Biology subjects

Oberg, K. C.

Publications and source records attributed to Oberg, K. C..

4 recordsLinked to original sources

bioRxiv2025

The 3' Region of the ZPA Regulatory Sequence (ZRS) is required for activity and contains a critical E-box

1BackgroundDuring 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. MethodsTo 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. ResultsWe 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. ConclusionsOur 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.

developmental biology↗

bioRxiv2025

An Open-Source Image Analysis Method for Quantifying Reporter Fluorescence

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.

developmental biology↗

bioRxiv2022

Failure of digit tip regeneration in the absence of suggests Lmx1b functions disparate from dorsoventral polarity

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of {Delta}LARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nails effect from the lack of DV polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative{Delta} LARM1/2 mutant blastemas reveals differential up-regulation of vascularization and connective tissue functional signatures in wild-type versus upregulation of inflammation in the mutant. These results, together with the finding of uniform Lmx1b expression in the wild-type blastema and in the dorsal dermis underneath the nail, indicate that, in addition of the nails effect, a direct role for Lmx1b in driving the progression of digit tip regeneration is likely.

developmental biology↗

bioRxiv2020

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-Patella Syndrome pathogenicity.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly, in mice Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of murine Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. Interestingly, in Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression in the limb and are necessary for Lmx1b-mediated limb dorsalization. Remarkably, we also report on two NPS patient families with normal LMX1B coding sequence, but loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

developmental biology↗