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Disruption of cardiac neural crest cells CNCCs results in congenital heart disease, yet we do not understand the cell fate dynamics as these cells differentiate to vascular smooth muscle cells.
Here we performed single-cell RNA-sequencing of NCCs from the pharyngeal apparatus with the heart in control mouse embryos and when Tbx1 , the gene for 22q We uncover three dynamic transitions of pharyngeal NCCs expressing Tbx2 and Tbx3 through differentiated CNCCs expressing cardiac transcription factors with smooth muscle genes.
These transitions are altered non-autonomously by loss of Tbx1. Further, inactivation of Tbx2 and Tbx3 in early CNCCs results in aortic arch branching defects due to failed smooth muscle differentiation. Loss of Tbx1 interrupts mesoderm to CNCC cell-cell communication with upregulation and premature activation of BMP signaling and reduced MAPK signaling, as well as alteration of other signaling, and failed dynamic transitions of CNCCs leading to disruption of aortic arch artery formation and cardiac outflow tract septation.
Yvanka de Soysa, Sanjeev S. Ranade, … Deepak Srivastava. Hiroko Nomaru, Yang Liu, … Bernice E. Neural crest cells NCCs are multipotent cells that migrate in three ordered streams from the rhombomeres in the neural tube to the pharyngeal apparatus, where they differentiate into many cell types 1.
The pharyngeal apparatus is a dynamic embryonic structure consisting of individual pharyngeal arches PA , forming in a rostral to caudal manner from mouse embryonic day E 8 to E A subset of pharyngeal NCCs migrate through the caudal pharyngeal arches, PA, and surround the pharyngeal arch arteries PAAs , while others continue to migrate to the cardiac outflow tract OFT , both differentiating to vascular smooth muscle cells 2.
The PAAs form in a similar, rostral to the caudal manner and are linked continuously with the cardiac OFT. The PAAs become remodeled to form the aortic arch and vascular branches that include the subclavian arteries. The fourth PAAs are particularly important in that the left fourth PAA is critical to form the aortic arch with the left subclavian artery and the right fourth PAA forms the branch to the right subclavian artery.
Differentiation of CNCCs into smooth muscle cells of the PAAs is important for the remodeling of the PAA 2. The OFT elongates by the addition of second heart field mesoderm cells and this is required for looping and septation of the OFT. It has been shown that CNCCs are required for the addition of second heart field mesoderm cells to expand the length of the OFT 3.
In addition, CNCCs are required for OFT septation by invading and allowing the endocardial cushions of the OFT to come into direct cell-cell contact, fuse, and form the aorticopulmonary septum 2 , 4 , 5. Complete ablation of NCCs from PA results in interruption of the aortic arch and arterial branching defects, as well as persistent truncus arteriosus of the OFT 6.
These NCCs in PA, are referred to as cardiac NCCs CNCCs based upon their position and known function in heart development as well as their differentiation to vascular smooth muscle. Understanding CNCC development is critical to determine the pathogenesis of human congenital heart defects such as those observed in 22q TBX1 , encoding a T-box transcription factor, is the major gene for congenital heart disease in 22q Although 22q Global inactivation of Tbx1 or conditional inactivation in the mesoderm using Mesp1 Cre 10 in the mouse results in neonatal lethality with a persistent truncus arteriosus 11 , 12 , 13 , in part due to failed CNCC development 9.
Therefore, one of the main functions of Tbx1 in the pharyngeal mesoderm is to signal to CNCCs to promote their development.
In order to understand how CNCCs are affected non-autonomously in Tbx1 mutant embryos, it is essential to define their transcriptional signatures and cardiac fate acquisition in the normal situation between E8.
Previously, single-cell RNA-sequencing scRNA-seq of NCCs from early stages in the chick embryo identified expression of Tgif1 , Ets1 , and Sox8 being important for early CNCC identity and fate decisions However, these were early migrating mesenchymal NCCs that also have the potential to contribute to the craniofacial skeleton and other cell types.
Another seminal scRNA-seq study demonstrated that NCC fate choices are made by a series of sequential binary decisions in mouse embryos at E8. In another recent study using mouse scRNA-seq data, Chen and colleagues focus on CNCC differentiation from E However, this study did not include an analysis of the cardiac progenitor cells in the pharyngeal arches.
To uncover genetic signatures and dynamic transitions of CNCCs in the normal situation and when Tbx1 is inactivated, we performed scRNA-seq of NCCs from control and Tbx1 null mutant mouse embryos.
We found that smooth muscle cell fate acquisition is in part dependent on two other T-box genes, Tbx2 and Tbx3. When Tbx1 is inactivated, we found reduced cell deployment reduced presence of cells along with the failure of dynamic progression of CNCC maturation.
This is due to the disruption of cell-cell communication from mesodermal cells, resulting in the downregulation of MAPK signaling and upregulation and premature activation of the BMP pathway, as well as affecting other ligand—receptor interactions.
We performed scRNA-seq of the Wnt1-Cre;ROSA-EGFP genetic lineage 17 , 18 in the mouse pharyngeal apparatus at E8. These stages correspond to developmental time points when Tbx1 is highly expressed in cell types adjacent to NCCs.
At E8. At E9. At E We excluded PA1 in order to enrich for NCCs relevant to cardiac development. EGFP-positive NCCs were purified by FACS and the Chromium 10X platform was used to perform scRNA-seq and data from 36, NCCs of control embryos were obtained Supplementary Table 1. Unsupervised clustering was performed using Seurat software 19 and individual clusters were identified Fig.
a — c Wnt1-Cre;ROSA-EGFP genetic lineage tracing shows the distribution of NCCs within the pharyngeal region and outflow tract of E8. The region rostral to the white dotted line of the embryo at E8.
d — f Seurat UMAP Uniform Manifold Approximation and Projection plots with cluster annotations of scRNA-seq data of NCCs at E8. g — i Expression of genes at E8.
Twist1 expression was analyzed at E8. PA pharyngeal arch, OFT outflow tract, OV otic vesicle, SM-CNCCs smooth muscle CNCCs. Expression of Sox10 and Foxd3 , as well as expression of Twist1, Prrx1 , and Prrx2 were used to identify early migratory and mesenchymal NCCs, respectively 20 , We used Hox and Dlx Homeodomain genes to provide spatial anterior-posterior and proximal-distal context to different arches PA Using the Hox genes as a guide, at E9.
Further, at E Additional marker genes are shown in Supplementary Fig. Spatial localization was confirmed for expression of S ox10 , Hoxa2 , Hoxb3 , and Twist1 by wholemount RNAscope in situ hybridization Fig.
In addition, to anterior-posterior spatial localization of the cells, we identified their proximal-distal location in the PAs with Dlx2 , Dlx5 , and Dlx6 Supplementary Fig.
NCCs of the neural tube were identified based on the expression of Zic1 and Zic2 genes and additional neural gene markers Supplementary Figs.
Differentiated NCCs of the OFT and PAAs express smooth muscle genes such as smooth muscle actin, Acta2 23 , Acta2 is a representative marker gene of progenitor smooth muscle cells, but it is not exclusive to smooth muscle cell types.
Early markers of smooth muscle cells in addition to Acta2 are Tagln , Myl9 , Myh9 , Rgs5 , and Cnn1 25 , 26 , We used Tagln Transgelin , Cnn1 Calponin , and Rgs5 Regulator of G Protein Signaling 5 as markers Figs.
CNN1 is a basic smooth muscle protein 28 , RGS5 is expressed in vascular smooth muscle and pericytes 29 and TAGLN is ubiquitously expressed in vascular smooth muscle cells and is an early marker of smooth muscle differentiation This cell population also expresses additional markers of smooth muscle progenitors Supplementary Data 3.
By combining results from Wnt1-Cre;GFP lineage tracing experiments, immunofluorescence, and RNAscope experiments of the early smooth muscle genes cluster C14, Fig.
Furthermore, we performed co-immunostaining at E Supplementary Fig. a UMAP plots of scRNA-seq data with genes that mark CNCCs identified by expression of Acta2 , Isl1 , Gata3 , Hand2 , Msx2 , Tbx2 , and Tbx3 with respect to Hoxb3 PA3 and Dlx5 distal PA1, 2, 3 expression red arrows.
Blue arrows show the expression of Gata3 , Hand2 , Msx2 , Tbx2 , and Tbx3 in the distal part of PA3. b , c Immunostaining on traverse sections through Wnt1-Cre;ROSA-EGFP embryos showing Cre -activated EGFP green , ACTA2, TAGLN, and ISL1 protein expression.
Nuclei blue are labeled with DAPI. Arrowheads indicate the expression of Gata3 , Isl1 , and Msx2 in CNCCs within the OFT. g Wholemount RNAscope analysis of Wnt1-Cre;ROSA-EGFP embryos at 20 and 24 somites where the position of the OFT is indicated white arrowhead. h PHATE potential of heat-diffusion for affinity-based transition embedding map of NCCs in clusters C1, C3, C4, C5, and C14 using Louvain clustering.
i PHATE map colored by cell fate probabilities, showing how each cell is likely to transition to SM-CNCCs as defined by CellRank software yellow represents high cell fate probabilities.
k PHATE maps with an expression of marker genes of CNCCs at different states of differentiation towards smooth muscle Acta2 expressing cells.
NT neural tube, end endoderm, OFT outflow tract, PA pharyngeal. a Seurat UMAP plots of scRNA-seq data from NCC populations with the expression of marker genes. Nuclei stained with DAPI are in blue. ISL1 is expressed in smooth muscle cells of the OFT cushions arrowheads.
Isl1 and Gata3 are expressed in NCCs within the OFT white arrowheads in c and at the level of the dorsal aortic sac wall and aortic sac protrusion blue arrowheads in c. Gata3 but not Isl1 is expressed in the more proximal part of OFT red arrowheads in c.
Cnn1 and Rgs5 are expressed in smooth muscle cells of the OFT white arrowheads in d and e. Expression of TAGLN is in smooth muscle cells of the OFT blue arrowheads and PAAs expressing TBX2 white arrowheads. Tbx2 and Gata3 expression overlaps in the dorsal wall of the OFT white arrowheads in g.
Tbx2 and Tbx3 are expressed in the mesenchyme dorsal to the aorta surrounding the PAAs at the level of the PA white arrowheads in h and i. j Schematic representation of a transverse section at the level of the OFT summarizing Tbx2 , Tbx3 , Isl1 , Gata3 , Acta2 , and Tagln expression in CNCCs at E NT neural tube, end endoderm, PAA pharyngeal arch artery.
To further delineate molecular signatures of CNCCs, we evaluated genes that were co-expressed in the SM-CNCCs cluster C14, Fig. We identified known transcription factor genes involved in cardiac development, but not all of these are known to have a function in CNCCs development, including Tbx2, Tbx3, Msx2, Isl1 , Gata3 , and Hand2 , at E9.
These genes were not only expressed in SM-CNCCs of the OFT but also in other cell clusters, such as NCCs in the distal PA expressing Dlx5 at E9.
Further, RNAscope in situ analysis confirmed the expression of Gata3 , Isl1 , and Msx2 in CNCCs within the OFT at E9. Gata3 was expressed in a larger domain of the OFT than Isl1 Fig. When taken together, at this time point, CNCCs of the OFT derived from the Wnt1-Cre;GFP genetic lineage and expressing Acta2 , Tagln , Cnn1 , Rgs5 , Isl1 , and Gata3 are smooth muscle progenitors.
Tbx2 and Tbx3 were widely expressed in pharyngeal NCCs at E9. Tbx2 and Tbx3 were expressed immediately lateral and dorsal to Isl1 and Gata3 expressing CNCCs in embryos at E A Tbx2 and Gata3 co-expression domain was present between Tbx2 and Gata3 expressing CNCCs Fig.
Together, this suggests that CNCCs downregulate Tbx2 and Tbx3 expression and activate Isl1 and Gata3 expression when entering the OFT. In addition, Tbx2 and Tbx3 , but not Isl1 and Gata3 , were expressed in NCCs surrounding the PAAs that are differentiating to smooth muscle, expressing ACTA2 and TAGLN at E Both ISL1 and TBX2 proteins were expressed in smooth muscle cells of the OFT and PAAs, respectively ACTA2 or TAGLN; Fig.
Expression of Tbx2 , Tbx3 , Isl1 , Gata3 , Tagln , and Acta2 in NCCs at E A subset of pharyngeal NCCs, expressing Hoxb3 that serves as a marker for PA as well as Tbx2 and Tbx3 , will form the CNCCs cluster C2 and we refer to these cells as cardiopharyngeal NCCs CP-NCCs; Fig.
Therefore, CNCCs can be subdivided into four populations based on position and expression of cardiac or smooth muscle genes, referred to as CP-NCCs contained in clusters C1, C3, C5, E9. We noted earlier that some CNCCs were located in clusters from PA1 and PA2 at E9.
Consistent with this, at E9. At late E9. These data are consistent with evidence from a previous report 31 , that NCCs from anterior arches also contribute to the developing heart.
To uncover CNCC fate dynamics at E9. We discovered genes that were progressively activated during the transition from pharyngeal NCCs to SM-CNCCs, which are candidate cardiac lineage driver genes Fig.
Our analysis indicates that CNCCs progressively activate Tbx2 , Tbx3 , Msx2, Hand2 , Gata3 , and Isl1 expression during their commitment towards SM-CNCCs at E9. To understand how NCCs progress at E The cell fate probability map from CellRank identified cells with a high potential to differentiate to smooth muscle fates from cluster C2 and C3 to C10; Fig.
The PAGA plots further indicated that some pharyngeal NCCs cluster C2 are CP-NCCs and they transition to OFT-CNCCs cluster C3 that then transition to SM-CNCCs cluster C10; blue color fraction in the pie chart; Fig.
This data also indicates that a small fraction of CP-NCCs may directly differentiate to smooth muscle cells blue fraction in the pie chart in C2 indicates a high probability of a fraction of cells in C2 to transition directly to C10 , in agreement with Tbx2 and Tbx3 expression in PAA-CNCCs at E We next identified genes whose expression correlates with SM-CNCC fate acquisition Fig.
a CellRank UMAP plot directed by RNA velocity and cell-cell similarity for clusters C2, C3, C4, and C10 from the feature plot in Fig. b UMAP plot for cell fate probabilities of CNCCs differentiating towards smooth muscle cells.
c Directed PAGA plot of NCCs. Pie charts show summarized fate probabilities of individual clusters, with blue representing the proportion of cells in each cluster with high probability to become smooth muscle CNCCs of a cluster, C e UMAP plots showing expression of marker genes in CNCCs.
f GO enrichment analysis of four groups of genes between Bmp4 and Gata6 defined by their pseudotime; Supplementary Data 6. Example of genes for each selected GO: biological processes are provided. The size of the dots indicates the adjusted p value by Bonferroni and Hochberg correction Hypergeometric test ; adjusted p value are indicated in Supplementary Data 6.
We mined the literature focusing on genes encoding transcription factors and signaling molecules, which traditionally have roles in cardiac development and modulating cell fate at E9. Representative genes, with a focus on transcription factors, signaling genes, and early smooth muscle markers, were ordered according to their expression peak in pseudotime Figs.
While some are known to function in NCCs for heart development Sema3c , Msx2 , Hand1 , Hand2 , Gata4 , Gata6 , Nrp1 , Prdm6 , and Smad7 , we found others that are newly recognized that may be required in NCCs for heart development, such as Tbx2 and Tbx3 stars; Figs.
We generated lineage driver gene sets at E Next, we performed a Gene Ontology GO enrichment analysis of each set using ToppGene Suite 33 to understand the function of the genes in each group Fig. Our analysis indicates that the initially activated genes of pharyngeal NCCs that include some CP-NCCs, are associated with general embryonic and mesenchyme development processes e.
Then cell division cell cycle genes are highly expressed, consistent with the expansion of pharyngeal NCCs during development 34 , together with cardiac development genes.
Furthermore, we integrated our scRNA-seq data from control embryos at E8. Cardiac fate trajectory analysis of the integrated data provides confirmatory information of our results from individual data analysis as progressive activation of the same specific genes during cardiac fate acquisition were found.
Thus, here we identified a specific CNCC transcriptomic signature at E9. We additionally identified transcription factor and signaling genes such as Dkk1 , Gata3 , Foxf1 , Mef2c , Isl1 , Isl2 , Tbx2 , Tbx3 , Rgs5, Bambi , and Smad6 , among others, which have not yet been considered as CNCC transition markers Supplementary Data 4, 5.
Tbx2 and Tbx3 are expressed in multiple tissue types within the pharyngeal apparatus and global inactivation of both genes lead to early embryonic lethality with severe cardiac defects 35 , 36 , 37 , We performed intracardiac ink injection, arterial branching analysis, and histological analysis at E n — q Immunofluorescence on coronal sections of controls at E Nuclei are stained with DAPI.
The bottom panels are a high magnification of the dashed regions in n — q. RSA right subclavian artery, RCC right common carotid, LCC left common carotid, BA brachiocephalic artery, Ao aorta, AoA aortic arch, LSA left subclavian artery, PT pulmonary trunk, E esophagus, PAA pharyngeal arch artery.
The right subclavian artery and aortic arch are formed from the right and left 4 th PAA, respectively. The failure of differentiation ranges from almost no expression of ACTA2 Fig. This variation might explain the partial penetrance of defects in mutant embryos.
Bmp4 and Foxf1 have been identified as regulators of smooth muscle cell differentiation in other organs We found that Bmp4 and Foxf1 expression was activated temporally after Tbx2 and Tbx3 expression during cardiac fate acquisition Fig. In Tbx1 null mutant embryos, the caudal pharyngeal apparatus is hypoplastic and unsegmented at E9.
Further, CNCCs fail to enter the shortened cardiac OFT, leading to a persistent truncus arteriosus later in development 9. We found that Tbx1 was not noticeably expressed in NCCs Supplementary Fig.
To understand how the absence of Tbx1 non-autonomously affects the development of CNCCs, we performed scRNA-seq of NCCs isolated from the microdissected pharyngeal region plus the heart of Tbx1 null mutant embryos at E9. We obtained sequencing data from 11, NCCs Supplementary Table 1 and integrated scRNA-seq data from control and Tbx1 null embryos using RISC Robust Integration of scRNA-seq software 40 Fig.
Even though there were visibly fewer NCCs in the pharyngeal apparatus Fig. We confirmed that most of the cells in each cluster of the integrated control and Tbx1 null scRNA-seq dataset shown in Fig.
This indicates that although affected, the general transcriptional profiles of the CNCCs are still conserved between control and Tbx1 null embryos. We then compared the proportion of cells in each cluster among the total number of NCCs in each dataset.
As expected, there was a reduction in the relative proportion of NCCs in Tbx1 null embryos as compared to controls, as shown in Fig. In addition, there was an increase in the relative proportion of NCCs in the distal part of PA1 and PA2 C3; 1. The increase in the proportion of NCCs is consistent with previous data that cells from PA2 abnormally migrate to PA1 in Tbx1 null embryos at this stage 9.
DAPI is in blue. NCCs from the region between the two dashed lines were used for scRNA-seq. b RISC UMAP plot of integrated scRNA-seq data from NCCs of control and Tbx1 null embryos. c UMAP plots colored by clusters from control left and Tbx1 null embryos right.
d Stack bar graph shows proportions of NCCs in indicated clusters divided by the total number of cells in control or Tbx1 null embryos. Additional information on statistical analysis is provided in the Source Data files. e , f Scatter plots show differential gene expression in cluster C8 e and in cluster C4 f from control and Tbx1 null embryos.
Note the upregulation of genes involved in the BMP pathway red and the downregulation of genes in the MAPK pathway purple. g Violin plots showing differential expression in control purple versus Tbx1 null red for Msx2 , Bambi , Spry1 , and Myc in clusters C4 and C8.
PA pharyngeal arch, OV otic vesicle, OFT outflow tract. We examined the data to identify differentially expressed genes DEGs in mutant versus control embryos at E9. One of the most notable changes in Tbx1 null embryos was an increase in the expression of genes that act downstream of BMP signaling in proximal PA2 and PA3 clusters C4, C8; Fig.
This includes increased expression of Msx2 , Msx1 , Bambi , Dkk1 , Smad7 , Id2 , and Id3. Our analysis also revealed a downregulation of the expression of genes in the MAP kinase mitogen-activated protein kinase signaling pathway, including Spry2 , Spry4 , Myc , Foxo1 , and Lyn Fig.
Signaling by BMP 41 and growth factors activating the MAPK pathway 42 , 43 are two signaling pathways known to be critical in NCCs for cardiac development. The increase in expression of Msx2 downstream in the BMP pathway , as well as reduced expression of Spry1 MAPK pathway in scRNA-seq data of Tbx1 null embryos, was observed in feature plots Fig.
We confirmed reduced MAPK signaling in pharyngeal NCCs of Tbx1 null embryos at E9. Expression of BMP downstream genes, Msx2 and Bambi , were expanded dorsally in Tbx1 null mutant embryos by wholemount RNAscope in situ and 3D reconstruction Fig.
These results were confirmed by RNAscope assays on traverse sections of control and Tbx1 null embryos Fig. Normally, BMP signaling is most active in the ventral part of the pharyngeal apparatus near the OFT.
NCCs migrate from the dorsal to ventral parts of the pharyngeal apparatus. Our results indicate that the BMP pathway is prematurely activated during their migration in a more dorsal region of the pharyngeal apparatus compared to control embryos. These data suggest that altered BMP and MAPK signaling might affect NCC development in Tbx1 null embryos.
A schematic representation of expanded and premature BMP signaling activity and reduced NCCs migrating to the shortened OFT in Tbx1 null embryos at E9. a UMAP plots show the expression level purple of Msx2 and Spry1 in NCCs split by control and Tbx1 null embryos. Digitally generated sections are shown in b.
Arrowheads show reduced expression of pERK in NCCs in the pharyngeal region of Tbx1 null embryo. c Msx2 , Bambi , and Egfp wholemount RNAscope in situ hybridization with DAPI of control and Tbx1 null embryos. Msx2 and Bambi expression is absent in the proximal part of the PA2 and PA3 of control embryos white arrowheads.
f Schematic representation of control left and Tbx1 null right sections at E9. PA pharyngeal arch, OFT outflow tract, NT neural tube, end endoderm, LV left ventricle.
During the formation of the heart, NCCs receive critical signaling from adjacent mesodermal cells Fig. We investigated cell-cell communication and how it is disrupted in the absence of Tbx1 at single-cell resolution using CellChat software a Schematic representation of a transverse section showing signaling arrows from pharyngeal mesoderm cells blue to NCCs yellow in the caudal pharyngeal apparatus.
b Bubble plot shows representative cell-cell signaling from Mesp1 Cre derived mesodermal cells to NCCs that were significantly altered Wilcox rank two-sided test without multiple test correction, p value indicated by the size of dot and color; right in Tbx1 mutant embryos.
Each dot represents a ligand—receptor pair interaction Y-axis between a specific cluster in the mesoderm cells and NCCs X-axis. Clusters include anterior and posterior second heart fields aSHF, pSHF and the multilineage progenitors MLP. d UMAP plots showing Isl1 expression in control and Tbx1 cKO cells.
e — m UMAP plots showing the expression of ligand genes including Wnt5a e , Wnt2 f , Sema3c g , Pdgfa h , Nrg1 i , Fgf10 j , Edn3 k , Bmp4 l , and Bmp7 m in control and Tbx1 cKO embryos.
Arrows indicate cell clusters with gene expression changes in Tbx1 cKO embryos. n Wholemount RNAscope in situ hybridization of Wnt1-Cre;ROSA-EGFP control lefts panels and Tbx1 null right panels embryos at E9.
Representative digital sections are shown on the right of 3D reconstruction pictures. Arrowheads show expression of Bmp4 in cells adjacent to the pharyngeal NCCs in Tbx1 null embryos. PA pharyngeal arch. Inactivation of Tbx1 in the mesoderm results in similar pharyngeal hypoplasia and altered NCC distribution as in global null embryos, implicating the pharyngeal mesoderm as being critical to signal to NCCs To identify Tbx1 -dependent signals from the mesoderm to NCCs, we investigated existing scRNA-seq data from Mesp1 Cre control and Tbx1 conditional null embryos at E9.
The scRNA-seq from two replicates of Mesp1 Cre control and Tbx1 conditional null embryos were integrated using RISC software as described We focused on mesodermal subpopulations, expressing Tbx1 , that are adjacent to the NCCs, including the anterior and posterior second heart field aSHF; pSHF 47 , We also included a critical Tbx1 -dependent multilineage progenitor population MLP in the pharyngeal mesoderm required for cell fate progression to the aSHF and pSHF We examined signaling to NCCs in clusters corresponding to migrating NCCs of the future PA C9 , distal part of PA C3 , mesenchyme of PA2 C8 and PA3 C4 , and CNCCs of the OFT C12 in integrated scRNA-seq data from control and Tbx1 null embryos at E9.
Representative results of ligand—receptor pairs altered when Tbx1 is inactivated are shown in Fig. Affected ligands in the mesoderm include Wnt5a, Wnt2, Sema3c, Pdgfa Nrg1, Fgf8, Fgf10, Bmp4 , and Edn3 and others.
To validate relationships, we analyzed integrated Mesp1 Cre data Fig. Isl1 , is a critical gene required for OFT development 49 , and it is expressed in the MLPs, aSHF, and pSHF Fig.
We examined expression changes in feature plots of Wnt5a , Wnt2 , Sema3c , Pdgfa , Nrg1 , Fgf10 , and Edn3 as well as other genes Fig.
These genes were altered in expression in the cell types specified and, in the direction of altered signaling decreased or increased in the mutant embryos , as indicated in Fig. Reduced expression of Wnt5a 50 , 51 , Fgf8 46 , 52 , 53 , Fgf10 54 , 55 , Sema3c 56 , and Nrg1 46 ligands and increase of Wnt2 57 are consistent with previous in vivo studies of Tbx1 mutant embryos; however, these were not known with respect to cell-cell communication to NCCs.
With these data, we show, on a single-cell level, that signaling to NCCs is altered in Tbx1 mutant embryos. We also identified ligand—receptor pairs that were not previously connected to NCCs or Tbx1. Two ligand genes, Edn3 and Pdgfa were identified in our cell-cell communication analysis Figs.
Edn3 encodes an endothelin ligand important in cell migration. Pdgfa encodes a growth factor regulating cell survival, proliferation, and migration and PDGF signaling is required in NCC development To our knowledge, there is no report of Edn3 and Pdgfa having been investigated in relation to Tbx1.
We investigated CellChat results for the BMP and MAPK pathways that were altered in NCCs when Tbx1 was inactivated. We performed RNAscope in situ hybridization for expression of Bmp4 and Bmp7 Fig. Bmp4 is expressed in the pharyngeal mesoderm and Bmp7 is expressed in the pharyngeal mesoderm and endoderm adjacent to the NCCs.
Expression of these two ligands were maintained in Tbx1 null embryos as compared to control embryos at E9. Fgf8 and Fgf10 are ligands in FGF signaling that act through the MAPK pathway and it is well known that they are reduced in expression in Tbx1 mutant embryos 46 , 52 , 53 , 54 , 55 Fig.
Similarly, we found that Fgf8 and Fgf10 were reduced in expression in the mesoderm and signaling to NCCs was altered Fig. It is known that FGF signals to NCCs during embryonic development 42 , 59 , To further understand how the contribution of NCCs to the OFT is altered in the absence of Tbx1 , we performed scRNA-seq of NCCs in Tbx1 null embryos at E We integrated scRNA-seq data from two control replicates 21, cells and two Tbx1 null replicates 17, cells using RISC software Fig.
a The NCCs between the dotted lines and heart in control and Tbx1 null embryos were used for scRNA-seq. b RISC UMAP plot of integrated scRNA-seq data from NCCs of two replicates of control and Tbx1 null embryos. c UMAP plots of pharyngeal and heart clusters from control left and Tbx1 null right embryos.
d Stack bar graph shows proportions of NCCs in selected clusters divided by the total cell number in control or Tbx1 null replicates. A two-sided proportion z -test was used to evaluate cell proportion differences between control and Tbx1 null embryos for the two replicates separately if the two Tbx1 null show consistent increase or decrease compared to control.
e UMAP plot colored by cell fate probabilities. f UMAP plots showing expression level of Tbx2 , Tbx3 , Isl1, Gata3 , Msx2 , and Dkk1 in NCCs from control or Tbx1 null embryos in pharyngeal NCC clusters.
Msx2 , Gata3 , and Dkk1 show increased expression in cluster C4. g Violin plots showing differential expression level of Msx2 , Bambi , Gata 3, and Tbx2 in cluster C4 in control purple and Tbx1 null red embryos.
h — j GO analysis for upregulated genes in C4 h , downregulated genes in C4 i , and upregulated genes in C10 j. Hypergeometric test was used, adjusted p value by Bonferroni—Hochberg correction. There is reduced ISL1-positive NCCs arrowheads.
Expression of TBX2 in NCCs is lateral to the pharyngeal endoderm end; white arrowheads. There are fewer CNCCs expressing ACTA2 within the OFT of Tbx1 null embryos red arrowhead. NT neural tube, end endoderm, OFT outflow tract.
Our analysis showed that the cell fate probabilities point from pharyngeal NCCs containing CP-NCCs C4 and OFT-CNCCs C10 toward SM-CNCCs C13 as shown in Fig. We generated a list of DEGs in clusters C4 and C10, between control and mutant embryos at E Msx2 , Bambi , Gata3 , and Dkk1 were increased and abnormally expressed in pharyngeal NCCs cluster C4 in Tbx1 null embryos Fig.
This suggests that an abnormal upregulation of BMP signaling occurred in the absence of Tbx1 , consistent with data at E9. GO analysis of the upregulated genes in pharyngeal NCCs cluster C4 confirms the upregulation of BMP signaling in Tbx1 null embryos Fig.
By GO analysis, we found downregulation in the expression of genes involved in embryonic organ development and mesenchyme development in pharyngeal NCCs that contain CP-NCCs Fig. We also noted that there was an upregulation of genes that inhibit the cell cycle progression of OFT-CNCCs Fig.
By immunostaining, we confirmed the overall reduction in the number of CNCCs within the OFT and NCCs in the pharyngeal region of Tbx1 null embryos Fig.
Supporting the scRNA-seq data, immunostaining experiments indicated that TBX2 and likely TBX3 expression was maintained in NCCs located in the lateral part of the pharyngeal apparatus Fig. We also noticed that there was normal differentiation of the CNCCs within the OFT of Tbx1 null embryos despite that there are fewer cells Fig.
Together this suggests a reduction but not a complete failure of cardiac fate progression between pharyngeal NCCs and OFT-CNCCs states in the absence of Tbx1. In this report, we identified the cell type signatures and cell fate dynamics of CNCCs. We focused on pharyngeal NCCs in the mouse at developmental stages when Tbx1 , the gene for 22q We determined the mechanisms by which Tbx1 non-autonomously regulates CNCC maturation at a single-cell level and found that altered BMP and FGF-MAPK signaling, together with other signaling, may contribute to cardiovascular malformations when Tbx1 is inactivated.
We also uncovered genes and ligand—receptor pairs with respect to cell-cell communication from mesoderm to NCCs that add to our understanding of CNCC fate progression.
NCCs are multipotent and differentiate into many cell types, including smooth muscle cells. Through examination of transitional dynamics along with embryonic localization by in situ analysis, we uncovered three main transition states from pharyngeal to smooth muscle expressing CNCC derivatives, termed CP-NCCs, OFT-CNCCs, and SM-CNCCs as shown in the model in Fig.
a Colors indicate cell fate progression of pharyngeal NCCs light yellow towards vascular smooth muscle cells red. The graph shows the relative change of biological gene ontology terms during the three transitions of CNCCs to smooth muscle cells.
Alteration of signaling in the mesoderm in the absence of Tbx1 results in abnormal premature activation of the BMP pathway during NCC deployment and migration light blue inhibitory arrow; red, up arrow , a decrease of MAPK signaling in NCCs light blue arrow; red down arrow , and alteration of other signaling pathways including upregulation of WNT signaling in developing NCCs.
The CP-NCCs express Hox and Dlx genes, as well as those implicated in cardiac development, including Tbx2 and Tbx3 , as well as other genes Fig.
A role of Tbx2 and Tbx3 in CNCCs in PAA development have not been previously described, and we found that inactivation results in the partially penetrant abnormal aortic arch and arterial branching, indicating besides function as markers of CP-NCCs, they have a role in smooth muscle differentiation.
Another reason for partial penetrance in mutant embryos could be because mice with a mixed genetic background were used in this study. These cells are required for OFT development, as determined by conditional inactivation studies in NCCs of Hand2 66 , Msx1 , and Msx2 Expression of Isl1 in CNCCs contributing to the OFT is consistent with a dual lineage tracing study We also identified genes not previously connected to CNCCs that include Dkk1 , Foxf1 , Rgs5 , Isl2 , and Gata3 , as well as newly recognized genes for cardiovascular development.
Supporting their requirement, conditional deletion of Gata6 in NCCs results in septation defects of the OFT Abnormal activation of Smad7 in the NCCs derived from Wnt1-Cre genetic lineage leads to pharyngeal arch and cardiac OFT defects We additionally found genes not yet connected to these cells, including Meox1 , Bambi , and Smad6.
We found that the progression of CNCC fate toward smooth muscle of the OFT is associated with progressive downregulation of genes involved in pharyngeal embryonic development and progressive increase in cardiac specification genes, consistent with maturation of the unipotent cellular state to smooth muscle cells Fig.
Development of NCCs in the pharyngeal apparatus is regulated by cell-cell signaling, in part from the pharyngeal mesoderm, as uncovered by studies of Tbx1 mutant embryos 45 , In global null or Mesp1 Cre mediated Tbx1 conditional null embryos, there is altered deployment of CNCCs and reduced contribution to the OFT, leading to a persistent truncus arteriosus 45 , Our results suggest that NCCs are produced normally in the neural tube in the absence of Tbx1 , but they fail to migrate to the caudal pharyngeal arches and OFT, as well as to fail to progress to more mature states, as shown in our model in Fig.
We suggest that failure to migrate and progress in development is due in part to disrupted signaling from adjacent mesodermal cells to the NCCs as shown in Fig. Here, we identified several receptor-ligand interactions that are disrupted by comparing scRNA-seq data from NCCs and the Mesp1 Cre lineages.
Using CellChat software 44 , we identified signaling interactions from the mesoderm to NCCs and their disruption in Tbx1 mutant embryos, at a single-cell level. We identified a reduction of FGF ligand expression Fgf8 and Fgf10 in the mesoderm with reduced MAPK signaling in NCCs, as shown in the model in Fig.
This confirms previous studies showing that inactivation of Tbx1 results in reduced expression of Fgf8 and Fgf10 ligands in the mesoderm 55 , It is known that loss of FGF ligands indirectly results in NCC mediated cardiovascular anomalies 42 , 59 , This function is affected significantly by the MAPK signaling pathway that is critical for NCC development and migration Further, FGF signaling is required downstream of Tbx1 , both genetically and mechanistically 52 , Finally, our bioinformatic analysis shows a reduction of downstream effector genes in the MAPK pathway Myc, Spry1, and Spry2.
When taken together, our results support an essential role of mesodermal TBX1-FGF signaling to NCCs that is required for normal heart development Fig.
Inactivation of a BMP antagonist gene, Dullard in NCCs resulted in similar cardiac OFT defects as in Tbx1 null mutant embryos 41 implicating a mechanistic connection between an increase in BMP signaling and OFT defects. The CellChat analysis showed that there is an increase of Bmp4 and Bmp7 expression in the adjacent mesoderm relative to the expression of specific Bmp receptor genes.
Although our RNAscope analysis of Bmp4 and Bmp7 did not show an obvious increase in the expression of these two ligands, they continue to be strongly expressed in the mesoderm of Tbx1 null mutant embryos suggesting that the source of BMP ligands is in adjacent cells that interact with NCCs.
In the future, utilization of organoids and cell culture systems will help dissect the mechanism of how BMP interacts with NCCs. Based upon this analysis, we propose that the inactivation of Tbx1 results in an alteration of paracrine signaling from the mesoderm, including FGF and BMP, separately contributes to CNCC migration and fate progression, as shown in Fig.
However, these are not the only signaling pathways that are affected by inactivation of Tbx1. We found an increase in Wnt2 expression in the mesoderm by CellChat analysis.
We previously found that global Tbx1 inactivation 76 or inactivation in the aSHF using Mef2c-AHF-Cre , resulted in increased Wnt2 expression Therefore, canonical WNT signaling also functions downstream of Tbx1 , and this may also affect NCC function.
The CellChat analysis also revealed downregulation of Nrg1-Erbb3 , implicating altered Neuregulin to ERBB3 signaling in CNCCs in Tbx1 mutant embryos. Neuregulin is important for the migration of NCCs, acting as a chemoattractant and chemokinetic molecule 77 and it is involved in heart development.
It has been shown recently that Nrg1 is a direct transcriptional target gene of TBX1 in the multilineage progenitors MLPs in the mesoderm Therefore, alteration of Neuregulin signaling in Tbx1 mutant embryos could, in part, explain the failed cardiac contribution of the NCCs.
There was other signaling affected, some of which has not been investigated with respect to Tbx1 or mesoderm-NCC signaling, such as identified by alteration in expression of Edn3 or Pdgf a genes, which can be pursued in the future.
Together, our analyses indicate that a combination of important signaling from the pharyngeal mesoderm to NCCs are affected in Tbx1 mutant embryos and could contribute to the failure of fate progression of CNCCs.
One of the reasons for the disruption of NCCs in Tbx1 mutant embryos is failed segmentation of the caudal pharyngeal arches by the endoderm. Alternatively, disruption of NCCs could affect the pharyngeal endoderm resulting in failed segmentation.
A previous study by Veitch and colleagues showed that after the ablation of NCCs in the chick embryo, pharyngeal segmentation still took place This suggests that pharyngeal segmentation defects in Tbx1 null embryos is not due to a reduced number of defective NCCs.
There are many reasons for failed segmentation of the caudal pharyngeal apparatus due to Tbx1 inactivation. The tissue-specific functions of Tbx1 in this process has been evaluated.
Loss of Tbx1 in the pharyngeal endoderm autonomously affects segmentation of the caudal pharyngeal apparatus leading to reduced contribution of CNCCs The pharyngeal endoderm is also an important source of signaling during development, including FGF ligands 59 , that could potentially affect NCCs development.
It was found that Tbx1 expression in the mesoderm is necessary, non-autonomously, for pharyngeal segmentation 45 , suggesting that defects in signaling from the adjacent mesoderm that expresses Tbx1 , cause failed segmentation.
Loss of Tbx1 in the mesoderm also leads to failed CNCC migration and fate progression. The Tbx1 expression domain in the pharyngeal ectoderm also serves as a signaling center for non-autonomous signaling 64 , It will be interesting to evaluate how the exchange of signaling between the pharyngeal epithelia and NCCs are affected in the absence of Tbx1.
In conclusion, in this report, we identified the transcriptional signature that defines the CNCCs and identified the gene expression dynamics that regulate CNCC fate progression into the smooth muscle of the OFT and PAAs.
In addition, we highlight the direct alteration of FGF, BMP, and other signaling from the mesoderm to NCCs, most likely leading to failed OFT septation in the absence of Tbx1 at a single-cell level. Together our results allow a better understanding of the normal development of CNCCs and provide insights into the origin of congenital heart defects associated with defective NCCs and 22q The IACUC protocol number is Mice were maintained on a mixed Swiss-Webster genetic background.
After recombination of the genomic sequences between the LoxP sites, the reading frame and T-Box domain of Tbx2 and Tbx3 are both disrupted Embryos were not evaluated for sex, because we studied developmental processes prior to sexual development.
Mice and embryos resulting from the different crosses were genotyped from DNA extracted from the tips of toes or yolk sacs. The individual PCR primers and assays used in the experiments are described in the reports on individual mouse alleles. Embryos were collected at different embryonic days dated from the day of the vaginal plug E0.
For each experiment, a representative result was presented from at least three analyzed embryos. Embryos were progressively dehydrated in ethanol, then xylene, and embedded in paraffin Paraplast X-tra, Sigma P The embryos were sectioned to the μm thickness and sections were deparaffinized in xylene and progressively rehydrated in an ethanol series.
After cooling at room temperature, sections were washed in PBS containing 0. Embryonic sections were imaged using a Zeiss Axio Imager M2 microscope with ApoTome.
Images were analyzed using Zen software from Zeiss, ImageJ, and Adobe Photoshop. After progressive rehydration in 1x PBS, embryos were incubated in 1x PBS containing 0. Embryos were washed in 0.
Embryos were imaged as a Z-stack using a Nikon CSU-W1 spinning disk confocal microscope. Images were analyzed using ImarisViewer 9. Wholemount RNAscope was performed using RNAscope Multiplex Fluorescent Detection Reagents v2 kit Advanced Cell Diagnostics, ref Embryos were progressively rehydrated in 1x PBS containing 0.
After three washes in 0. C2 and C3 probes were revealed following the previous steps and using HRP-C2 for C2 probes and HRP-C3 for C3 probes. Nuclei were stained overnight with DAPI.
Wholemount embryos were imaged as Z-stacks using a Leica SP5 confocal microscope or a Nikon CSU-W1 spinning disk confocal microscope, after being cleared with BABB or not. RNAscope was performed on μm sections mounted on SuperFrost Plus slides Fisher Scientific, following the RNAscope Multiplex Fluorescent Reagent Kit v2 assay protocol from Advanced Cell Diagnostics.
Sections were imaged using a Zeiss Axio Imager M2 microscope with ApoTome. Acquisitions were processed using Zen software from Zeiss, ImageJ, and Adobe Photoshop. Probes used for RNAscope: Egfp , C3 , Tbx2 , Hoxb3 , Dlx2 , Bambi , Hoxa2 , Gata3 C2 , Isl1 , C2 , Tbx3 C2 , Sox10 C2 , Msx2 C2 , Meox1 C2 , Dlx5 C3 , Twist1 , Rgs5 C3 , Cnn1 , Bmp4 , and Bmp7 They were progressively dehydrated in ethanol and incubated in xylene prior to embedding in paraffin.
Sections were imaged using a Zeiss Axioskop 2 plus microscope and analyzed using Zen software and Adobe Photoshop software. Fetuses at E Immediately after filling the left ventricle and arterial branches, the heart and aortic arch with arterial branches were imaged using a Leica MZ stereomicroscope.
Images were analyzed using Zen software and Adobe Photoshop software. For E8. The head of the embryo was conserved at E8. The head of the embryos containing NCCs not relevant to the cardiovascular system were removed at E9.
The head and PA1 were removed at E Cells were FACS Fluorescence Activated Cell Sorting purified using a BD FACSAria II BD Biosciences system and BD FACSDiva 8. The FACS gating strategy is exemplified in Supplementary Fig. One scRNA-seq experiment was performed for control and Tbx1 null embryos at E9.
Each sample that was submitted for scRNA-seq was a pool of two or more embryos of the same genotype Supplementary Table 1. Sequencing of the DNA libraries was performed using an Illumina HiSeq system Genewiz Company, South Plainfield, NY, USA scRNA-seq at E9.
CellRanger v6. All the samples passed quality control measures for Cell Ranger version 6. Individual scRNA-seq sample data were analyzed using Seurat V4. After individual samples were analyzed by Seurat for clustering, the data were integrated by RISC software v1.
The integrated data were re-clustered by RISC, using parameters adjusted to match the cell type clusters in the Seurat results. Cell compositions were computed from the integrated cell clusters and used for the two-proportion Z -test as implemented in the R prop. test function to evaluate the statistical significance in changes between control and null datasets.
At stage E9. At stage E CellRank v1. The analysis used RNA velocity from Velocyto v0.
Nodes represent cell groups and edge weights quantify the connectivity between groups. We see a strong bifurcation of CD8 cytotoxic cells and Tregs into For each pair of clusters, PAGA connectivity is the ratio of the number of inter-edges between the clusters normalized with the number of inter-edges expected MLPs are localized to the caudal pharyngeal apparatus. a Single-cell embedding graph with PAGA plot colored by clusters with abbreviated names
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