KEY PUBLICATIONS

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...

Shown are the dynamic patterns of the MECOM, LEF1, and TCF7 footprints in mouse and chicke

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development...

Direct comparison of the Grem1 expression domain in forelimb buds of the most relevant CRM

Reinhardt R., Gullotta F., Nusspaumer G., Ünal E., Ivanek R., Zuniga A. and Zeller R.

The key molecular interactions governing vertebrate limb bud development are a paradigm to study the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the molecular signatures of the early limb bud mesenchymal progenitors (LMPs) that ultimately give rise to the chondrogenic condensations of the future limb skeletal elements...

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond

Frédéric Laurent, Ausra Girdziusaite, Julie Gamar1, Iros Barozzi, Marco Osterwalder, Jennifer A. Akiyama, Joy Lincoln, Javier Lopez-Rios, Axel Visel, Aimée Zuniga and Rolf Zeller.

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation...

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Dev

Aimée Zuniga

The molecular analysis of limb bud development in vertebrates continues to fuel our understanding of the gene regulatory networks that orchestrate the patterning, proliferation and differentiation of embryonic progenitor cells. In recent years, systems biology approaches have moved our understanding of the molecular control of limb organogenesis to the next level by incorporating next generation ‘omics’ approaches, analyses of chromatin architecture, enhancer-promoter interactions and gene network simulations based on quantitative datasets into experimental analyses...

Next generation limb development and evolution: old questions, new perspectives.png

Lopez-Rios J., Duchesne A., Speziale D., Andrey G., Peterson K.A., Germann P., Ünal E., Liu J., Floriot S., Barbey S., Gallard Y., Müller-Gerbl M., Courtney A.D., Klopp C., Rodriguez S., Ivanek R., Beisel C., Wicking C., Iber D., Robert B., McMahon A.P., Duboule D. and Zeller R.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme...

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.png

Osterwalder M., Speziale D., Shoukry M., Mohan R., Ivanek R., Kohler M., Beisel C., Wen X., Scales S.J., Christoffels V.M., Visel A., Lopez-Rios J., and Zeller R.

 The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting Gli3and Tbx3 expression...

HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Ear

Lopez-Rios J., Speziale D., Robay D., Scotti M., Nusspaumer G., Osterwalder M., Galli A., Holländer G., Kmita M. and Zeller R.

  

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G1–S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate...

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Probst S., Kraemer C., Demougin P., Sheth R., Martin GR, Shiratori H., Hamada H., Iber D., Zeller R., Zuniga A.

The essential roles of SHH in anteroposterior (AP) and AER-FGF signalling in proximodistal (PD) limb bud development are well understood. In addition, these morphoregulatory signals are key components of the self-regulatory SHH/GREM1/AER-FGF feedback signalling system that regulates distal progression of limb bud development. This study uncovers an additional signalling module required for coordinated progression of limb bud axis development. Transcriptome analysis using Shh-deficient mouse limb buds revealed that the expression of proximal genes was distally extended from early stages onwards, which pointed to a more prominent involvement of SHH in PD limb axis development. In particular, retinoic acid (RA) target genes were upregulated proximally, while the expression of the RA-inactivating Cyp26b1 enzyme was downregulated distally, pointing to increased RA activity in Shh-deficient mouse limb buds...

SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid cle

Antonella Galli , Dimitri Robay, Marco Osterwalder, Xiaozhong Bao, Jean-Denis Bénazet, Muhammad Tariq, Renato Paro, Susan Mackem, and Rolf Zeller

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shhexpression, which results in a skeletal phenotype identical to Shh deficient limb buds...

Distinct Roles of Hand2 in Initiating Polarity and Posterior Shh Expression during the Ons

Bénazet J.D., Bischofberger M., Tiecke E., Gonçalves A., Martin J.F., Zuniga A., Naef F. and Zeller R.

Embryogenesis depends on self-regulatory interactions between spatially separated signaling centers, but few of these are well understood. Limb development is regulated by epithelial-mesenchymal (e-m) feedback loops between sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist Gremlin1 (GREM1). By combining mouse molecular genetics with mathematical modeling, we showed that BMP4 first initiates and SHH then propagates e-m feedback signaling through differential transcriptional regulation of Grem1 to control digit specification...

REPORT A Self-Regulatory System of Interlinked Signaling Feedback Loops Controls Mouse Lim

Zuniga A., Michos O., Spitz F., Haramis A.P.G., Panman L., Vintersten K., Klasen C., Mansfield W., Kuc S., Duboule D., Dono R. and Zeller R.

The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial–mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ldalleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ldalleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis...

Mouse limb deformity mutations disrupt a global control region within the large regulatory

teWelscher P., Zuniga, A, Kuijper S., Drenth T., Goedemans H, Meijlink F. and Zeller R.

Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly...

Progression of Vertebrate Limb Development Through SHH-Mediated Counteraction of GLI3.png

Zuniga A., Haramis A.G., McMahon A.P. and Zeller R.

Outgrowth and patterning of the vertebrate limb are controlled by reciprocal interactions between the posterior mesenchyme (polarizing region) and a specialized ectodermal structure, the apical ectodermal ridge (AER). Sonic hedgehog (SHH) signalling by the polarizing region modulates fibroblast growth factor (FGF)4 signalling by the posterior AER, which in turn maintains the polarizing region (SHH/FGF4 feedback loop). Here we report that the secreted bone-morphogenetic-protein (BMP) antagonist Gremlin4 relays the SHH signal from the polarizing region to the AER. Mesenchymal Gremlin expression is lost in limb buds of mouse embryos homozygous for the limb deformity (ld) mutation, which disrupts establishment of the SHH/FGF4 feedback loop...

Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds