The CNGa-expressing clock neurons that have been functionally characterized rep-resent a subset of the cry1 and hiomt expression domains. As shown in fig.6.6D and fig.6.8D,F, these genes form nested expression domains, where hiomt>cry1>CNGa.
Moreover, only a subset of the CNGa expressing cells is constituted by cells ex-pressing c-ops1 (like the “canonical” cPRCs and the asymmetric serotoninergic cell, compare fig.6.4). This implies that the clock region includes several cell types, with different specializations.
The availability of PrImR for the 48hpf stage (Tomer et al., 2010) allows the dissec-tion of these individual cell types, based on averaged expression patterns (par. 1.2.2).
In order to compare the results of my analysis with data across species, I enriched the lab dataset with genes involved in circadian processes (cfr. table 6.1) or transcrip-tion factors expressed in the vertebrate pineal-SCN system (table 7.1 and appendix D). This, together with similar efforts ongoing in the lab, provided a dataset of 180 genes for the analysis; of these, 49 were differentially expressed in the hiomt+ region.
To identify individual cell types, I have taken advantage of the presence of many
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Table 7.1: Transcription factors of the vertebrate pineal, retina and SCN/anterior hypothalamus anal-ysed in Platynereis.
vertebrate gene ID Platynereis ortholog expression in vertebrates brn1, brn2 and brn4 brn1/2/4 habenula, hypothalamus
brn3 brn3 specific marker of habenula and retinal ganglion cells
bsx bsx pineal, arcuate nucleus of the hypothalamus
crx/otx5 otx pineal and retinal PRCs
er81/etv1 er81/erm/pea3 specific marker of habenula
eya genes eya mouse pineal (not clearly described in other species)
foxD3 foxD specific marker of zebrafish pineal
gfi-1 sens specific marker of parapineal projection neurons and retinal gan-glion cells
islet genes islet pineal projection neurons and retina
lhx1 lhx1/5 specific marker of SCN
lhx2 lhx2/9 pineal, habenula and SCN
lhx3 lhx3/7 zebrafish pineal projection neurons
neuroD neuroD general PRCs marker, expressed also in the habenula, but not in SCN
not/flh not pineal and parapineal, both PRCs and projection neurons nr2e1/tll tll nr2e1:broad in nervous system, including hypothalamus and eye
field
pax4, pax6 pax6 pineal, not clear if it is also expressed in the habenula
rfx4 rfx4/6 specific marker of SCN
rx genes rx hypothalamic precursors, differentiated pineal and retinal PRCs tbx2, tbx3 tbx2/3 pineal and parapineal, eye field, hypothalamus
Expression of transcription factors in the vertebrate structures involved directly or indirectly in circa-dian entrainment (additional expression domains are not indicated). An extended version of this table can be found in Appendix D. Abbreviations: PRC=photoreceptor, SCN=suprachiasmatic nucleus of the hypothalamus.
genes with a small expression domain, and I used those to subdivide the hiomt expression domain in 15 “non-overlapping minimal synexpression groups”. Here, these synexpression groups are defined “cell types”: in many cases, this definition matches reality since very often I found genes demarcating single cells; otherwise, it refers to groups of adjacent cells that cannot be distinguished further morpholog-ically and molecularly, using the current dataset. The entire set of these minimal synexpression groups is represented in fig. 8.3.
Some of these cell types became of immediate interest because of the expression of key transcription factors, known in vertebrates for being involved in the develop-ment of the pineal organ and the SCN.
As shown in Chapter 5, the development of the deep brain ciliary photoreceptors
Rx. However, some other “famous” eye genes, present in both the pineal and the retina, like otx and pax6, are not expressed in these cells at differentiation stages.
Therefore, I investigated the expression of transcription factors that are specifically expressed in the pineal but not in the retina. One of the most specific pineal markers in the developing vertebrate brain is not; however, this transcription factor does not have any role in determining cell fates, but it is required to sustain the proliferation of pineal progenitors and to induce the expression of proneural genes (Masai et al., 1997; Cau, 2003). In Platynereis, none of the c-ops1+ cells of the dorsal brain expresses not, but a small group of not+ cells develops adjacent to the cPRCs.
A second transcription factor, tbx2b, has been shown to be critical for pineal and parapineal development in zebrafish (Snelson et al., 2008b). In contrast to not, this gene has a function in the specification of the photoreceptor fate (Alvarez-Delfin et al., 2009). Consistently, in Platynereis tbx2/3 is expressed in the asymmetric sero-toninergic cell and in the lineage of the cPRCs (“blue lineage” in par. 3.4).
The transcription factor bsx is also known to play a critical role in the final dif-ferentiation of pineal photoreceptors (D’Autilia et al., 2010), but is never expressed in the retina. The lineage analysis (par. 3.4) shows that bsx expression is very dy-namic during development, and one of its early expression sites is the lineage of the asymmetric serotoninergic cell. After differentiation, this cell expresses senseless (sens), a specific marker of retinal ganglion cells and parapineal projection neurons (Dufourcq et al., 2004; Wallis, 2003; Del Bene et al., 2007). In the dorsal brain, sens is one of the few transcription factors showing asymmetric expression (Appendix B).
Sens is a well known specifier of Drosophila R8 rhabdomeric photoreceptors (Pepple et al., 2008).
Thus, the asymmetric serotoninergic cell has some properties of both photorecep-tors (c-ops1, cry1, bsx, tph, hiomt, tbx2/3) and projection neurons (sens) of the retina
Figure 7.1 (on the next page): Subdivision of the hiomt expression domain in minimal synexpression groups.The panel shows the minimal synexpression groups identified for molecular fingerprint anal-ysis. The images are PrImR averages plotted on the average axonal scaffold. None of the minimal synexpression group corresponds to the complete expression pattern of one gene; see Methods, par.
??for details. The color code and the letters correspond to fig. 7.2. A. Dorsal bsx cells. B. Lateral DLamide cells. C. Asymmetric 5HT cell. D. cPRCs. E. Lateral not cells. F. Lateral otp cells. G. er81-vtn-c-ops1 cells. H. lhx3/7-sox2 cells. I. Lateral 5HT cells. J. dach deep cells. K. lhx1/5 cells. L. six4 cells. M. pdf cells. N. deep rfx4/6 cells. O. cpa-otp cells. P. Adult eyes pigment cells (AE-PCs). Q.
Adult eyes photoreceptor cells (AE-PRCs).
A B C
D E F
G H I
J K L
M N O
P Q
time it sends a projection to the brain serotoninergic plexus, located underneath the apical organ. In comparison, the cPRCs do not express neither sens (they do not have long axons) nor bsx and tph. The different expression profiles of these two types of c-ops1+ is correlated to their different clonal origin, and might be also correlated to differences in their functions.
The “not cells” (fig. 7.1E), which are the only cells of the 48hpf brain express-ing not, are adjacent to the ciliary photoreceptors and located just above the lateral serotoninergic cells (see also Appendix B). At 48hpf, there are 3 not-expressing cells on each side, and their number increases at later stages. These cells are demarcated also by the expression of foxD (compare also Appendix B), the Platynereis ortholog of the foxD3 gene, another very specific marker of the fish pineal cells1 (Gilmour et al., 2002). The “not cells” express also a third transcription factor, very restricted in both Platynereis and vertebrates: rfx4/6. However, in vertebrates this gene does not demarcate the pineal but the suprachiasmatic nucleus of the hypothalamus. The coexpression of these specific markers in one cell type of Platynereis brain makes it very hard to infer a clear relationship with the cell types of vertebrates.
At 48hpf the gene bsx demarcates a specific group of differentiated cells in the CNGa domain, here called dorsal bsx cells (fig. 7.1A). In Platynereis bsx is not as specific as the other markers described above, since at 48hpf bsx expression includes the whole apical region of the episphere, and two expression domains between the larval and the adult eyes. However, the dorsal bsx cells are the only ones coexpress-ing CNGa and cry1. Like the not cells, these cells express perops but not c-ops1.
The molecular fingerprint of the lateral serotoninergic cells is also interesting for evolutionary comparisons. These cells lie immediately adjacent to the ciliary photoreceptors, and are located in a deep position. Considering that they lack apical specializations, they are probably interneurons; consistently, they do not express any opsin. The lateral serotoninergic cells are the only cells in the hiomt domain to express rx, otx and pax6 at differentiation stages; the same genes are present already in their progenitors during development (par. 3.4). There are few cell types in the brain that coexpress these three transcription factors. Rx is necessary for pax6
1It needs to be said that the foxD expression is less interesting than other genes, like not, since the Platynereis foxD gene is the ortholog of all the vertebrate foxD genes, including foxD1, which is expressed in the hypothalamus. This is a clear example where the absence of a 1:1 correspondence of ortholog genes makes the molecular fingerprint comparison more difficult.
expression in these cells, and might be able to induce or maintain otx expression (Chapter 5). Obviously, the presence of rx, otx and pax6 is reminiscent of the retina and the pineal, but this is certainly not enough to infer homology of cell types.
However, exactly for this reason it will be very interesting in the future to find out what is the role of these cells in the brain, and how they better compare with cell types in other organisms.
Finally, many hiomt expressing cells are present in a more ventral position, under-neath the so called “crescent cell” of the apical organ. Many of these cells are sensory and release neuropeptides, like DLamide (Conzelmann et al., 2011), and project di-rectly to the prototroch. Among these, there are also cells expressing c-ops1 but not the CNGa channel, which have been described previously (Tessmar-Raible et al., 2007). Additional cells expressing hiomt at lower levels “wrap” around the apical or-gan; however, since these cells do not express CNGa, they have not been investigated in big detail.
7.2. Hierarchical clustering identifies two major subgroups of hiomt+ cells