Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

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.

Single-image Bayesian Restoration and Multi-image Super-resolution Restoration for B-mode Ultrasound Using an Accurate System Model Involving Correlated Nature of the Speckle Noise.

B-mode ultrasound is an essential part of radiological examinations due to its low cost, safety, and portability, but has the drawbacks of the speckle noise and output of most systems is two-dimensional (2D) cross sections. Image restoration techniques, using mathematical models for image degradation and noise, can be used to boost resolution (deconvolution) as well as to reduce the speckle. In this study, new single-image Bayesian restoration (BR) and multi-image super-resolution restoration (BSRR) methods are proposed for in-plane B-mode ultrasound images. The spatially correlated nature of the speckle was modeled, allowing for examination of two different models for BR and BSRR for uncorrelated Gaussian (BR-UG, BSRR-UG) and correlated Gaussian (BR-CG, BSRR-CG). The performances of these models were compared with common image restoration methods (Wiener filter, bilateral filtering, and anisotropic diffusion). Well-recognized metrics (peak signal-to-noise ratio, contrast-to-noise ratio, and normalized information density) were used for algorithm free-parameter estimation and objective evaluations. The methods were tested using superficial tissue (2D scan data collected from volunteers, tissue-mimicking resolutions, and breast phantoms). Improvement in image quality was assessed by experts using visual grading analysis. In general, BSRR-CG performed better than all other methods. A potential downside of BSRR-CG is increased computation time, which can be addressed by the use of high-performance graphics processing units (GPUs).

Image-based modelling of organogenesis.

One of the major challenges in biology concerns the integration of data across length and time scales into a consistent framework: how do macroscopic properties and functionalities arise from the molecular regulatory networks-and how can they change as a result of mutations? Morphogenesis provides an excellent model system to study how simple molecular networks robustly control complex processes on the macroscopic scale despite molecular noise, and how important functional variants can emerge from small genetic changes. Recent advancements in three-dimensional imaging technologies, computer algorithms and computer power now allow us to develop and analyse increasingly realistic models of biological control. Here, we present our pipeline for image-based modelling that includes the segmentation of images, the determination of displacement fields and the solution of systems of partial differential equations on the growing, embryonic domains. The development of suitable mathematical models, the data-based inference of parameter sets and the evaluation of competing models are still challenging, and current approaches are discussed.

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.

Smad4 is required to induce digit ray primordia and to initiate the aggregation and differentiation of chondrogenic progenitors in mouse limb buds.

SMAD4 is an essential mediator of canonical TGFß/BMP signal transduction and we inactivated Smad4 in mouse limb buds from early stages onward to study its functions in the mesenchyme. While this Smad4 inactivation did not alter the early Sox9 distribution, prefiguring the chondrogenic primordia of the stylopod and zeugopod, it disrupted formation of all Sox9-positive digit ray primordia. Specific inactivation of Smad4 during handplate development pointed to its differential requirement for posterior and anterior digit ray primordia. At the cellular level, Smad4 deficiency blocked the aggregation of Sox9-positive progenitors, thereby preventing chondrogenic differentiation as revealed by absence of collagen type II. The progressive loss of SOX9 due to disrupting digit ray primordia and chondrogenesis was paralleled by alterations in genes marking other lineages. This pointed to a general loss of tissue organization and diversion of mutant cells toward non-specific connective tissue. Conditional inactivation of Bmp2 and Bmp4 indicated that the loss of digit ray primordia and increase in connective tissue were predominantly a consequence of disrupting SMAD4-mediated BMP signal transduction. In summary, our analysis reveals that SMAD4 is required to initiate: (1) formation of the Sox9-positive digit ray primordia; and (2) aggregation and chondrogenic differentiation of all limb skeletal elements.