The immunostainings with anti-acetylated tubulin antibody at 48hpf showed ad-ditional defects in brain morphogenesis. The most significant phenotypes could be identified in the dorsal brain, where the asymmetric axon was either not connected properly to the prototroch nerve ring, or was “duplicated” (fig. 5.1). Thus, to reveal additional effects of rx on brain development, I investigated the expression of genes preferentially expressed in the dorsal episphere.
One of the genes chosen is the transcription factor tbx2/3; this gene has a peculiar expression domain, since it appears in the apical organ and in the most dorsal cell populations, including the midline, at about 15hpf, where it partially overlaps with
A B
C D
24hpf
stctrlMO stctrlMO
rxin1ex2MO rxin1ex2MO
24hpf
24hpf 24hpf
tbx2/3 tbx2/3
tbx2/3 tbx2/3
Figure 5.7: Knockdown of rx affects the expression of tbx2/3. A. tbx2/3 expression of a 24hpf larva injected with stctrl MO, apical view. B. tbx2/3 expression of a 24hpf larva injected with stctrl MO, ventral view of the larva in A. C. tbx2/3 expression of a 24hpf larva injected with rxin1ex2 MO, apical view. The dorsal expression of tbx2/3 is severely reduced. The apical organ expression is not affected.
Moreover, tbx2/3 expression is expanded in the lateral brain. D. tbx2/3 expression of a 24hpf larva injected with rxin1ex2 MO, ventral view of the larva in C. The ventral expression of tbx2/3 is mostly the same as in the control larva, with the exception of the expression in the stomodeum.
rx expression (cfr. par.3.4). Interestingly, in rxin1ex2 MO injected larvae at 24hpf the expression of tbx2/3 was reduced in the dorsal brain, but not in the apical organ, while the expression was expanded in lateral-ventral domains (fig. 5.7A,C). The specificity of this phenotype was evaluated using the trunk expression of tbx2/3 as an internal control: in this way, it was possible to estimate the number of misde-velopers or severe retardation events, and exclude these embryos from the analysis (fig. 5.7B, D). Moreover, showing that only a fraction of the injected larvae had no specific phenotypes, this estimate gave confidence on other phenotypes described previously, where the genes analyzed (like c-ops1) are restricted to the brain or even to few cells of the brain.
The tbx2/3 phenotypes were characterized by a general decrease of the total cell number in the brain. This raised the question whether Rx activity in the brain during early development is required to keep the proliferative state of neural progenitors.
and otx expressing cells after Rx gain of function. To test this hypothesis, I checked the expression of the proneural transcription factor neurogenin (ngn) at 24hpf. In con-trol larvae, ngn expression is enriched in the lateral dorsal episphere, where there is still sustained proliferation at 24hpf (as shown by live imaging and EdU experi-ments, cfr. Chapter 3). In rxin1ex2 MO injected larvae, the expression of ngn was generally disorganized, and severely reduced, especially in the lateral dorsal do-mains. This further suggests that Rx has a role in maintaining the proliferative state of neural progenitors, and it is consistent with its extremely dynamic expression pattern throughout development.
Table 5.3: Effects of Rx knockdown on the expression of brain markers.
exp. ID stage phenotype rxin1ex2 MO stctrl MO p-value
25.4, tbx2/3 ish 24hpf brain expr. affected 64.7%, n=11 0
normal tbx2/3 expr. 35.3%, n=6 100%, n=23 5.353e-06
misdevelopers n=13 0
54.18, tbx2/3 ish 24hpf brain expr. affected 28.6%, n=8 0
normal tbx2/3 expr. 71.4%, n=20 100%, n=22 0.006385
misdevelopers n=4 0
6.4, ngn ish 24hpf brain and trunk affected 25%, n=1 0 brain affected, trunk normal 75%, n=3 0
normal ngn expr. 0 100%, n=11 0.0007326
misdevelopers n=1 0
58.18, ngn ish 24hpf brain and trunk affected 32.4%, n=11 0
brain affected, trunk normal 52.9%, n=18 12.1%, n=4
normal ngn expr. 14.7%, n=5 87.9%, n=29 8.975e-10
misdevelopers n=2 0
The table shows the analysis of phenotypes in different biological replicas. The number in “exp. ID”
is a code for each injection session. The p-value is calculated after Fischer’s exact test. Misdevelopers are included for completeness, but not considered for the statistical test. ish=in situ hybridization.
The experiments discussed above are the first gene functional experiments ever attempted in Platynereis. These experiments could establish some roles of Rx in the development of ciliary photoreceptors and serotoninergic cells, cell types that belong to the larval circadian system. A similar role for Rx has been shown in vertebrates, where this transcription factor is essential for eye development, but also important for the correct development and functioning of the hypothalamus and the pineal.
However, further experiments in Platynereis are needed to understand more precisely
A
24hpfB C
stctrlMO ngn rxin1ex2MO ngn rxin1ex2MO ngn
24hpf 24hpf
Figure 5.8: Knockdown of rx reduces the expression of ngn. A. ngn expression in a 24hpf larva, injected with stctrl MO, apical view. B. and C. ngn expression in a 24hpf larvae, injected with rxin1ex2 MO, apical view. The pattern of ngn expression is disorganized and overall reduced compared to controls.
the developmental roles of Rx. Indeed, the use of morpholinos to knock down gene expression has very well known issues about specificity, which are aggravated by using them in a new model system, where functional studies were never performed so far. For this reason, this description of Rx function is very conservative and does not include (numerous) other phenotypes that were observed after WMISH on morphant larvae. The development of mutant lines with the ZFN technology will allow to achieve a more complete description of Rx loss of function phenotypes, and together will offer the opportunity to test Rx function with complementary techniques (like RNA-seq and ChIP-seq), which require larger numbers of larvae.