Muscle precursors need to be correctly positioned during embryonic development for proper body movement. In zebrafish, a subset of hypaxial muscle precursors from the anterior somites undergo longrange migration, moving away from the trunk in three streams to form muscles in distal locations such as the fin. We mapped long-distance muscle precursor migrations with unprecedented resolution using live imaging. We identified conserved genes necessary for normal precursor motility (six1a, six1b, six4a, six4b and met). These genes are required for movement away from somites and later to partition two muscles within the fin bud. During normal development, the middle muscle precursor stream initially populates the fin bud, then the remainder of this stream contributes to the posterior hypaxial muscle. When we block fin bud development by impairing retinoic acid synthesis or Fgfr function, the entire stream contributes to the posterior hypaxial muscle indicating that muscle precursors are not committed to the fin during migration. Our findings demonstrate a conserved muscle precursor motility pathway, identify dynamic cell movements that generate posterior hypaxial and fin muscles, and demonstrate flexibility in muscle precursor fates.
We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.
Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.
We currently have little understanding of the mechanisms by which hematopoietic stem and progenitor cells (HSPCs) gain a selective advantage in patients with clonal hematopoiesis and other myeloid neoplasms. The chemokine CXCL8 is elevated in a subset of patients with myeloid neoplasms. Our previous work in zebrafish has discovered a novel role for cxcl8 and its receptor, cxcr1, in supporting colonization of HSPCs within the sinusoidal endothelial cell niche of the embryonic zebrafish known as the caudal hematopoietic tissue (CHT). We hypothesized that mosaic overexpression of cxcl8 in a population of HSPCs during development would alter HSPC-niche interactions, selectively favor HSPCs expressing cxcl8 and lead to their expansion in adults. To test this hypothesis, we microinjected DNA constructs encoding cxcl8-2A-GFP or GFP alone under the control of the HSPC-specific Runx1+23 enhancer into zebrafish embryos at the single-cell stage. Time lapse fluorescence video microscopy and single-cell tracking was performed on HSPCs within the CHT. Overexpression of cxcl8 nearly doubled the amount of time HSPCs resided within the CHT when compared to expression of GFP alone as a control (cxcl8: 4.94 ± 0.86 h vs GFP: 2.54 ± 0.18 h, p=0.01, N=142 tracked cells). Substitution of WT cxcl8 with a mutant cxcl8 construct lacking the ELRCXC motif required for receptor binding reduced these effects (WT cxcl8: 6.6 ± 0.48 h vs ELRCXC-cxcl8: 5.3 ± 0.33 h, p=0.02, N=355 tracked cells). To observe HSPC-niche interactions, kdrl:mCherry endothelial cell reporter zebrafish were microinjected with Runx1+23:cxcl8-2A-GFP or Runx1+23:GFP DNA constructs. The percent of time individual HSPCs spent closely interacting with a single group of CHT endothelial cells (endothelial cell cuddling) was quantified over the period from 52 to 72 hours post-fertilization. Overexpression of cxcl8 by HSPCs increased HSPC-endothelial cell cuddling time by 30% (cxcl8: 87% vs GFP: 57%, p=0.001). To directly test competition between wild type and cxcl8 overexpressing HSPCs, zebrafish embryos were microinjected with a 1:1 molar ratio of Runx1+23:cxcl8-2A-mCherry and Runx1+23:clover DNA. Single cxcl8-2A-mCherry+ and clover+ competitor cells were tracked by time-lapse fluorescence confocal microscopy. HSPCs expressing cxcl8 resided longer within the CHT than competitor HSPCs when quantified over the period from 72 to 96 hours post-fertilization (cxcl8: 4.0 ± 0.20 h vs competitor: 2.5 ± 0.25 h, p=2.0 x 10-6, n=426 tracked cells). Single cell RNA-sequencing (scRNA-seq) of zebrafish embryos with mosaic expression of cxcl8 in HSPCs showed upregulation of cxcl12a in endothelial cells compared to endothelial cells from control embryos (p=5.19 x 10-3), suggesting a possible mechanism to explain the increased CHT residency time. Zebrafish with mosaic expression of Runx1+23:cxcl8 were raised to adulthood and the kidney marrow cells were analyzed by flow cytometry. Compared to clutchmate controls, Runx1+23:cxcl8 mosaic transgenics had a higher hematopoietic progenitor/precursor to lymphocyte ratio, suggesting a mild differentiation block and possible lineage skewing (cxcl8: 2.0 ± 0.15 vs control: 1.6 ± 0.10, p=0.048, N=25 animals). Taken together, these data support a model in which pre-malignant HSPC clones aberrantly express cxcl8 and acquire a selective advantage over normal clones through enhanced interactions with the endothelial cell niche. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.
Hall that loose use of the term Amyoplasia could lead to confusion. Indeed, other than capitalizing this term in the title, an AJHG-style standard at the time, we used the term amyoplasia with a lowercase ''a,'' rather than use the term Amyoplasia, throughout our manuscript to describe the state of having partial or complete absence of skeletal muscles in a body segment. 1,2 Moreover, we agree that Amyoplasia is, in most cases, unlikely to be due to germline mutations. But we do think it reasonable to hypothesize that somatic mosaicism for large-effect alleles underlies the condition in some persons with Amyoplasia. Such an observation would be consistent with the lack of familial cases. As we also noted, reports of ''familial amyoplasia'' accompanied by identification of the underlying pathogenic variant do exist. [3][4][5] But the ''amyoplasia'' in such reports is typically limited to the upper or lower limbs, 6 which is again why we stated that ''at least in some families with amyoplasia [lower case], large-effect risk alleles appear to be segregating.'' Addressing a couple of additional points more precisely will further clarify the issue for readers. First, we don't know how many affected persons we reported had complete absence of the skeletal muscles of the ankle and foot. The one individual we described with this finding was the only person who underwent sophisticated imaging and pathological exam of the affected limb. Although we don't think the complete absence of skeletal muscles in a limb segment is likely to be a common finding, it was striking enough to draw attention to it, particularly because the zebrafish model we reported points toward a potential mechanism for muscle loss in persons with pathogenic MYLPF variants. Moreover, although one or several muscles have been reportedly absent in individuals with various arthrogryposis conditions, we are not aware of other reports of absence of all of the muscles of a limb segment. If such persons exist, we would be delighted to study them. Finally, we would like to emphasize that this work was a collaborative effort among multiple groups: the Bamshad group contributed the human clinical genetic analyses, and the Amacher group contributed the zebrafish model and analysis.
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