A Modified SMART-Seq Method for Single-Cell Transcriptomic Analysis of Embryoid Body Differentiation.

Embryoid bodies (EBs) are aggregate of cells that contain three embryonic germ layers. They can be formed by direct differentiation from pluripotent embryonic stem cells (ESCs), which serves as a useful model for understanding early embryo development. Due to the mixture of different cell types, it is necessary to investigate EBs at the single-cell level. Here, we describe a robust and straightforward method for single-cell gene expression profiling during mouse EB differentiation from mouse ESCs (mESCs). The protocol is modified from a widely used method in the SMART-seq family, which only requires standard molecular biology techniques and lab equipment. It allows for accurate 3' counting of transcript at the single-cell level, which helps reveal cellular identities during EB formation. Combined with perturbation experiments, the method provides an opportunity for mechanistic studies of embryo development at the single-cell level.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

Wdr47 Controls Neuronal Polarization through the Camsap Family Microtubule Minus-End-Binding Proteins.

Neurons require proper polarization for precise positioning and axon-dendrite formation. Their intrinsic regulators and underlying mechanisms are poorly understood. Here, we show that Wdr47 is a key polarization regulator. Wdr47-deficient newborn mice die of suffocation due to central nervous system defects including axonal tracts agenesis and slowed radial migration. Wdr47 deficiency represses the multipolar-bipolar transition of cortical neurons, reduces neurite tip-directed microtubule dynamics, and causes multi-axon formation. Overexpression of Wdr47 in wild-type neurons inhibits axon specification and neutralizes Taxol-induced neurite overgrowth and axon overproduction. Wdr47 interacts with the Camsap family of microtubule minus-end-binding proteins; associates with microtubules through Camsap3, whose gene disruption also causes multi-axons; and promotes Camsap3 accumulation in neurites of unpolarized neurons. Furthermore, Camsap overexpression rescues the polarization defects of Wdr47-deficient neurons. Our results indicate that Wdr47 facilitates proper neurite remodeling through Camsaps to fine-tune regional microtubule dynamics and organization during early stages of neuronal polarization.

Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat.

Motile cilia and flagella are microtubule-based organelles important for cell locomotion and extracellular liquid flow through beating. Although axonenal dyneins that drive ciliary beat have been extensively studied in unicellular Chlamydomonas, to what extent such knowledge can be applied to vertebrate is poorly known. In Chlamydomonas, Dynein-f controls flagellar waveforms but is dispensable for beating. The flagellar assembly of its heavy chains (HCs) requires its intermediate chain (IC) IC140 but not IC138. Here we show that, unlike its Chlamydomonas counterpart, vertebrate Dynein-f is essential for ciliary beat. We confirmed that Wdr78 is the vertebrate orthologue of IC138. Wdr78 associated with Dynein-f subunits such as Dnah2 (a HC) and Wdr63 (IC140 orthologue). It was expressed as a motile cilium-specific protein in mammalian cells. Depletion of Wdr78 or Dnah2 by RNAi paralyzed mouse ependymal cilia. Zebrafish Wdr78 morphants displayed ciliopathy-related phenotypes, such as curved bodies, hydrocephalus, abnormal otolith, randomized left-right asymmetry, and pronephric cysts, accompanied with paralyzed pronephric cilia. Furthermore, all the HCs and ICs of Dynein-f failed to localize in the Wdr78-depleted mouse ependymal cilia. Therefore, both the functions and subunit dependency of Dynein-f are altered in evolution, probably to comply with ciliary roles in higher organisms.

Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation.

Cilia are cellular protrusions containing nine microtubule (MT) doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions. Cilia require the central pair (CP) apparatus, consisting of two CP MTs covered with projections of CP proteins, for planar strokes. How the CP MTs of such '9 + 2' cilia are constructed, however, remains unknown. Here we identify Spef1, an evolutionarily conserved microtubule-bundling protein, as a core CP MT regulator in mammalian cilia. Spef1 was selectively expressed in mammalian cells with 9 + 2 cilia and specifically localized along the CP. Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6, resulting in rotational beat of the ependymal cilia. Spef1, which binds to MTs through its N-terminal calponin-homologous domain, formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs. Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus. We propose that Spef1 bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.