Fossil evidence for a pharyngeal origin of the vertebrate pectoral girdle.

The origin of vertebrate paired appendages is one of the most investigated and debated examples of evolutionary novelty1-7. Paired appendages are widely considered as key innovations that enabled new opportunities for controlled swimming and gill ventilation and were prerequisites for the eventual transition from water to land. The past 150 years of debate8-10 has been shaped by two contentious theories4,5: the ventrolateral fin-fold hypothesis9,10 and the archipterygium hypothesis8. The latter proposes that fins and girdles evolved from an ancestral gill arch. Although studies in animal development have revived interest in this idea11-13, it is apparently unsupported by fossil evidence. Here we present palaeontological support for a pharyngeal basis for the vertebrate shoulder girdle. We use computed tomography scanning to reveal details of the braincase of Kolymaspis sibirica14, an Early Devonian placoderm fish from Siberia, that suggests a pharyngeal component of the shoulder. We combine these findings with refreshed comparative anatomy of placoderms and jawless outgroups to place the origin of the shoulder girdle on the sixth branchial arch. These findings provide a novel framework for understanding the origin of the pectoral girdle. Our evidence clarifies the location of the presumptive head-trunk interface in jawless fishes and explains the constraint on branchial arch number in gnathostomes15. The results revive a key aspect of the archipterygium hypothesis and help reconcile it with the ventrolateral fin-fold model.

Cell segmentation in images without structural fluorescent labels.

High-content screening (HCS) provides an excellent tool to understand the mechanism of action of drugs on disease-relevant model systems. Careful selection of fluorescent labels (FLs) is crucial for successful HCS assay development. HCS assays typically comprise (a) FLs containing biological information of interest, and (b) additional structural FLs enabling instance segmentation for downstream analysis. However, the limited number of available fluorescence microscopy imaging channels restricts the degree to which these FLs can be experimentally multiplexed. In this article, we present a segmentation workflow that overcomes the dependency on structural FLs for image segmentation, typically freeing two fluorescence microscopy channels for biologically relevant FLs. It consists in extracting structural information encoded within readouts that are primarily biological, by fine-tuning pre-trained state-of-the-art generalist cell segmentation models for different combinations of individual FLs, and aggregating the respective segmentation results together. Using annotated datasets that we provide, we confirm our methodology offers improvements in performance and robustness across several segmentation aggregation strategies and image acquisition methods, over different cell lines and various FLs. It thus enables the biological information content of HCS assays to be maximized without compromising the robustness and accuracy of computational single-cell profiling.