The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Origin and diversification of fibroblasts from the sclerotome in zebrafish.

Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes.

Temporal cell fate determination in the spinal cord is mediated by the duration of Notch signalling.

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors generate both early-born Kolmer-Agduhr" (KA") interneuron and late-born V3 interneuron populations. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 progenitors receive higher total levels of Notch response, but they collect this response over a longer duration compared to KA" progenitors. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA" and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA" cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling, not simply the level, mediates cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA" and V3 interneurons. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny.

Dual function of perivascular fibroblasts in vascular stabilization in zebrafish.

Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts markedly reduces collagen deposition around endothelial cells, resulting in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors.

Single cell dynamics of embryonic muscle progenitor cells in zebrafish.

Muscle stem cells hold a great therapeutic potential in regenerating damaged muscles. However, the in vivo behavior of muscle stem cells during muscle growth and regeneration is still poorly understood. Using zebrafish as a model, we describe the in vivo dynamics and function of embryonic muscle progenitor cells (MPCs) in the dermomyotome. These cells are located in a superficial layer external to muscle fibers and express many extracellular matrix (ECM) genes, including collagen type 1 α2 (col1a2). Utilizing a new col1a2 transgenic line, we show that col1a2+ MPCs display a ramified morphology with dynamic cellular processes. Cell lineage tracing demonstrates that col1a2+ MPCs contribute to new myofibers in normal muscle growth and also during muscle regeneration. A combination of live imaging and single cell clonal analysis reveals a highly choreographed process of muscle regeneration. Activated col1a2+ MPCs change from the quiescent ramified morphology to a polarized and elongated morphology, generating daughter cells that fuse with existing myofibers. Partial depletion of col1a2+ MPCs severely compromises muscle regeneration. Our work provides a dynamic view of embryonic muscle progenitor cells during zebrafish muscle growth and regeneration.

Stereotypic generation of axial tenocytes from bipartite sclerotome domains in zebrafish.

Development of a functional musculoskeletal system requires coordinated generation of muscles, bones, and tendons. However, how axial tendon cells (tenocytes) are generated during embryo development is still poorly understood. Here, we show that axial tenocytes arise from the sclerotome in zebrafish. In contrast to mouse and chick, the zebrafish sclerotome consists of two separate domains: a ventral domain and a previously undescribed dorsal domain. While dispensable for sclerotome induction, Hedgehog (Hh) signaling is required for the migration and maintenance of sclerotome derived cells. Axial tenocytes are located along the myotendinous junction (MTJ), extending long cellular processes into the intersomitic space. Using time-lapse imaging, we show that both sclerotome domains contribute to tenocytes in a dynamic and stereotypic manner. Tenocytes along a given MTJ always arise from the sclerotome of the adjacent anterior somite. Inhibition of Hh signaling results in loss of tenocytes and enhanced sensitivity to muscle detachment. Together, our work shows that axial tenocytes in zebrafish originate from the sclerotome and are essential for maintaining muscle integrity.

Coordination of cytochrome c oxidase gene expression in the remodelling of skeletal muscle.

Many fish species respond to low temperature by inducing mitochondrial biogenesis, reflected in an increase in activity of the mitochondrial enzyme cytochrome c oxidase (COX). COX is composed of 13 subunits, three encoded by mitochondrial (mt)DNA and 10 encoded by nuclear genes. We used real-time PCR to measure mRNA levels for the 10 nuclear-encoded genes that are highly expressed in muscle. We measured mRNA levels in white muscle of three minnow species, each at two temperatures: zebrafish (Danio rerio) acclimated to 11 and 30°C, goldfish (Carassius auratus) acclimated to 4 and 35°C, and northern redbelly dace (Chrosomus eos) collected in winter and summer. We hypothesized that temperature-induced changes in COX activity would be paralleled by COX nuclear-encoded subunit transcript abundance. However, we found mRNA for COX subunits showed pronounced differences in thermal responses. Though zebrafish COX activity did not change in the cold, the transcript levels of four subunits decreased significantly (COX5A1, 60% decrease; COX6A2, 70% decrease; COX6C, 50% decrease; COX7B, 55% decrease). Treatments induced changes in COX activity in both dace (2.9 times in winter fish) and goldfish (2.5 times in cold fish), but the response in transcript levels was highly variable. Some subunits failed to increase in one (goldfish COX7A2, dace COX6A2) or both (COX7B, COX6B2) species. Other transcripts increased 1.7-100 times. The most cold-responsive subunits were COX4-1 (7 and 21.3 times higher in dace and goldfish, respectively), COX5A1 (13.9 and 5 times higher), COX6B1 (6 and 10 times higher), COX6C (11 and 4 times higher) and COX7C (13.3 and 100 times higher). The subunits that most closely paralleled COX increases in the cold were COX5B2 (dace 2.5 times, goldfish 1.7 times) and COX6A2 (dace 4.1 times, goldfish 1.7 times). Collectively, these studies suggest that COX gene expression is not tightly coordinated during cold-induced mitochondrial remodelling in fish muscle. Further, they caution against arguments about the importance of transcriptional regulation based on measurement of mRNA levels of select subunits of multimeric proteins.