Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

/pdf/spatial-reconstruction-of-single-cell-gene-expression-data-5583gr0ht4.pdf

Spatial reconstruction of single-cell gene expression data

Genetics and analysis of quantitative traits

In vivo imaging of embryonic vascular development using transgenic zebrafish.

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

https://anatomypubs.onlinelibrary.wiley.com/doi/epdf/10.1002/dvdy.21343

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio

We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

Stages of embryonic development of the zebrafish.

The shaping of pharyngeal cartilages during early development of the zebrafish.

Mechanisms underlying the morphogenesis of skeletal shape are largely not understood, mainly because the ability to study the dynamic processes of cellular behavior giving rise to cartilage and bone in living embryos has been limited. In recent years, techniques have emerged that make the study of tissue formation possible in vivo (Beis and Stainier, 2006; Field et al., 2003; Koster and Fraser, 2004), suggesting that skeletal morphogenesis can be similarly studied. The zebrafish is an ideal model organism for studying skeletal development because embryos and larvae are small and transparent, enabling the study of organogenesis in the living organism. Zebrafish cartilage and bone elements develop early and have distinct morphologies, and the genetic mechanisms underlying skeletal formation are shared with other vertebrates (Yelick and Schilling, 2002).

Transgenic lines are especially valuable for analysis of living embryos, including time-lapse confocal microscopy (Cooper et al., 2005; Glickman et al., 2003; Smith et al., 2008). Transgenic fish lines have already been produced that express GFP in cells that give rise to, amongst other tissues, cartilage elements in the head including Tg(−1252sox10:GFP)ba5 (Dutton et al., 2008) and Tg(foxp2-enhancerA:EGFP)zc42 (Bonkowsky et al., 2008). A transgenic line expressing an observable marker of osteoblasts would help us explore osteoblast behavior specifically during the formation of intramembranous bony elements, which have no cartilaginous precursor, and for studying the induction of osteoblasts in perichondrium during endochondral ossification.

Sp7 is a zinc-finger-containing transcription factor expressed in osteoblasts and not chondrocytes, making it an excellent marker for studying osteoblasts (Nakashima et al., 2002). Recently, the promoter of sp7 has been shown to drive mCherry in osteoblasts of medaka fish allowing for the analysis of osteoblast behavior in the forming skeleton of this species (Renn and Winkler, 2009). This medaka sp7 regulatory sequence has been used to drive fluorescent marker expression in zebrafish (Hammond and Schulte-Merker, 2009; Spoorendonk et al., 2008), however, there exists no transgenic line using the regulatory region of sp7 in zebrafish to drive a fluorescent marker in zebrafish.

We used BAC-mediated transgenesis to drive EGFP under the control of sequence upstream of sp7 in a zebrafish BAC. In the case of zebrafish sp7, we do not know the regulatory elements necessary for gene transcription. Therefore, an advantage of using BACs for transgenesis is that they often contain large genomic clones that include the essential regulatory elements of a gene of interest. The presence of large inserts of a zebrafish-specific sequence makes it likely that the BAC contains sequence essential to drive a transgene in an expression pattern consistent with the endogenous gene. We injected the BAC into embryos to generate stable transgenic lines expressing GFP in osteoblasts. We show here the native expression pattern of sp7 by in situ hybridization in whole-mounts and sections, and compare it to the expression of GFP in the Tg(sp7:EGFP)b1212 transgenic line. We found that GFP expression reproduces endogenous sp7 gene expression, indicating that this line will be an excellent tool for future study of the dynamic behavior of cells that create the skeleton.

/pdf/zebrafish-sp7-egfp-a-transgenic-for-studying-otic-vesicle-2iylrtk4bt.pdf

Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration

How do developmental mechanisms evolve to control changing skeletal morphology, the shapes and sizes of individual bones? We address this question with studies of the opercle (OP), a large facial bone that has undergone marked morphological evolution in the ray-finned fish. Attributes for developmental analysis motivated us to examine how OP shape and size evolve and develop in threespine sticklebacks, a model system for understanding vertebrate evolution. We find that when Alaskan anadromous fish take up permanent residence in lakes, they evolve smaller and reshaped OPs. The change is a reduction in the amount of bone laid down along one body axis, and it arises at or shortly after the onset of OP development. A quantitative trait locus is present on linkage group 19 that contributes in a major way to this phenotype.

/pdf/evolution-and-development-of-facial-bone-morphology-in-2ovzriy1ah.pdf

Evolution and development of facial bone morphology in threespine sticklebacks

Endothelin 1 (Edn1), a secreted peptide expressed ventrally in the primordia of the zebrafish pharyngeal arches, is required for correct patterning of pharyngeal cartilage development. We have studied mutants and morpholino-injected larvae to examine the role of the Edn1 signal in patterning anterior pharyngeal arch bone development during the first week after fertilization. We observe a remarkable variety of phenotypic changes in dermal bones of the anterior arches after Edn1 reduction, including loss, size reduction and expansion, fusion and shape change. Notably, the changes that occur appear to relate to the level of residual Edn1. Mandibular arch dermal bone fusions occur with severe Edn1 loss. In the dorsal hyoid arch, the dermal opercle bone is usually absent when Edn1 is severely reduced and is usually enlarged when Edn1 is only mildly reduced, suggesting that the same signal can act both positively and negatively in controlling development of a single bone. Position also appears to influence the changes: a branchiostegal ray, a dermal hyoid bone normally ventral to the opercle, can be missing in the same arch where the opercle is enlarged. We propose that Edn1 acts as a morphogen; different levels pattern specific positions, shapes and sizes of bones along the dorso-ventral axis. Changes involving Edn1 may have occurred during actinopterygian evolution to produce the efficient gill-pumping opercular apparatus of teleosts.

/pdf/endothelin-1-mediated-regulation-of-pharyngeal-bone-4d7esuiq57.pdf

Bonnie Ullmann

Papers

Stages of embryonic development of the zebrafish.

The shaping of pharyngeal cartilages during early development of the zebrafish.

Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration

Evolution and development of facial bone morphology in threespine sticklebacks

Endothelin 1-mediated regulation of pharyngeal bone development in zebrafish.