Lee Y et al. (2023), Solute carrier (SLC) expression reveals skeleto...

ECB-ART-51698

Dev Biol 2023 Nov 21;503:68-82. doi: 10.1016/j.ydbio.2023.08.004.

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Solute carrier (SLC) expression reveals skeletogenic cell diversity.

Lee Y , Tjeerdema E , Kling S , Chang N , Hamdoun A .

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Within the developing embryo is a microcosm of cell type diversity. Single cell RNA-sequencing (scRNA-seq) is used to reveal cell types, typically by grouping cells according to their gene regulatory states. However, both across and within these regulatory states are additional layers of cellular diversity represented by the differential expression of genes that govern cell function. Here, we analyzed scRNA-seq data representing the late gastrula stage of Strongylocentrotus purpuratus (purple sea urchin) to understand the patterning of transporters belonging to the ABC and SLC families. These transporters handle diverse substrates from amino acids to signaling molecules, nutrients and xenobiotics. Using transporter-based clustering, we identified unique transporter patterns that are both shared across cell lineages, as well as those that were unique to known cell types. We further explored three patterns of transporter expression in mesodermal cells including secondary mesenchyme cells (pigment cells and blastocoelar cells) and skeletogenic cells (primary mesenchyme cells). The results revealed the enrichment of SMTs potentially involved in nutrient absorption (SLC5A9, SLC7A11, SLC35F3, and SLC52A3) and skeletogenesis (SLC9A3, SLC13A2/3/5, and SLC39A13) in pigment cells and blastocoelar cells respectively. The results indicated that the strategy of clustering by cellular activity can be useful for discovering cellular populations that would otherwise remain obscured.

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	Fig. 2. Localization of SMT genes in the late gastrula embryo. A) Localization of SMTs by Hybridization Chain Reaction (HCR result is magenta, nuclei are gray). ABCC10 and SLC18A1 appear localized to the apical ectoderm. ABCG12, SLC5A9, and SLC7A11 are expressed in pigment cells. SLC35F3 is expressed in pigment cells, midgut and hindgut. SLC43A2 is expressed in the left coelomic pouch. SLC5A11 is expressed in PMCs. Numbers represent the embryos observed to have the respective HCR pattern out of total number of embryos. B) SMT genes enriched in the pigment cells, SLC5A9, SLC35F3, and SLC52A3 are expressed in gut compartments in 72 hpf embryos. SLC5A9 is expressed in the foregut and the hindgut. SLC35F3 and SLC52A3 are expressed in the midgut.
	Fig. 3. Clustering of cells in the gastrula stage embryo based on SMT expression.Fiogure shows UMAP of clustering of cells based on SMTs. A) Figure shows UMAP plot of the late gastrula dataset showing the 19 SMT AP clusters discovered (numbered 0 to 18), each representing a population of cells expressing a unique profile of SMT genes. B) Dot plot shows the differential expression of SMT genes across the discovered AP clusters. The x-axis lists clusters, and the y-axis lists names of SMT gene names, general class of the substrate transported by the SMT, and the Entrez GeneID for the specific transcript. Circle sizes represent percentage of cells within a cluster expressing a corresponding gene of interest, and the color gradient corresponds to the average log2FC value of the gene of interest in a cluster compared to that of other clusters for the same gene. Alternating dark gray and light gray bars on the right highlight the clusters for which the corresponding genes met or exceeded the thresholds for being expressed in at least 20% of the cells in a cluster and had average log2FC values of at least 0.5.
	Fig. 4. SMT AP clusters 8, 15 and 16 map to pigment and mesenchyme cellsEach of the four panels represents a cell identity from those in Fig. S1A, while numbers on the x-axis represent activity pattern clusters from Fig. 3A and B. While some cell types, (e.g., pigment and mesenchyme) are dominated by one or two activity patterns others (e.g., ectoderm) are composed of cells corresponding to multiple activity patterns. A) 394/413 (orange bar, 95.4%) pigment cells (Pigment 1, 2) map to cells from a single SMT AP cluster 8. B) 96/185 (green bar, 51.8%) and 57/185 (blue bar, 30.8%) of primary/secondary mesenchyme cells (Primary/secondary mesenchyme 1, 2) map to SMT AP clusters 15 and 16, respectively. C, D) 1871/4811 (38.9%), 1255/4811 (26.1%), and 829/4811 (17.2%) of aboral ectoderm/neural cluster cells (Aboral ectoderm/neural clusters 1–5) map to SMT AP clusters 0, 1, and 4, respectively. 600/2276 (26.4%), 443/2276 (19.5%), 354/2276 (15.6%) of oral ectoderm cells (Oral ectoderm 1–4) map to SMT AP clusters 2, 5, and 6 respectively.
	Fig. 5. Identification of genes that are highly co-expressed in cells with SMT APs 8, 15 and 16.Dot plot shows the differential expression of skeletogenic and pigment cell genes as positive controls, top expressed SMT genes, and other top expressed markers in cells mapped to SMT AP 8, 15 and 16 clusters. The x-axis lists clusters named by cell mappings and are color-coded to correspond to the cell populations represented by bars in Fig. 4. The y-axis lists names of gene names and the Entrez GeneID for the specific transcript. Circle sizes represent percentage of cells within a cluster expressing a corresponding gene of interest, and the color gradient corresponds to the average log2FC value of the gene of interest in a cluster compared to that of other clusters for the same gene. Alternating dark gray and light gray bars on the right highlight functional gene categories. Colored squares to the left of each gene name indicate results from prior studies which indicate localization of mRNA or corresponding protein for these genes.
	Fig. 6. Blastocoelar cell marker Prox1 colocalizes with MMP18 and matrilysin in 24 and 48 hpf embryosIn 24 hpf embryos, Prox1 is coexpressed with MMP18 and matrilysin in the NSM. In 48 hpf embryos, Prox1 is coexpressed with MMP18 and matrilysin in blastocoelar cells (BCs). In both 24 or 48 hpf embryos, cells expressing SM50 do not express Prox1, MMP18, or matrilysin.
	Fig. 7. Cells expressing SMT APs 8, 15, and 16 correspond to pigment, primary mesenchyme and blastocoelar cells. A) Genes from SMT AP cluster 16, COLP2alpha and col1a(V)chain, do not colocalize with gcm in aboral NSM cells. In late gastrula embryos, COLP2alpha and col1a(V)chain are co-expressed in SMCs budding off the archenteron. In prism stage embryos, COLP2alpha and col1a(V)chain continue to co-localize in cells which may be blastocoelar cells. Meanwhile, gcm is expressed in pigment/aboral NSM cells and is not co-expressed with either COLP2alpha or col1a(V)chain in either stage. In late gastrula embryos, SM50 is expressed in the two ventrolateral PMC clusters. In prism stage embryos, SM50 is expressed along the location of the spicule rods, and at the tips COLP2alpha+ and col1a(V)chain+ cells can be seen making contact in an alternating pattern. SM50+ cells align with thin filopodia that have signal for COLP2alpha+ and col1a(V)chain+ but adjustment of brightness and contrast to clearly view the segregation between gcm+ cells and COLP2alpha+ or col1a(V)chain+ cells have eliminated those signals in these images. B) Genes from SMT AP clusters 15 and 16 are spatially segregated at the late gastrula (48–52 hpf) and prism (72–84 hpf) stages into skeletogenic and presumptive non-skeletogenic mesenchyme. In late gastrula embryos, MMP18 and thrombospondin-1 are co-expressed in SMCs budding off the archenteron. In prism stage embryos, thrombospondin-1 expression is sparse, and MMP18 is expressed in cells which appear to form a line of cells resembling the positions of spicule forming PMCs. In late gastrula embryos, msp130 and angiopoietin 1-like co-localize and are expressed in the two ventrolateral PMC clusters. In prism stage embryos, msp130 and angiopoietin 1-like continue to co-localize in PMCs along the two spicule rods. In prism stage embryos, combinations of localization merges among MMP18, angiopoietin-1 like, and msp130 show a pattern of cells along the location of the spicule rods alternating between MMP18+ cells and msp130+ or angiopoietin 1-like+ cells.
	Fig. 8. Localization patterns of cells expressing skeletogenic genes in SMT AP clusters 15 and 16.SMCs expressing col1a(V)chain are located alongside PMCs expressing COLP3alpha. In late gastrula embryos, COLP3alpha is expressed in the two ventrolateral PMC clusters. Col1a(V)chain is expressed in cells which may be blastocoelar cells but can also be seen in cells which appear to be a part of the ventrolateral PMC clusters. COLP3alpha and col1a(V)chain do not co-localize, but calumenin co-localizes with the two collagen genes separately. In prism stage embryos, COLP3alpha is expressed in cells along the spicule rods. Col1a(V)chain is expressed in cells with long filopodia in the positions expected of PMCs. Co-expression of calumenin and col1a(V)chain is less pronounced compared to the co-expression calumenin and COLP3alpha. In merged images containing the spatial expression patterns of all three genes, COLP3alpha+ and calumenin+ PMCs are aligned along col1a(V)chain+ filopodia.
	Fig. 9. SLC13A2 and SLC26A5 expression shows similar alternating cell patterns along spicule domains.In late gastrula embryos, SLC13A2 is expressed in putative SMCs adjacent to and in contact with PMCs expressing SLC26A5. SLC13A2 and SLC26A5 do not colocalize, but calumenin co-localizes with the two SMT genes separately. In prism stage embryos, SLC13A2 co-localizes with calumenin in cells along the locations of spicules in an interspersed manner. SLC26A5 and calumenin are clearly colocalized in cells along the locations of spicule rods.