Biology subjects

Schneider, R. A.

Publications and source records attributed to Schneider, R. A..

8 recordsLinked to original sources

bioRxiv2026

High efficiency gene editing and phenotype generation in avian embryos using lipid nanoparticles as a vector delivery platform

Birds are a longstanding developmental biology model because easy access to their fertilized eggs allows for direct observation and experimental manipulation during any stage of embryonic growth prior to hatching. However, functional genetics studies in avian embryos continue to rely primarily on in ovo electroporation and viral transduction, each of which is limited by variable efficiency, procedure-induced embryonic defects, substantial embryo mortality, and ineffective delivery of gene editing reagents such as CRISPR/Cas9. Here we establish lipid nanoparticle (LNP)-mediated delivery as a reproducible alternative for manipulating gene expression in avian embryos with high viability. Using the chicken embryo (Gallus gallus), we targeted migrating cranial NCCs, which contribute extensively to the craniofacial skeleton. We optimized LNP formulation and injection timing and frequency to build a robust and adaptable pipeline that offers efficient delivery of plasmid DNA and RNA cargos. We show that LNP delivery can support the piggyBac transposon system, enabling persistent reporter expression and long-term labeling of cranial NCC derivatives. To test if LNP-mediated delivery of RNA-based CRISPR/Cas9 components can achieve efficient somatic genome editing in ovo, we targeted Runt-related transcription factor 2 (RUNX2), which is required for osteogenesis. Embryos with RUNX2 edits in their cranial NCCs show a severe loss and disruption of craniofacial ossification on the targeted side of their craniofacial skeletons, demonstrating the potential of this approach for rapid functional interrogation of gene function in vivo. We also show that LNPs can mediate transfection in domestic pigeon (Columba livia) and Pekin duck (Anas platyrhynchos) embryos, indicating this method can be deployed across avians as a vector delivery platform for studying development, disease, and evolution.

developmental biology↗

bioRxiv2025

Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development

The neural crest is a vertebrate innovation central to craniofacial development and evolution. While the gene regulatory networks guiding neural crest development are well characterized, the mechanisms generating species-specific craniofacial diversity remain poorly understood. Marsupials provide a unique model for studying neural crest plasticity, having evolved accelerated patterns of craniofacial development during embryogenesis. This adaptation arises in response to marsupials being born altricial after a short gestation yet require well-developed mouthparts to attach to a teat and continue development in the pouch. However, how marsupials achieve this heterochronic shift in neural crest development is largely unknown. In this study, we investigate the cellular and molecular mechanisms underlying their distinct heterochrony, revealing that marsupials produce dense pre-migratory aggregates of neural crest cells which undergo collective migration as epithelial-like sheets, potentially facilitating rapid establishment of the facial prominences. These cellular behaviours are unique amongst amniotes but resemble patterns in anamniotes which similarly exhibit accelerated craniofacial development to support early feeding. Marsupials appear to have evolved a similar mechanism of neural crest migration to facilitate their developmental heterochrony. These findings suggest that vertebrate neural crest migration may be shaped by the pace of craniofacial development during embryogenesis rather than phylogeny, providing new perspectives on neural crest plasticity and the developmental mechanisms driving craniofacial diversity across vertebrates.

developmental biology↗

bioRxiv2021

Differential regulation of SHH signaling and the developmental control of species-specific jaw size through neural crest-mediated Gas1 expression

Developmental control of jaw size is crucial to prevent disease and facilitate evolution. We have shown that species-specific differences in jaw size are established by neural crest mesenchyme (NCM), which are the jaw progenitors that migrate into the mandibular primordia. NCM relies on multiple signaling molecules including Sonic Hedgehog (SHH) to mediate interactions with mandibular epithelium that facilitate outgrowth of the jaws. SHH signaling is known to promote outgrowth and so we tested if differential regulation of the SHH pathway can account for species-specific variation in mandibular primordia size. We analyze gene expression of SHH pathway members in duck, chick, and quail, and find higher transcriptional activation in the larger mandibular primordia of duck relative to those of chick and quail. We generate quail-duck chimeras and demonstrate that such activation is NCM-mediated. Gain- and loss-of-function experiments reveal a species-specific response to SHH signaling, with the target Gas1 being most sensitive to manipulations. Gas1 overexpression and knockdown in NCM alters cell number and/or mandibular primordia size. Our work suggests that NCM-mediated changes in SHH signaling may modulate jaw size during development, disease, and evolution. Summary StatementWe have determined that Gas1, which is a component of the SHH signaling pathway, plays a key role in the development and evolution of jaw size.

developmental biology↗

bioRxiv2021

Species-specific deployment of Runx2 isoforms and differential regulation of target genes during avian jaw development and evolution

Developmental regulation of bone formation in the jaw skeleton is essential to species-specific adaptation. The jaws are derived from neural crest mesenchyme (NCM), a progenitor population that directs skeletal patterning by exerting temporal and spatial control over molecular and cellular programs for osteogenesis. One important NCM-mediated gene is Runx2, which is a transcription factor required for osteoblast differentiation. RUNX2 protein binds many target genes involved in the deposition and resorption of bone. To determine the extent to which changes in Runx2 structure, function, and expression underlie the evolution of the jaw skeleton, we compare Runx2 across vertebrates and within birds. Runx2 contains two alternative promoters, tandem repeats of glutamine and alanine with variable lengths in different species, a conserved DNA-binding domain, an exon that is alternatively spliced, as well as two possible C-termini. Such alternative splicing produces eight potential isoforms that show distinct stage- and species-specific patterns in the jaw primordia of chick, quail and duck embryos. We also find that certain isoforms are strongly induced by TGF{beta} signaling whereas others are not. Overexpressing Runx2 isoforms in NCM reveals that some are transcriptionally activating, while others are repressive. But context appears to be relevant since species-specific polymorphisms in the promoter of target genes like Mmp13, can modulate the effects of different isoforms. Overall, our study indicates that the structure and species-specific deployment of Runx2 isoforms affect the transcriptional activity of target genes in ways that may have played a generative and regulatory role in the evolution of the avian jaw skeleton.

developmental biology↗

bioRxiv2020

Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution

Developmental control of jaw length is critical for survival. The jaw skeleton arises from neural crest mesenchyme and previously we demonstrated that these progenitors upregulate bone-resorbing enzymes including Matrix metalloproteinase 13 (Mmp13) when generating short quail beaks versus long duck bills. Inhibiting bone resorption or Mmp13 increases jaw length. Here, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of, and sensitivity to Transforming Growth Factor-Beta (TGF{beta}) signaling than duck; where mediators like SMADs and targets like Runx2, which bind Mmp13, become elevated. Inhibiting TGF{beta} signaling decreases bone resorption. We discover a SMAD binding element in the quail Mmp13 promoter not found in duck and single nucleotide polymorphisms (SNPs) near a RUNX2 binding element that affect expression. Switching the SNPs and SMAD site abolishes TGF{beta}-sensitivity in the quail Mmp13 promoter but makes duck responsive. Thus, differential regulation of TGF{beta} signaling and Mmp13 promoter structure underlie avian jaw development and evolution.

developmental biology↗

bioRxiv2020

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.View Full Text

developmental biology↗

bioRxiv2018

Developmental Mechanisms Linking Form and Function During Jaw Evolution

How does form arise during development and change during evolution? How does form relate to function, and what enables embryonic structures to presage their later use in adults? To address these questions, we leverage the distinct functional morphology of the jaw in duck, chick, and quail. In connection with their specialized mode of feeding, duck develop a secondary cartilage at the tendon insertion of their jaw adductor muscle on the mandible. An equivalent cartilage is absent in chick and quail. We hypothesize that species-specific jaw architecture and mechanical forces promote secondary cartilage in duck through the differential regulation of FGF and TGF{beta} signaling. First, we perform transplants between chick and duck embryos and demonstrate that the ability of neural crest mesenchyme (NCM) to direct the species-specific insertion of muscle and the formation of secondary cartilage depends upon the amount and spatial distribution of NCM-derived connective tissues. Second, we quantify motility and build finite element models of the jaw complex in duck and quail, which reveals a link between species-specific jaw architecture and the predicted mechanical force environment. Third, we investigate the extent to which mechanical load mediates FGF and TGF{beta} signaling in the duck jaw adductor insertion, and discover that both pathways are mechano-responsive and required for secondary cartilage formation. Additionally, we find that FGF and TGF{beta} signaling can also induce secondary cartilage in the absence of mechanical force or in the adductor insertion of quail embryos. Thus, our results provide novel insights on molecular, cellular, and biomechanical mechanisms that couple musculoskeletal form and function during development and evolution.

developmental biology↗

bioRxiv2017

Tissue Folding by Mechanical Compaction of the Mesenchyme

Many tissues fold during development into complex shapes. Engineering this process in vitro would represent an important advance for tissue engineering. We use embryonic tissue explants, finite element modeling, and 3D cell patterning techniques to show that a mechanical compaction of the ECM during mesenchymal condensation can drive tissue folding along programmed trajectories. The process requires cell contractility, generates strains at nearby tissue interfaces, and causes specific patterns of collagen alignment around and between condensates. Aligned collagen fibers support elevated tensions that promote the folding of interfaces along paths that can be predicted by finite element modeling. We demonstrate the robustness and versatility of this strategy for sculpting tissue interfaces by directing the morphogenesis of a variety of folded tissue forms from engineered patterns of mesenchymal condensates. These studies provide insight into the active mechanical properties of the embryonic mesenchyme and establish entirely new strategies for more robustly directing tissue morphogenesis ex vivo, without genetic engineering.

bioengineering↗