Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating.

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.

Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.SIGNIFICANCE STATEMENT Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias.

Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure.

Hepatoblasts comprise a niche for fetal liver erythropoiesis through cytokine production.

In mammals, definitive erythropoiesis first occurs in fetal liver (FL), although little is known about how the process is regulated. FL consists of hepatoblasts, sinusoid endothelial cells and hematopoietic cells. To determine niche cells for fetal liver erythropoiesis, we isolated each FL component by flow cytometry. mRNA analysis suggested that Dlk-1-expressing hepatoblasts primarily expressed EPO and SCF, genes encoding erythropoietic cytokines. EPO protein was detected predominantly in hepatoblasts, as assessed by ELISA and immunohistochemistry, and was not detected in sinusoid endothelial cells and hematopoietic cells. To characterize hepatoblast function in FL, we analyzed Map2k4(-/-) mouse embryos, which lack hepatoblasts, and observed down-regulation of EPO and SCF expression in FL relative to wild-type mice. Our observations demonstrate that hepatoblasts comprise a niche for erythropoiesis through cytokine secretion.

Variation in mesodermal and hematopoietic potential of adult skin-derived induced pluripotent stem cell lines in mice.

Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine. Use of iPSC lines for future hematotherapy will require examination of their hematopoietic potential. Adult skin fibroblast somatic cells constitute a source of iPSCs that can be accessed clinically without ethical issues. Here, we used different methods to compare mesodermal and hematopoietic potential by embryoid body formation of five iPSC lines established from adult mouse tail-tip fibroblasts (TTFs). We observed variation in proliferation and in expression of genes (Brachyury, Tbx1, Gata1, Klf1, Csf1r) and proteins (Flk1, Ter119 and CD45) among TTF-derived lines. 256H18 iPSCs showed highest proliferation and most efficient differentiation into mesodermal and hematopoietic cells, while expression levels of the pluripotency genes Oct3/4, Sox2, Klf4 and Nanog were lowest among lines analyzed. By contrast, the 212B2 line, transduced with c-Myc, showed lowest proliferation and differentiation potential, although expression levels of Oct3/4, Sox2 and Klf4 were highest. Overall, we find that mesodermal and hematopoietic potential varies among iPSCs from an identical tissue source and that c-Myc expression likely underlies these differences.