Mammalian hematopoietic stem cells (HSCs) colonize the bone marrow during late fetal development, and this becomes the major site of hematopoiesis after birth. However, little is known about the early postnatal bone marrow niche. We performed single-cell RNA sequencing of mouse bone marrow stromal cells at 4 days, 14 days, and 8 weeks after birth. Leptin-receptor-expressing (LepR+) stromal cells and endothelial cells increased in frequency during this period and changed their properties. At all postnatal stages, LepR+ cells and endothelial cells expressed the highest stem cell factor (Scf) levels in the bone marrow. LepR+ cells expressed the highest Cxcl12 levels. In early postnatal bone marrow, SCF from LepR+/Prx1+ stromal cells promoted myeloid and erythroid progenitor maintenance, while SCF from endothelial cells promoted HSC maintenance. Membrane-bound SCF in endothelial cells contributed to HSC maintenance. LepR+ cells and endothelial cells are thus important niche components in early postnatal bone marrow.
Cranial neural crest cells are a multipotent cell population that generate all the elements of the pharyngeal cartilage with differentiation into chondrocytes tightly regulated by temporal intracellular and extracellular cues. Here, we demonstrate a novel role for miR-27, a highly enriched microRNA in the pharyngeal arches, as a positive regulator of chondrogenesis. Knock down of miR-27 led to nearly complete loss of pharyngeal cartilage by attenuating proliferation and blocking differentiation of pre-chondrogenic cells. Focal adhesion kinase (FAK) is a key regulator in integrin-mediated extracellular matrix (ECM) adhesion and has been proposed to function as a negative regulator of chondrogenesis. We show that FAK is downregulated in the pharyngeal arches during chondrogenesis and is a direct target of miR-27. Suppressing the accumulation of FAK in miR-27 morphants partially rescued the severe pharyngeal cartilage defects observed upon knock down of miR-27. These data support a crucial role for miR-27 in promoting chondrogenic differentiation in the pharyngeal arches through regulation of FAK.
The bone marrow contains peripheral nerves that promote haematopoietic regeneration after irradiation or chemotherapy (myeloablation), but little is known about how this is regulated. Here we found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR+) stromal cells is required to maintain nerve fibres in adult bone marrow. In nerveless bone marrow, steady-state haematopoiesis was normal but haematopoietic and vascular regeneration were impaired after myeloablation. LepR+ cells, and the adipocytes they gave rise to, increased NGF production after myeloablation, promoting nerve sprouting in the bone marrow and haematopoietic and vascular regeneration. Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signalling in LepR+ cells, and potentially in adipocytes, increasing their production of multiple haematopoietic and vascular regeneration growth factors. Peripheral nerves and LepR+ cells thus promote bone marrow regeneration through a reciprocal relationship in which LepR+ cells sustain nerves by synthesizing NGF and nerves increase regeneration by promoting the production of growth factors by LepR+ cells.
Abstract Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.
Unlike the adult mammalian retina, Müller glia (MG) in the adult zebrafish retina are able to dedifferentiate into a "stem cell"-like state and give rise to multipotent progenitor cells upon retinal damage. We show that miR-216a is downregulated in MG after constant intense light lesioning and that miR-216a suppression is necessary and sufficient for MG dedifferentiation and proliferation during retina regeneration. miR-216a targets the H3K79 methyltransferase Dot1l, which is upregulated in proliferating MG after retinal damage. Loss-of-function experiments show that Dot1l is necessary for MG reprogramming and mediates MG proliferation downstream of miR-216a. We further demonstrate that miR-216a and Dot1l regulate MG-mediated retina regeneration through canonical Wnt signaling. This article reports a regulatory mechanism upstream of Wnt signaling during retina regeneration and provides potential targets for enhancing regeneration in the adult mammalian retina.
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