Distribution of cells originating from the 24 hpf MHB in the adult brain
The her5 transcription factor is known to be an early marker of the midbrain-hindbrain domain in zebrafish [11, 12]. Indeed, the zebrafish MHB is well established at 24 hours post-fertilization (hpf), and at this stage, her5 expression is restricted to the MHB ([13]; see also Fig. 2a, b, Additional file 1: Movie S1.). In sagittal sections, the her5 expression territory appears as a thin band, extending from dorsal to ventral. To follow the fate of these her5-expressing progenitors, we crossed two transgenic lines: Tg(her5:ERT2CreERT2) and Tg(βact:lox-stop-lox-hmgb1:mCherry) (Fig. 2d). We verified that the Cre expression is limited to the MHB at 24 hpf, recapitulating the her5 expression (Fig. 2c). Thus, after tamoxifen treatment at 24 hpf, cells expressing her5 and their progenies express mCherry, and we can interpret that all the mCherry-positive cells observed at later stages (after induction) are derived from the MHB.
Movie S1. Expression of her5 in the 24 hpf zebrafish embryo. Expression of mCherry in a transgenic line Tg(her5:mCherry) is shown in magenta, and DiD fiber labeling indicating the morphology of the embryo is shown in gray. (MP4 1999 kb)
As expected, in the brains of adult fish (3 months or older), mesencephalic structures such as the tectum (TeO), the torus semicircularis (TS), and the tegmental area were massively mCherry positive after induction with tamoxifen at 24 hpf (Fig. 3a–c). There was no labeling in forebrain structures such as the hypothalamus or the pretectum (Hy and PT respectively; Fig. 3a, b).
However, some unexpected structures exhibited mCherry-positive cells. The preglomerular nucleus (PG; Fig. 3a’) is a sensory relay nuclear complex that is considered to be part of the posterior tuberculum of the diencephalon. The PG is continuous with a nucleus named the torus lateralis (TLa; Fig. 3b). TLa is classified as part of the tegmentum (thus midbrain) by some authors [14, 15], while as a diencephalic (thus forebrain) by others [10, 16]. The cluster of mCherry-positive cells is continuous from PG to IL through TLa.
The IL is usually considered as part of the hypothalamus [14] because of its location posterior to the hypothalamus proper. Indeed, this structure develops as a lateral elongation of the teleost LR (which corresponds to the amniote 3V in the hypothalamus). However, our data showing a massive of mCherry-positive cells in IL (Fig. 3b, c, Additional file 2: Figure S1) suggest that a large part of this structure is formed by cells originating from the MHB. The diffuse nucleus of the inferior lobe [10] (DIL; Fig. 3b’, c’) is the most labeled area. Numerous mCherry-positive cells are also found in the area corresponding to the central nucleus of the inferior lobe (CIL; Fig. 3c’). Interestingly, most of the outer region of the IL is mCherry positive, but the inner part, close to the LR, is mCherry-negative (Fig. 3b’, c’). The dorso-medial structure called the corpus mamillare (CM) is also mCherry-negative (Fig. 3c’).
Migration of the cells originating from the MHB during development
Our data on the adult brain show that IL has a mesencephalic component. In order to decipher the development and anatomy of this structure in further details, we followed the mCherry-positive cells in whole brains at different stages after induction at 24 hpf.
At 3 days post-fertilization (dpf), all the mCherry-positive cells are still located around the MHB, and there is no labeling at the level of the forebrain (Fig. 4a–g, Additional file 3: Movie S2). This confirms the specificity of the molecular cell tracing method and the absence of leaky expression during the induction process. It further supports that the anterior structures, in which we observe mCherry expression at later stages, are composed of the progeny of cells originating from the MHB exclusively.
Movie S2. Localization of the mCherry-positive cells in the 3 dpf larval head of Tg(her5:ERT2CreERT2;βact:lox-stop-lox-hmgb1:mCherry) zebrafish treated with tamoxifen at 24 hpf (3D visualization of Fig. 4a–d). mCherry-positive cells are shown in magenta, and YOYO-1, a nuclear marker, is shown in green. (MP4 5759 kb)
At 3 dpf, we could not identify the IL. In the lateral view (Fig. 4e), the MHB progenies (cells which were expressing her5 at 24 hpf) extend in a triangular cluster, wider in the dorsal part. The IL was first observable at 5 dpf (Fig. 4h, i; arrows), being more remarkable at 7 dpf (Fig. 4j, k; arrows). MHB progenies extend ventrally in the outer surface of IL (Fig. 4i, k).
From late larval to juvenile stages, we could clearly identify the IL as a ventral protuberance with mCherry cells. Observation of global mCherry expression in the whole brain confirms a continuity of the mCherry labeling between the IL and more dorsal midbrain structures known to be part of the tectum and tegmentum (Fig. 5a–d, Additional file 4: Movie S3). This is also visible in frontal sections at 19 dpf, in which mCherry-positive cells form a continuous strip from the dorsal tectum to the ventral IL (Fig. 5f). At this stage, frontal sections of the IL already resemble those in adult, both anteriorly (Fig. 5e–g) and posteriorly (Fig. 5h–j). At 5 weeks post-fertilization (wpf), the IL continues to grow and appears as a ventral protuberance (Additional file 5: Figure S2).
Movie S3. Localization of the mCherry-positive cells in the 14 dpf larval brain of Tg(her5:ERT2CreERT2;βact:lox-stop-lox-hmgb1:mCherry) zebrafish treated with tamoxifen at 24 hpf (3D visualization of Fig. 5a–d). mCherry-positive cells are shown in magenta, and DiD fiber labeling is shown in gray. (MP4 2602 kb)
Formation of the IL in relation to the lateral recess
In order to better understand how the IL is formed around the LR, we performed a 3D reconstruction of the mCherry-positive cells in relation to the ventricular morphology (Fig. 6a–d, https://zenodo.org/record/2556246). ZO-1 immunostaining labels tight junctions of neuroepithelial cells [17, 18]; thus, it can be used to visualize the ventricular zones of the brain.
Teleosts possess two distinct hypothalamic recesses, LR and PR, which are already observable at 48 hpf [4, 7]. A vast extension of the LR is found at later stages of development, and in the 14 dpf brain, the LR elongates in a posterior direction close to the PR (Fig. 6c, https://zenodo.org/record/2556246).
3D visualization of mCherry-positive cells in relation to the ventricular zone clearly shows that the mCherry-positive cell cluster, which is continuous with the tectal region, covers the external part of the IL (Fig. 6a). In contrast, the ventricular zone of the LR is devoid of mCherry-positive cells (Fig. 6d, https://zenodo.org/record/2556246). In situ hybridization for ccna2, a cell proliferation marker, demonstrates that ventricular cells around the LR are in proliferation, whereas there is no proliferating cell in the external zone where mCherry-positive cells are found (Fig. 7a, a’). A closer look at the IL shows that the LR ventricular zone and the mCherry-positive external zone are separated by a cell-free fiber-rich zone (Fig. 7a’, b’, c; asterisks). Thus, IL is constituted of two anatomically distinct areas: the mCherry-negative ventricular zone is likely to be formed by the cells originating from the LR wall, while the mCherry-positive external zone is formed by the progeny of cells originating from the MHB.
Comparison between 14 dpf and adult IL (after induction at 24 hpf) shows that the relative size of the mCherry-positive external zone is significantly increased in the adult IL. At 14 dpf, there is only a thin layer of mCherry-positive cells (Fig. 7b’). In the adult (Fig. 7c), the mCherry-positive area is enlarged, forming a thicker mass laterally (which corresponds to the DIL). It is also worth noticing that the relative size of the whole IL is larger in adult and that the increase of the number of mCherry-positive cells largely contributes to the growth of IL mainly through its external portion.
Later maturation of IL
The her5 expression domain decreases in size during development but remains specific to the midbrain (Fig. 8a–c, Additional file 6: Movie S4 for 2 wpf; Fig. 8d, e, Additional file 7: Figure S3C for 4 wpf; Fig. 8f, g, Additional file 7: Figure S3F for 6 wpf; and Fig. 8h, i for 8 wpf). In the juvenile brain, her5 is expressed only in two cell clusters along the tectal ventricular zone: one more anteriorly (Fig. 8; yellow arrowheads) and another more posteriorly (Fig. 8; blue arrowheads), which are considered to be the alar part of the mesencephalon. A short-term tracing experiment showed that a few days after the tamoxifen induction, a few induced mCherry cells were observed at the two mesencephalic locations, but not in the forebrain (Additional file 8: Figure S4).
Movie S4. 14 dpf brain of Tg(her5:mCherry) zebrafish, with a focus on the cells expressing her5 endogenously (3D visualization of Fig. 8a–c). For better visualization, the brain is truncated anteriorly in the middle of the optic tectum (TeO) and posteriorly in the cerebellum (Cb). The section includes about half of IL ventrally. mCherry-positive cells are shown in green, and DiD fiber labeling is shown in magenta. The movie starts in frontal view. When switching to lateral/dorsal views, anterior is on the left, posterior is on the right. (MP4 2500 kb)
Observation of their progeny at 3 months post-fertilization (mpf) confirms that they contribute to the formation of IL and that IL continues to grow until late juvenile stages. The her5 progenies are restricted to the outer zone close to the surface of the IL (Fig. 8j–o). This suggests that cells are inserted at the periphery of the IL, all along development. Comparison of 3 mpf brains induced at 4 wpf (Fig. 8j, k), 6 wpf (Fig. 8l, m), and 8 wpf (Fig. 8n, o) clearly shows that induction at later stages results in less mCherry labeling. Thus, the growth of IL is slowing down over time, and IL is nearly mature around 8 wpf.
Comparison between zebrafish and cichlid IL
The IL is a brain structure observed in all teleost species investigated so far. We compared the general organization of the IL of zebrafish with another teleost species, a Malawian cichlid (Maylandia zebra). As it is the case for the zebrafish, cichlid IL is also divided into a cell-dense ventricular zone along the LR and a cell-sparse external zone, which are separated by a cell-free fiber zone (Fig. 9; asterisks).
Nonetheless, there are significant differences between zebrafish and cichlid IL. The relative size of the IL is much larger in cichlid. This is obvious by comparing the IL (the size indicated in green arrows in Fig. 9a, d) with the caudal zone of the hypothalamus (Hy; the size indicated in red arrows in Fig. 9a, d) that is located medially. It is also clear that the proportion of the external zone (mesencephalic part; the size indicated in green arrows in Fig. 9b, e) in comparison with the ventricular zone (hypothalamic part; the size indicated in red arrows in Fig. 9b, e) is much larger in the cichlid IL.
Thus, the organization of the IL changes significantly between young and mature zebrafish brains and also between different species of teleosts. Note that in the cichlid brain, the relative size of the pallium (dorsal telencephalon) is also much larger than in the zebrafish brain (compare Fig. 9c, f, also see the “Discussion” section).