Genetic Mechanism for the Cyclostome Cerebellar Neurons Reveals Early Evolution of the Vertebrate Cerebellum

Abstract

The vertebrate cerebellum arises at the dorsal part of rhombomere 1, induced by signals from the isthmic organizer. Two major cerebellar neuronal subtypes, granule cells (excitatory) and Purkinje cells (inhibitory), are generated from the anterior rhombic lip and the ventricular zone, respectively. This regionalization and the way it develops are shared in all extant jawed vertebrates (gnathostomes). However, very little is known about early evolution of the cerebellum. The lamprey, an extant jawless vertebrate lineage or cyclostome, possesses an undifferentiated, plate-like cerebellum, whereas the hagfish, another cyclostome lineage, is thought to lack a cerebellum proper. In this study, we found that hagfish Atoh1 and Wnt1 genes are co-expressed in the rhombic lip, and Ptf1a is expressed ventrally to them, confirming the existence of r1's rhombic lip and the ventricular zone in cyclostomes. In later stages, lamprey Atoh1 is downregulated in the posterior r1, in which the NeuroD increases, similar to the differentiation process of cerebellar granule cells in gnathostomes. Also, a continuous Atoh1-positive domain in the rostral r1 is reminiscent of the primordium of valvula cerebelli of ray-finned fishes. Lastly, we detected a GAD-positive domain adjacent to the Ptf1a-positive ventricular zone in lampreys, suggesting that the Ptf1a-positive cells differentiate into some GABAergic inhibitory neurons such as Purkinje and other inhibitory neurons like in gnathostomes. Altogether, we conclude that the ancestral genetic programs for the formation of a distinct cerebellum were established in the last common ancestor of vertebrates.

Keywords: Purkinje cells; cerebellum evolution; cyclostome; evolutionary developmental biology (EvoDevo); granule cells of cerebellum; hagfish; lamprey; rhombic lip.

FIGURE 1

Development of the cerebellum in mammals, based on Fujiyama et al., 2009 (A). (A) Dorsal view of the rhombomeres. The rhombomere 1 (r1) gives rise to the cerebellum, while r2–8 generates the cochlear nuclei and precerebellar nuclei. (A’) Sagittal view of the rhombomere 1. Granule precursor cells, generated in the r1 rhombic lip, migrate tangentially to form the external germinal layer (EGL). The Purkinje precursor cells originating from the ventricular zone secrete Shh, which is involved in the proliferation of the granule cells. The granule cells in EGL then migrate ventrally to form the granular layer. (A”) Transverse view of rhombomere 1 showing the rhombic lip as the source for excitatory neurons, and the ventricular zone where the inhibitory neurons arise. (B–H) Upper and lower rhombic lip gene expression patterns in the hagfish, E. burgeri. (B) Illustration of a sagittal section of the anterior part of a stage 53 embryo. (left) Rhombomere 1 (Upper rhombic lip) is identified as a negative region of both Otx and Hox genes (Oisi et al., 2013; Pascual-Anaya et al., 2018). (right) Anterior-posterior level of transverse sections shown in (C–H) are indicated in a sagittal illustration. (C–H) Section in situ hybridizations for hagfish Wnt1, Atoh1 and Ptf1a genes showing their expression patterns in r1 (C,E,G) and posterior to r1 (D,F,H). (C’–H’) Higher magnification images of the rhombic lip expression patterns shown in (C–H). (I) Comparison of the upper rhombic lip gene expression patterns in the shark (S. torazame), lamprey (L. camtschaticum), and hagfish (E. burgeri) based on Sugahara et al. (2017) and the present study. di, diencephalon; hy, hypothalamus; m, mesencephalon; n, notochord; ot, otic capsule; tel, telencephalon; r1, rhombomere 1; V, trigeminal nerve. Scale bars: 200 μm (C–H), 50 μm (C’–H’).

FIGURE 2

Temporal Atoh1 expression pattern in the lamprey hindbrain. Lateral (A–F) and dorsal views (A’–F’). Dashed lines indicate the MHB. Atoh1 expression starts at stage 23.5 in the rostral half of the rhombic lip (B) and subsequently expands throughout the rhombic lip during stages 24–26 (C–E). After stage 27 (not shown), the expression is restricted only to the rostral border of the rhombomere 1 (F). Note that the apparent asymmetrical staining in (B) does not reflect the actual difference in expression, but is caused by the staining process. e, eye; gf, complex of facial/lateral line ganglion; ov, otic vesicle.

FIGURE 3

Expression of genes involved in neuron differentiation during lamprey late embryonic stages. (A,A’) Atoh1 expression is restricted to the anterior r1 in stage 27 embryo (arrow heads). Dashed lines indicate the MHB. (B,B’) NeuroD expression is observed behind the Atoh1 expression domain (arrowheads). Note that (A’,B’) are not at the same rostrocaudal positions in r1. The apparent asymmetrical staining in (B’) does not reflect the actual difference in expression, but is caused by the slightly oblique cutting of the sections. (C,D) In situ hybridization of both OtxA and Hox2α probes in stage 27 in lateral view (C) and dorsal view (D). OtxA is expressed only anterior to the midbrain, whereas Hox2α is expressed caudally from r2. Dashed lines indicate the borders of r1 identified as “Otx–Hox free” domain. (E) High magnifications of the panel (A–C) in r1 region. (F,F’) Ptf1a-B expression at the ventricular zone at stage 26 (arrowheads). (G,G’) GAD is expressed in the hindbrain at the same dorsal level than –but lateral to– the Ptf1a expression domains (black arrowheads) and also in a ventral domain (white arrowheads). (H) Comparison of Atoh1 and NeuroD expression patterns in the hindbrain between paddlefish, Polyodon spathula (stage 42, based on Butts et al., 2014c), and lamprey. Note that although the lamprey hindbrain does not show a ‘diamond shape’ of the rhombic lip during embryonic stages, the topological relationship of these gene expression patterns is comparable in both species. cb, cerebellum primordium; gV, trigeminal ganglion; ph, pharynx; va, valvula cerebelli. See Figures 1,2 for other abbreviations. Scale bars, 100 μm (A–D), 50 μm (A’,B’,F,F’,G,G’).

FIGURE 4

An evolutionary scenario for the vertebrate cerebellum, based on Chaplin et al. (2010) and Butts et al. (2014c) and the present study. Note that the internal migration of postmitotic granule cells has been observed in teleosts but not in the paddlefish. Therefore, it is equally possible that this migration of granule cells was acquired after the divergence of chondrichthyans and osteichthyans, with paddlefish losing this ability secondarily. Alternatively, it is possible that teleosts acquired the migration independently from amniotes. val, valvula cerebelli; EGL, external granule cell layer.