Cranial nerve fasciculation and Schwann cell migration are impaired after loss of Npn-1

Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon–axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A–Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1^−/− and Npn-1^Sema− mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon–Schwann cell interactions.     

Adult human CD133/1 kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin⁺ and CD133/1⁺ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1⁺ cells. Isolated CD133/1⁺ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1⁺ progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca²⁺ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca²⁺ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.     

Differential expression of tumor-associated genes and altered gut microbiome with decreased Akkermansia muciniphila confer a tumor-preventive microenvironment in intestinal epithelial Pten-deficient mice

Phosphatase and tensin homolog (Pten) antagonizes PI3K-Akt signaling; therefore, Pten impairment causes tumorigenesis. However, the correlation between Pten deficiency and colon cancer has remained elusive due to numerous opposite observations. To study this correlation, we examined whether Pten deficiency in intestinal epithelial cells (IECs) induces tumorigenesis.With mucosal biopsies of human colon cancer and normal colon, Pten mRNA was evaluated by quantitative PCR. Using IEC-specific Pten knockout mice (Pten^ΔIEC/ΔIEC), we examined the mitotic activity of IECs; and Pten^ΔIEC/ΔIEC; Apc^min/+ mice were generated by combining Pten^ΔIEC/ΔIEC with Apc^min/+ mice. Tumor-associated gene was evaluated by micro-array analysis. Fecal microbiome was analyzed through 16S rRNA gene sequencing.We found that Pten mRNA level was reduced in human colon cancer relative to normal tissues. Augmented chromatids, increased Ki-67 and PCNA expression, and enhanced Akt activation were identified in IECs of Pten^ΔIEC/ΔIEC mice compared to Pten^+/+ littermate. Combining Pten^ΔIEC/ΔIEC with Apc^min/+ condition caused rapid and aggressive intestinal tumorigenesis. However, Pten^ΔIEC/ΔIEC mice did not develop any tumors. While maintaining the tumor-driving potential, these data indicated that IEC-Pten deficiency alone did not induce tumorigenesis in mice. Furthermore, the expression of tumor-promoting and tumor-suppressing genes was decreased and increased, respectively, in the intestine of Pten^ΔIEC/ΔIEC mice compared to controls. The abundance of Akkermansia muciniphila, capable of inducing chronic intestinal inflammation, was diminished in Pten^ΔIEC/ΔIEC mice compared to controls.These findings suggested that altered tumor-associated gene expression and changed gut microbiota shape a tumor-preventive microenvironment to counteract the tumor-driving potential, leading to the tumor prevention in Pten^ΔIEC/ΔIEC mice.