Dagmar Iber

Branch Mode Selection during Early Lung Development

Many organs of higher organisms, such as the vascular system, lung, kidney, pancreas, liver and glands, are heavily branched structures. The branching process during lung development has been studied in great detail and is remarkably stereotyped. The branched tree is generated by the sequential, non-random use of three geometrically simple modes of branching (domain branching, planar and orthogonal bifurcation). While many regulatory components and local interactions have been defined an integrated understanding of the regulatory network that controls the branching process is lacking. We have developed a deterministic, spatio-temporal differential-equation based model of the core signaling network that governs lung branching morphogenesis. The model focuses on the two key signaling factors that have been identified in experiments, fibroblast growth factor (FGF10) and sonic hedgehog (SHH) as well as the SHH receptor patched (Ptc). We show that the reported biochemical interactions give rise to a Schnakenberg-type Turing patterning mechanisms that allows us to reproduce experimental observations in wildtype and mutant mice. The kinetic parameters as well as the domain shape are based on experimental data where available. The developed model is robust to small absolute and large relative changes in the parameter values. At the same time there is a strong regulatory potential in that the switching between branching modes can be achieved by targeted changes in the parameter values. We note that the sequence of different branching events may also be the result of different growth speeds: fast growth triggers lateral branching while slow growth favours bifurcations in our model. We conclude that the FGF10-SHH-Ptc1 module is sufficient to generate pattern that correspond to the observed branching modes.

SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling

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. Further genetic and molecular analysis established that Cyp26b1 expression is regulated by AER-FGF signalling. During initiation of limb bud outgrowth, the activation of Cyp26b1 expression creates a distal ‘RA-free’ domain, as indicated by complementary downregulation of a transcriptional sensor of RA activity. Subsequently, Cyp26b1 expression increases as a consequence of SHH-dependent upregulation of AER-FGF signalling. To better understand the underlying signalling interactions, computational simulations of the spatiotemporal expression patterns and interactions were generated. These simulations predicted the existence of an antagonistic AER-FGF/CYP26B1/RA signalling module, which was verified experimentally. In summary, SHH promotes distal progression of limb development by enhancing CYP26B1-mediated RA clearance as part of a signalling network linking the SHH/GREM1/AER-FGF feedback loop to the newly identified AER-FGF/CYP26B1/RA module.

The control of branching morphogenesis

Many organs of higher organisms are heavily branched structures and arise by an apparently similar process of branching morphogenesis. Yet the regulatory components and local interactions that have been identified differ greatly in these organs. It is an open question whether the regulatory processes work according to a common principle and how far physical and geometrical constraints determine the branching process. Here, we review the known regulatory factors and physical constraints in lung, kidney, pancreas, prostate, mammary gland and salivary gland branching morphogenesis, and describe the models that have been formulated to analyse their impacts.

Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs

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. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

Digit patterning during limb development as a result of the BMP-receptor interaction

Turing models have been proposed to explain the emergence of digits during limb development. However, so far the molecular components that would give rise to Turing patterns are elusive. We have recently shown that a particular type of receptor-ligand interaction can give rise to Schnakenberg-type Turing patterns, which reproduce patterning during lung and kidney branching morphogenesis. Recent knockout experiments have identified Smad4 as a key protein in digit patterning. We show here that the BMP-receptor interaction meets the conditions for a Schnakenberg-type Turing pattern, and that the resulting model reproduces available wildtype and mutant data on the expression patterns of BMP, its receptor, and Fgfs in the apical ectodermal ridge (AER) when solved on a realistic 2D domain that we extracted from limb bud images of E11.5 mouse embryos. We propose that receptor-ligand-based mechanisms serve as a molecular basis for the emergence of Turing patterns in many developing tissues.

Axis Patterning by BMPs: Cnidarian Network Reveals Evolutionary Constraints

Summary BMP signaling plays a crucial role in the establishment of the dorso-ventral body axis in bilaterally symmetric animals. However, the topologies of the bone morphogenetic protein (BMP) signaling networks vary drastically in different animal groups, raising questions about the evolutionary constraints and evolvability of BMP signaling systems. Using loss-of-function analysis and mathematical modeling, we show that two signaling centers expressing different BMPs and BMP antagonists maintain the secondary axis of the sea anemone Nematostella. We demonstrate that BMP signaling is required for asymmetric Hox gene expression and mesentery formation. Computational analysis reveals that network parameters related to BMP4 and Chordin are constrained both in Nematostella and Xenopus, while those describing the BMP signaling modulators can vary significantly. Notably, only chordin, but not bmp4 expression needs to be spatially restricted for robust signaling gradient formation. Our data provide an explanation of the evolvability of BMP signaling systems in axis formation throughout Eumetazoa.

Kidney branching morphogenesis under the control of a ligand–receptor-based Turing mechanism

The main signalling proteins that control early kidney branching have been defined. Yet the underlying mechanism is still elusive. We have previously shown that a Schnakenberg-type Turing mechanism can recapitulate the branching and protein expression patterns in wild-type and mutant lungs, but it is unclear whether this mechanism would extend to other branched organs that are regulated by other proteins. Here, we show that the glial cell line-derived neurotrophic factor-RET regulatory interaction gives rise to a Schnakenberg-type Turing model that reproduces the observed budding of the ureteric bud from the Wolffian duct, its invasion into the mesenchyme and the observed branching pattern. The model also recapitulates all relevant protein expression patterns in wild-type and mutant mice. The lung and kidney models are both based on a particular receptor-ligand interaction and require (1) cooperative binding of ligand and receptor, (2) a lower diffusion coefficient for the receptor than for the ligand and (3) an increase in the receptor concentration in response to receptor-ligand binding (by enhanced transcription, more recycling or similar). These conditions are met also by other receptor-ligand systems. We propose that ligand-receptor-based Turing patterns represent a general mechanism to control branching morphogenesis and other developmental processes.

An interplay of geometry and signaling enables robust lung branching morphogenesis

Early branching events during lung development are stereotyped. Although key regulatory components have been defined, the branching mechanism remains elusive. We have now used a developmental series of 3D geometric datasets of mouse embryonic lungs as well as time-lapse movies of cultured lungs to obtain physiological geometries and displacement fields. We find that only a ligand-receptor-based Turing model in combination with a particular geometry effect that arises from the distinct expression domains of ligands and receptors successfully predicts the embryonic areas of outgrowth and supports robust branch outgrowth. The geometry effect alone does not support bifurcating outgrowth, while the Turing mechanism alone is not robust to noisy initial conditions. The negative feedback between the individual Turing modules formed by fibroblast growth factor 10 (FGF10) and sonic hedgehog (SHH) enlarges the parameter space for which the embryonic growth field is reproduced. We therefore propose that a signaling mechanism based on FGF10 and SHH directs outgrowth of the lung bud via a ligand-receptor-based Turing mechanism and a geometry effect.

Dynamic scaling of morphogen gradients on growing domains

Developmental mechanisms are highly conserved, yet act in embryos of very different sizes. How scaling is achieved has remained elusive. Here we identify a generally applicable mechanism for dynamic scaling on growing domains and show that it quantitatively agrees with data from the Drosophila wing imaginal disc. We show that for the measured parameter ranges, the Dpp gradient does not reach steady state during Drosophila wing development. We further show that both, pre-steady-state dynamics and advection of cell-bound ligand in a growing tissue can, in principle, enable scaling, even for non-uniform tissue growth. For the parameter values that have been established for the Dpp morphogen in the Drosophila wing imaginal disc, we show that scaling is mainly a result of the pre-steady-state dynamics. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for dynamic scaling of morphogen gradients in growing developmental systems.

Integrin activation—the importance of a positive feedback

Integrins mediate cell adhesion and are essential receptors for the development and functioning of multicellular organisms. Integrin activation is known to require both ligand and talin binding and to correlate with cluster formation but the activation mechanism and precise roles of these processes are not yet resolved. Here mathematical modeling, with known experimental parameters, is used to show that the binding of a stabilizing factor, such as talin, is alone insufficient to enable ligand-dependent integrin activation for all observed conditions; an additional positive feedback is required.