A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells

Abstract

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

Keywords: Calcitonin; Endoderm; Evolution; Neural crest; Neuroendocrine.

Fig. 1.

Chick C-cells derive from endoderm, not neural crest. (A,A′) At E10, the chick UB is located between the esophagus and the carotid artery, at the level of the nodose ganglion. (B,C) Chick C-cells co-express calcitonin (CT) (B) and tyrosine hydroxylase (TH) (C). (D) Neural crest lineage tracing was performed by isotopic unilateral grafting of GFP⁺ neural fold into a wild-type host embryo. In this example, a graft was performed with neural fold from the otic vesicle to somite 7. (E-E″) In grafted embryos, we recovered GFP⁺ cells in and around the UB, but we observed no colocalization of GFP and CT. (F) Endodermal lineage tracing was performed by microinjecting CM-DiI into the pharyngeal cavity of chick embryos at Hamburger Hamilton stage 11. (G-G″) In 3/5 labeled embryos, we recovered CM-DiI within the UB at E8.5, with colocalization of CM-DiI and CT indicating endodermal origin of chick C-cells. Dashed lines indicate the ultimobranchial body. ca, carotid artery; e, esophagus; ng, nodose ganglion; ot, otic vesicle; S1-S7, somites 1-7; tr, trachea; ub, ultimobranchial body. Scale bars: 100 μm (A); 50 μm (A′); 25 μm (B,C,E,G).

Fig. 2.

C-cells of the larval zebrafish develop in the absence of neural crest. (A-B) The UB of a 10 dpf zebrafish is located between the gut and the heart, at the axial level of the sinus venosus (A). It is recognizable as a distinct cluster of cells beneath the muscle layer of the gut (A′), and by its expression of calca (B). Dashed lines indicate the ultimobranchial body. (C,D) 7 dpf wild-type zebrafish (C) possess calca⁺ C-cells within their UB (D). (E,F) 7 dpf tfap2a^mob;foxd3^mos mutants (E) lack neural crest cells, but still possess calca⁺ C-cells in their UB (F). ub, ultimobranchial body. Scale bars: 20 μm (A,A′,B,D,F); 1 mm (C,E).

Fig. 3.

Zebrafish C-cells derive from pharyngeal endoderm, not neural crest. (A-A″) The UB of adult zebrafish stains positively for calcitonin (CT) by immunofluorescence (A). The Sox10>dsRed line labels all neural crest derivatives with DsRed. We found no DsRed⁺ cells within the UB of adult fish of this line, despite strong labeling of other neural crest-derived strictures on the same sections – e.g. Tyrosine Hydroxylase (TH)-positive neurons of the sympathetic ganglia (see inset boxes in A-A″). (B-B″) sox17>mCherry zebrafish embryos were treated with 4OHT during gastrulation to indelibly label the endodermal lineage with mCherry, and then grown to adult. We observed complete colocalization of mCherry and CT within the UB of adult fish, indicating endodermal origin of zebrafish UB C-cells. Dashed lines indicate the ultimobranchial body. ub, ultimobranchial body. Scale bars: 50 μm (A,B).

Fig. 4.

Skate C-cells derive from pharyngeal endoderm. (A,A′) In a stage (S)32 skate embryo, the developing UB is located within connective tissue between the fifth gill arch and the pectoral girdle. (B) The UB of a stage 33 skate embryo, with Calca expression in C-cells. (C,D) Microinjection of CM-DiI into the pharyngeal cavity of a S18 skate embryo (C) specifically labels the pharyngeal endoderm (D). (E) Maximum intensity projection of the UB of a S32 skate embryo that received CM-DiI labeling of pharyngeal endoderm at S18. There is abundant CM-DiI labeling throughout the UB. Dashed lines indicate the ultimobranchial body. (F,G) Colocalization of CM-DiI and Calca expression within the UB of S32 skate embryos indicate endodermal origin of C-cells in the skate. Insets show magnification of boxed areas. ga2-5, gill arches 2-5; pg, pectoral girdle; ub, ultimobranchial body. Images in panels C and D are reproduced from Rees et al. (2023) under the terms of a CC-BY 4.0 license. Scale bars: 200 μm (A); 50 μm (A′); 25 μm (B,D-G); 500 μm (C).

Fig. 5.

Putative C-cell homologs within the pharynx of invertebrate chordates. (A,B) Adult Ciona intestinalis (A). Sections in the plane indicated by the dashed line reveal a branchial sac that is perforated by slits, with an endostyle on one side and the gut on the other (B). (C) HCR for Ci-CT reveals expression in the endodermally-derived wall of the branchial sac (note autofluorescence within the gut). Asterisks in B and C indicate the papillae of the branchial sac. (D-E′) Expression of Ci-CT localizes to distinct clusters of cells within the papilla of the branchial sac. No expression was observed within the endostyle. (F,G) An adult amphioxus, Branchiostoma lanceolatum (F). Sections in the plane indicated by the dashed line reveal a pharyngeal cavity containing a series of pharyngeal bars separated by pharyngeal slits (G). A lower magnification image of the section in G is shown in Fig. S5A. (H) Each pharyngeal bar consists of atrial cells, ciliated cells and pharyngeal cells, and is supported by an acellular skeletal rod. (I) HCR for amphioxus CTFP1-3 reveals co-expression of all three paralogs within the atrial cells of each pharyngeal bar. Dashed lines indicate CTFP-expressing atrial cells. ac, atrial cells; cc, ciliated cells; es, endostyle; gt, gut; pb, pharyngeal bar; pc, pharyngeal cells; s, pharyngeal slit; sk, skeletal rod. Scale bars: 1 mm (A); 250 μm (B,C); 50 μm (D,E); 2 mm (F); 75 μm (G-I).

Fig. 6.

Evolution and homology of chordate C-cells. C-cells were broadly distributed throughout the endoderm-derived lining of the pharynx in the last common ancestor of chordates. These C-cells then became localized into a discrete ultimobranchial body in non-mammalian vertebrates, with that ultimobranchial body fusing with the thyroid gland of mammals.