Kerby C. Oberg

LIM Homeobox 9 (Lhx9) is a transcription factor involved in vertebrate limb development. Despite conserved anterior expression, its distal expression is species‐specific. In chickens, distal expression is restricted to the anterior mesoderm; in mice, it extends into posterior mesoderm. We identified and characterized a limb‐specific Lhx9‐ associated cis‐ regulatory module, LARR9A. In chicken limb bioassays, chicken LARR9A was active in the distal anterior limb mesoderm, while murine LARR9A was active both anteriorly and posteriorly. HAND2, a posteriorly expressed transcription factor, complements anterior chicken LHX9 expression. Anterior ectopic Hand2 expression in chicken limbs inhibited LHX9 expression and chicken LARR9A activity. We identified an additional HAND2 binding site (E‐box 5′C) in chicken LARR9A that was absent in mice. Presence or absence of this E‐box is due to a single base‐pair variation (A/C) in a core nucleotide. Introduction of the E‐box into murine LARR9A excluded activity from the Hand2 domain in both chicken limb bioassays and transgenic mice. Although murine LARR9A exhibited posterior activity in chicken bioassays, it did not in transgenic mice indicating the need for additional posterior regulation to fully explain species‐specific Lhx9 expression. Nevertheless, our data suggest that a single base‐pair variation in LARR9A is associated with differential Hand2‐modulated repression.

Background: During development, Hand2 and Hoxd13 transcription factors (TFs) regulate Sonic hedgehog (Shh) expression in the zone of polarizing activity (ZPA) in the distal posterior limb mesoderm. The ZPA regulatory sequence (ZRS) is a conserved, limb-specific enhancer that controls Shh expression. The ZRS can be divided into 5′, central, and 3′ subdomains, each with an E-box site that can bind basic helix-loop-helix (bHLH) TFs like Hand2. In addition, two Hoxd13 sites are present in the 5′ and central subdomains. Hand2 purportedly binds the ZRS through the central E-box, and both Hand2 and Hoxd13 have been shown to activate the ZRS in vitro. We hypothesized that the central E-box was required for activity, while the other E-boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm.Methods: To identify the functional role of each subdomain, we generated three ZRS fragments (5′, central, and 3′) and combined fragment constructs to test subdomain collective contributions. Additionally, we disrupted the five binding sites, alone or in concert, using site-directed mutagenesis. All ZRS constructs were cloned into a GFP reporter and evaluated in an in vivo chicken limb bioassay. We validated our findings using select ZRS constructs in transgenic mice.Results: We found that the 3′ fragment was necessary for ZRS activity, while the 5′ and central fragments had no activity alone or when combined. However, combining the 3′ fragment with the 5′ fragment restored robust activity. Further, mutation of all five binding sites markedly reduced ZRS activity. Reinstating each of the Hoxd13 sites restored focal activity, while restoring the 5′ and central E-boxes had little effect. However, the 3′ E-box proved sufficient for robust activity even in the absence of the other four binding sites.Conclusion: Our data indicate that the ZRS 3′, not the central, subdomain is necessary for activity and contains the 3′ E-box that Hand2 likely uses to induce Shh expression, while the 5′ and central E-boxes appear to be inhibitory. Our data also suggest that the Hoxd13 binding sites promote localized activity within the ZPA.

Introduction: In vertebrate limb morphogenesis, wingless-related integration site (Wnt) proteins and fibroblast growth factors (Fgfs) secreted from the apical ectodermal ridge (AER) coordinate proximodistal outgrowth. Fgfs also sustain sonic hedgehog (Shh) in the zone of polarizing activity (ZPA). Shh directs anteroposterior patterning and expansion and regulates AER-Fgfs, establishing a positive regulatory feedback loop that is vital in sustaining limb outgrowth. The transcription factor LIM homeodomain 2 (Lhx2) is expressed in the distal mesoderm and coordinates AER and ZPA signals that control cellular proliferation, differentiation, and shaping of the developing limb. Yet how Lhx2 is transcriptionally regulated to support such functions has only been partially characterized.Methods/Results: We have identified two limb-specific cis-regulatory modules (CRMs) active within the Lhx2 expression domain in the limb. Chromatin conformation analysis of the Lhx2 locus in mouse embryonic limb bud cells predicted CRMs-Lhx2 promoter interactions. Single-cell RNA-sequencing analysis of limb bud cells revealed co-expression of several Fgf-related Ets and Wnt-related Tcf/Lef transcripts in Lhx2-expressing cells. Additionally, disruption of Ets and Tcf/Lef binding sites resulted in loss of reporter-driven CRM activity. Finally, binding of β-catenin to both Lhx2-associated CRMs supports the associated binding of Tcf/Lef transcription factors.Discussion: These results suggest a role for Ets and Tcf/Lef transcription factors in the regulation of Lhx2 expression through these limb-specific Lhx2-associated CRMs. Moreover, these CRMs provide a mechanism for Fgf and Wnt signaling to localize and maintain distal Lhx2 expression during vertebrate limb development.

Image analysis is a rapidly developing field that provides unique opportunities to characterize and quantify spatial information in images. We study cis -regulatory modules (CRMs), non-coding DNA regions that regulate gene expression, during development using fluorescent reporters in vivo. Characterizing CRM activity during development presents challenges including image segmentation into biologically relevant regions of interest that are not easily distinguishable via common segmentation methods, fluorophore band passing, and variable transfection undermine standardized analysis. To quantify and compare CRM activity levels, we compiled an open-source computer vision tool stack in the form of a Python -based Jupyter notebook and tested four analysis methods to assess their efficacy in quantifying CRM expression in limb development. This Jupyter notebook provides a reproducible, standardized workflow that can be adapted to numerous image analysis applications.

1 Background: During development, Hand2 and Hoxd13 transcription factors (TFs) regulate Sonic hedgehog (Shh) expression in the zone of polarizing activity (ZPA) in the distal posterior limb mesoderm. The ZPA regulatory sequence (ZRS) is a conserved, limb-specific enhancer that controls Shh expression. The ZRS can be divided into 5’, central, and 3’ subdomains, each with an E-box site that can bind basic helix-loop-helix (bHLH) TFs like Hand2. In addition, two Hoxd13 sites are present in the 5’ and central subdomains. Hand2 purportedly binds the ZRS through the central E-box, and both Hand2 and Hoxd13 have been shown to activate the ZRS in vitro . We hypothesized that the central E-box was required for activity, while the other E-boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm. Methods: To identify the functional role of each subdomain, we generated three ZRS fragments (5’, central, and 3’) and combined fragment constructs to test subdomain collective contributions. Additionally, we disrupted the five binding sites, alone or in concert, using site-directed mutagenesis. All ZRS constructs were cloned into a GFP reporter and evaluated in an in vivo chicken limb bioassay. We validated our findings using select ZRS constructs in transgenic mice. Results: We found that the 3’ fragment was necessary for ZRS activity, while the 5’ and central fragments had no activity alone or when combined. However, combining the 3’ fragment with the 5’ fragment restored robust activity. Further, mutation of all five binding sites markedly reduced ZRS activity. Reinstating each of the Hoxd13 sites restored focal activity, while restoring the 5’ and central E-boxes had little effect. However, the 3’ E-box proved sufficient for robust activity even in the absence of the other four binding sites. Conclusions: Our data indicate that the ZRS 3’, not the central, subdomain is necessary for activity and contains the 3’ E-box that Hand2 likely uses to induce Shh expression, while the 5’ and central E-boxes appear to be inhibitory. Our data also suggest that the Hoxd13 binding sites promote localized activity within the ZPA.

"Letter to the Editor. Interesting, rare case, but VACTERL?" published on 26 Aug 2024 by American Association of Neurological Surgeons.

The limb anatomy displays well‐defined dorsal and ventral compartments, housing extensor, and flexor muscles, which play a crucial role in facilitating limb locomotion and manipulation. Despite its importance, the study of limb dorsoventral patterning has been relatively neglected compared to the other two axes leaving many crucial questions about the genes and developmental processes implicated unanswered. This review offers a thorough overview of the current understanding of limb dorsoventral patterning, synthesizing classical literature with recent research. It covers the specification of dorsal fate in the limb mesoderm and its subsequent translation into dorsal morphologies—a process directed by the transcription factor Lmx1b. We also discuss the potential role of dorsoventral patterning in the evolution of paired appendages and delve into the involvement of LMX1B in Nail‐Patella syndrome, discussing the molecular and genetic aspects underlying this condition. Finally, the potential role of dorsoventral polarity in digit tip regeneration, a prominent instance of multi‐tissue regeneration in mammals is also considered. We anticipate that this review will renew interest in a process that is critical to limb function and evolutionary adaptations but has nonetheless been overlooked.

We report eight cases of complex radial polydactyly and/or ulnar dimelia each with a fistulous lesion of heterotopic respiratory mucosa in radial-anterior skin ranging from shoulder to wrist. Although speculative, the coincidence of these rare conditions suggests a mechanistic relationship.

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.