Thyroid and endostyle development in cyclostomes provides new insights into the evolutionary history of vertebrates

Abstract

Background: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century.

Results: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages.

Conclusions: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

Keywords: Atavism; Cyclostomes; Development; Endostyle; Evolution; Hagfish; Lamprey; Thyroid gland.

Fig. 1

Transition of the endostyle to the thyroid in lampreys and classical evolutionary hypothesis of the vertebrate thyroid gland. A Larval lampreys (lateral view) possess an exocrine endostyle (en), which transforms into thyroid follicles (thy) in the adult (ventral view). p, pharynx; ht, heart; or, orifice; bb, branchial basket; va, ventral aorta; hbm, hypobranchial muscle; tm, tongue muscle. Bars, 100 μm. B Classical evolutionary hypothesis of the relationship between the endostyle and the thyroid based on the previous studies. Although an endostyle-like thyroid primordium was observed in developing hagfish (Eptatretus stoutii) embryos in 1906, it has not been verified ever since [15]. The primitive endostyle has been retained only in modern lampreys but was independently lost in the gnathostome and possibly in hagfish.

Fig. 2

Development of the thyroid gland in the Eptatretus burgeri embryo. Dorsal (A, D) and lateral views (G) of fixed embryos immersed in methyl benzoate prior to paraffin embedding. B, B’, E, E’, H, H’ Midline sagittal sections of the hagfish thyroid. B, B’ Sections were stained using hematoxylin and eosin. E, E’, H, H’ Sections including thyroid primordia were stained via in situ hybridization of Tbx1/10A (E, E’) and Lhx3/4A (H, H’) with nuclear fast red in a previous study [17]. The area surrounded by dashed lines indicates thyroid primordium. Lateral (C, F, I) and frontal (C’, F’, I’) views of 3D reconstructed images. The ectoderm and endoderm are colored light blue and yellow, respectively. Dashed lines indicate the boundary between stomodeum and pharynx. Thyroid is colored red. Both cardiac field and blood vessel are transparently colored light red. pp, pharyngeal pouch; ect, ectoderm; nc, notochord; ot, otic vesicle; end, endoderm; heart, ht; ventral aorta, va; bs, bifurcation site of ventral aorta. See Fig. 1 for other abbreviations. Bars, 1 mm (B, E, H) and 200 μm (B’, E’, H’)

Fig. 3

Expression profiles of the genes involved in thyroid development in modern cyclostomes. A–I and J–U’ hagfish and lamprey embryos, respectively. V, W, X schematic of the Nkx2-1/2-4 expressing domain (colored red) in hagfish, lamprey, and catshark embryos when the expression of Nkx2-1/2-4 first detected in this study (stages 45, 23, and 24, respectively). A’, D’, G’, K’, L’, S’, T’ Filled and open arrowheads indicate the presence and absence of signals, respectively. K’, L’, M’, O’, P’, Q’, S’, T’, and U’ Transverse sections of hybridized embryos in the plane of the dashed lines. M’, Q’, U’ Diagram of gene expression in the endostyle is shown in the boxes. as, aortic sac; opm, vestigial oropharyngeal membrane; MHB, midbrain-hindbrain boundary; ma, mandibular arch; liv, liver; hyp, hypothalamus; ve, velum; pn, pronephros. See Figs. 1 and 2 for other abbreviations. Bars, 200 μm

Fig. 4

New alternative evolutionary scenario for the endostyle and thyroid gland. A–D Hypothetical independent evolutionary events that occurred during chordate evolution. Arrows indicate developmental sequence. A Acquisition of the primitive endostyle, which originated from the pharyngeal endoderm with broad expression of Nkx2-1/2-4 and subordinate endostyle GRN. B Thyroid follicles arise from a subset of epithelial cells in the endostyle with the acquisition of thyroid GRN including Nkx2-1/2-4, Pax2/5/8, and Hhex. Co-existence: the primitive endostyle and thyroid follicles are not mutually exclusive in this state. C Anatomy and developmental module of the primitive endostyle was lost in the LCA of modern vertebrates. Crown group of cyclostomes have retained the early and broad expression of Nkx2-1/2-4. C’ According to the classical hypothesis, the independent GRN of the primitive endostyle may have enabled the loss of its morphology without influencing thyroid development in each ancestor of gnathostomes, hagfishes, and possibly stem lampreys. D In the LCA of modern lampreys, an atavistic endostyle is secondarily acquired by recruiting a similar developmental module to form the endostyle. END, endostyle; THY, thyroid