sox transcription factors are involved in a number of developmental processes including joint formation. Sox9, Sox5, and Sox6 (the Sox‐trio) form a complex that promotes chondrogenesis. In joint‐associated cells, Sox9 may give rise to a subpopulation of cells expressing growth differentiation factor 5 (Gdf5). Gdf5, a secreted morphogen critical to joint development, regulates cartilage growth and tissue differentiation. Osteoarthritis (OA) is condition characterized by articular cartilage degradation and is a leading cause of disability worldwide. Gdf5 dysregulation has been identified as a risk factor for OA, and in the joints of OA patients, the Sox‐trio are downregulated. The link between Sox, Gdf5, and joint development is not fully understood. We previously identified an enhancer for Gdf5, known as Gdf5‐Associated Regulatory Region (GARR), and have identified possible Sox9 and 5/6 binding sites within GARR by in silico analysis. Thus, we hypothesized that Sox9 is required for Gdf5 upregulation through GARR, while other Sox transcription factors (Sox5/6) enhance or stabilize this interaction. To test this hypothesis, we analyzed publicly available single cell RNA sequencing (scRNA‐seq) data from mouse embryo (E12‐5‐E15.5) knee joints. Analysis revealed a strong correlation between Gdf5 and Sox9. Sox9 was upregulated in Gdf5 positive cells. Sox5 and 6 were upregulated in Gdf5 positive cells but had a lower correlation to Gdf5 expression indicating a weaker or indirect/secondary role in Gdf5 regulation. We also compared the activity of the wildtype GARR enhancer‐reporter construct to constructs with mutated Sox binding sites in developing chicken limbs to determine the role of these sites on enhancer activity. We found that in developing chicken embryo limbs, constructs with mutated Sox9 binding sites had a marked decrease in enhancer activity in the elbow but not in the autopod. Mutation of predicted Sox5/6 binding sites did not seem to affect activity relative to the non‐mutated control in the forelimb joints. Our findings suggest that Sox9 is a key regulator of elbow development through GARR‐mediated expression of Gdf5. The correlations identified in the transcriptomic data may be relevant only to the elbow and knee joints. Alternatively, the Sox transcription factors may be acting though other Gdf5‐associated enhancers in the autopod joints. Further investigation of enhancer activity in the presence of various combinations of Sox5/6/9 will throw more light on how they collectively affect Gdf5 expression as well as joint development and OA progression.

The effect of disease‐causing DVL1 mutations on chondrogenesis in chicken limb mesenchyme

The Wingless (WNT) signaling pathway is a key regulator of skeletal development. Perturbations in WNT signaling are associated with several skeletal disorders, including Robinow syndrome (RS). RS is a rare skeletal dysplasia characterized by deficiencies in facial and limb skeletogenesis. All identified pathogenic variants of RS occur in genes of the WNT signaling pathway. The majority of autosomal dominant RS patients (DRS) cases are attributed to mutations in Dishevelled‐1 (DVL1) (OMIM#1807000) and Dishevelled‐3 (DVL3) (OMIM#616894), a cytoplasmic phosphoprotein. All RS DVL1variants have frameshift mutation in the penultimate exon that substitutes an abnormal peptide for the C‐terminus of the protein. Previous research in our lab has shown the expression of avian‐ specific retroviruses containing human wild‐type DVL1 and RS DVL1 variants in the limb of the chicken embryo in vivo disrupts cartilage formation. In order to examine the possible role of WNT signaling in the dysregulation of chondrogenesis caused by DVL1, high‐density cultures of chicken limb mesenchyme were treated with DVL1 viruses and either TCS JNK6o, which antagonizes non‐canonical, JNK signaling or XAV939, a canonical pathway antagonist. Cultures were grown for eight‐days stained with Alcian blue for cartilage. The area occupied by cartilage was analyzed using two‐way ANOVA with Tukey’s post‐hoc test (Graphpad Prism v9.3). In the absence of drug treatment, wtDVL1 and RS DVL11519ΔTvariant significantly increased chondrogenesis compared to GFP controls (Fig. 1A,B). Cultures treated with TCS6o (Fig. 1A) did not significantly increase cartilage compared to non‐treated controls culture. The combination of wtDVL1 with TCS6o synergistically increased chondrogenesis. This suggests 1) that endogenous non‐canonical WNT signaling is not required for chondrogenesis, 2) that wtDVL1 has a degree of JNK signaling that when antagonized increases chondrogenesis and 3) that the variants are not interacting with JNK signaling. When canonical signaling was antagonized (Fig. 1B), chondrogenesis was significantly increased for all virus types compared to GFPcontrols. However, the effect of XAV939 on the DVL11519ΔT variant‐infected cultures did not further increase chondrogenesis over the virus alone. This suggest that endogenous canonical WNT signaling represses chondrogenesis and but the variant may have altered ability to respond to a decrease in canonical signaling. Misregulation of canonical and non‐canonical WNT signaling is tied to abnormal chondrogenesis and could be contributing to the shortened limbs observed in RS patients.

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.

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation.

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

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.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

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.

Lewis Wolpert (1929–2021)

Lewis Wolpert (1929–2021)

The Role of the SOX11 Transcription Factor in Regulating Growth Differentiation Factor‐5 During Joint Formation

Growth differentiation factor 5 (GDF5) is associated with the formation and maintenance of joints. Disruption of GDF5 expression is also linked to accelerated osteoarthritis. We have identified a GDF5 Associated Regulatory Region (GARR) that contains several Sox binding sites. The SOX11 transcription factor has been shown to upregulate GDF5 and we hypothesized that SOX11 is necessary for GARR mediated expression of GDF5 during joint development and maintenance. To test this hypothesis, we compared the expression of SOX11 and GDF5 in chicken limbs during development using in-situ hybridization, using MYOD, TNMD, and Col1A2 as muscle and tendon markers. We also generated a GARR reporter construct and mutated the Sox binding sites. The native and mutated GARR reporters were transfected into developing chick wings. Our results showed that in chicken, SOX11 expression primarily surrounds GDF5 expression in joint spaces at Hamburger-Hamilton stages (HH) 25-27. At HH 29, the expression of SOX11 and GDF5 colocalized in joint spaces. SOX11 expression was not confined to joints, but also found in muscles (MYOD) and tendons (TNMD, Col1A2). Disruption of the Sox binding sites greatly reduced GARR activity within joint spaces. Colocalization of SOX11 and GDF5 in joints shows that SOX11 may play a role in the regulation of GDF5 through GARR. Additionally, it may be a competency factor for the formation and organization of joint-related tissues. Decreased activity with mutant GARR suggests it is required for GARR activity. Further experiments are needed to clarify the role of SOX11 in regulating joints and joint associated tissues.

Effects of of disease‐causing DVL1 mutations on chondrogenesis in chicken limb mesenchyme

Robinow syndrome (RS) is a rare skeletal dysplasia characterized by skeletal phenotypes affecting the face and limbs. The majority of autosomal dominant RS patients (DRS) have mutations in Dishevelled-1 (DVL1) (OMIM#1807000) and Dishevelled-3 (DVL3) (OMIM#616894). DVL is a cytoplasmic phosphoprotein that transduces WNT(Wingless-related) signals. Any type of WNT signaling that involves Frizzled receptor binding also requires the binding of DVL to activate signaling. All RS DVL1 variants have frameshift mutations in exon 14 that produce an abnormal peptide that is over 200 amino acids long.. This peptide replaces the entire C-terminus and removes respective binding partners. It is not possible to predict whether this peptide causes a loss- gain-of-function or whether there may be novel functions. Previous in vivo studies in our lab showed that the ectopic expression of wtDVL did not affect the overall shape of the cartilage and limbs contained closely packed differentiated chondrocytes. The phenotypes of the variants were very different to wtDVL1 but similar to each other. There were pockets of undifferentiated fibroblasts between areas of differentiated chondrocytes thus shape was very irregular. These phenotypes were not providing clear answers as to which steps of chondrogenesis are affected. Our objective is to misexpress RS hDVL1 variants (DVL11519DT, DVL11529DG, DVL11615DA) and wtDVL1 in cells isolated from the limb bud and then assess the effect on different stages of chondrogenesis. High density micromass cultures were created from stage 24 chicken limb mesenchyme, infected with viruses and grown for 4, 6 or 8 days of culture. Cultures were photographed and the proportion of the culture occupied by cartilage was determined. Two-way ANOVA with Tukey's post-hoc test for multiple comparisons was carried out. All cultures formed nodules stained with Alcian blue by 4 days of culture. Then differences appeared. The GFP controls continued to differentiate into cartilage and there was a significant increased over the 8 day experimental period. The wtDVL1 started out similar to GFP but then chondrogenesis gradually decreased. By 8-days there was significantly less cartilage than at 4 days in wtDVL1-infected cultures. Two of the RS DVL1 variants stimulated chondrogenesis significantly compared to GFP and wtDVL1 by 8 days of culture (1519 and 1529). This indicates RS DVL1 variants caused a gain-of-function on chondrogenesis. In contrast, the 1615 variant failed to induce cartilage beyond the amount present at 4 days. This seems like the 1615 variant allowed the initial chondrogenesis to take place but then failed to support further differentiation. This unique phenotype could mean that there are interesting effects on downstream transcription of genes. that may indicate signalling differences for this particular variant. In conclusion DVL1 does not play a role in the initial condensation of cartilage but is regulating differentiation.

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.