Combined loss of Neurog1 and Neurod4 function disrupts habenular neurogenesis.

Whole-mount in situ hybridization against cxcr4b (A-D) or brn3a (F-H) showing the epithalamus of wild type (A,E), neurog1^hi1059 mutant embryos (B,F), neurod4 morpholino injected embryos (C,G) or neurog1^hi1059;neurod4 morphant embryos (D,H) at 36 (A-D) or 48 (E-H) hpf. The expression of cxcr4b and brn3a appears unaffected in neurod4 morpholino injected embryos (C, n = 13/13 and G, n = 14/15) as compared with the expression in wild type (A, n = 9/9 and E, n = 9/9); cxcr4b and brn3a expression is also unaffected in neurog1^hi1059 mutant (B, n = 12/14 and F, n = 9/11) or only slightly decreased in few cases (respectively n = 2/14 and n = 2/11; data not shown). In contrast, the expression of both genes is either abrogated or strongly reduced in neurog1^hi1059;neurod4 morphants (D, n = 16/16 and H, n = 12/14). Embryos are viewed dorsally with anterior up.

Pax6a but not Pax6b are required for habenular neurogenesis.

Whole-mount in situ hybridization against cxcr4b (A-D) or brn3a (E-H) showing the epithalamus of wild type (A,E), pax6b^sa86 mutant embryos (B,F), pax6a morpholino injected embryos (C,G) or pax6b^sa8;pax6a morphant embryos (D,H) at 36 (A-D) or 48 (E-H) hpf. Whereas the expression of cxcr4b and brn3a appears unaffected in pax6b^sa86 mutant embryos (B, n = 6/6 and F, n = 5/5) compared to the expression in wild type controls (A and E, n = 5/5), expression of both genes is abrogated after injection of morpholinos against pax6a in either wild type (C, n = 4/6 and G, n = 6/8) or pax6b^sa8 embryos (D and H, n = 4/4). Embryos are viewed dorsally with anterior up.

Expression of pax6a is unaffected in neurog1^hi1059;neurod4 morphant embryos.

Confocal sections (A,B) or 10μm maximum projections (A’,B’) of the head of wild type (A; n = 6/6) or neurog1^hi1059;neurod4 Mo injected embryos (B; n = 10/10) at 36 hpf after a whole-mount in situ hybridization against pax6a (red) and an immunostaining against HuC/D protein (green); cell nuclei staining (in grey) makes visible brain structures (A,B). As previously described, the neuronal marker HuC/D is expressed in the epithalamus, both in epiphyseal projection neurons (ep) and in habenular neurons (Hb, white brackets) located on either sides of the epiphysis. The expression of HuC/D is abrogated or strongly reduced in the habenular domain of neurog1^hi1059;neurod4 morphant embryos, while it is still detected in the telencephalon (Tel), in the epiphysis and in neurons of the tectum (Tc) (B,B’). On the contrary, no change is seen in the expression of pax6a in the same region, suggesting that the expression of neurog1 and neurod4 does not regulate pax6a during habenular neurogenesis. Embryos are viewed dorsally with anterior up. The expression of HuC/D and pax6a in neurog1^hi1059 mutants (n = 5/5) and neurod4 Mo injected embryos (n = 11/11) was similar to that observed in the non-injected controls (data not shown).

Habenular neurogenesis requires hedgehog signalling.

Whole-mount in situ hybridization against cxcr4b at 36 hpf (A-B’) or brn3a at 48 hpf (C-D’) showing heads (A-D) or the epithalamus (A’-D’) of wild type (A,A’,C,C’) or smo^hi229 embryos (B,B’,D,D’). The expression of cxcr4b and brn3a is detected in the habenulae of all the wild type siblings (n = 21/21 and n = 6/6, respectively). While residual expression of cxcr4b in the eyes (*) and brn3a in the tectum (blue bracket) is visible in smo^hi229 embryos, the expression of both genes is abrogated in the habenular nuclei (Black bracket, B,B’, n = 12/13 and D,D’, n = 6/6). Embryos are viewed dorsally with anterior up.

Hedgehog signalling is required continuously from 16hpf for correct habenular development.

Whole-mount in situ hybridization against cxcr4b at 36 hpf (A-B’) or brn3a at 48 hpf (C-D’) showing heads (A-D) or the epithalamus (A’-D’) of wild type (A,A’,C,C’) or smo^hi229 embryos (B,B’,D,D’). The expression of cxcr4b and brn3a is detected in the habenulae of all the wild type siblings (n = 21/21 and n = 6/6, respectively). While residual expression of cxcr4b in the eyes (*) and brn3a in the tectum (blue bracket) is visible in smo^hi229 embryos, the expression of both genes is abrogated in the habenular nuclei (Black bracket, B,B’, n = 12/13 and D,D’, n = 6/6). Embryos are viewed dorsally with anterior up.

Hh signalling is required for the expression of pax6a, neurog1 and neurod4 in the Habenular nuclei.

Confocal sections (A-F) or 15μm maximum projections (A’-F’) showing the heads of control treated embryos (A-A’, n = 9; C-C’, n = 6; E-E’, n = 6) or those treated from 16 hpf with cyclopamine (B-B’, n = 10, D-D’, n = 7, F-F’, n = 6) after a whole-mount in situ hybridization against pax6a (A,A’,B,B’), neurog1 (C,C’,D,D’) and neurod4 (E,E’,F,F’) (red) and immunostaining against HuC/D protein (green); cell nuclei staining (grey) makes visible brain structures in the confocal sections (A-F). The overall morphology of the head appears normal in cyclopamine treated embryos and the expression of HuC/D does not appear to be affected in the telencephalon (Tel), in the epiphysis (*) nor in the tectum (Tc). In contrast, HuC/D expression is absent or strongly reduced in the habenular domain (Hb, white brackets) of cyclopamine treated embryos. The expression of pax6a is strongly reduced (B-B’, n = 10/10) in the habenular domain (Hb, white brackets) of cyclopamine treated embryos. The expression of neurog1 and neurod4 is also abrogated specifically in the habenular domain of cyclopamine treated embryos (D-D’, n = 7/7, F-F’, n = 6/6). All embryos are at 36 hpf. Embryos are viewed dorsally with anterior up.

Autonomous reception of Hedgehog signals is required for habenular neurogenesis.

Confocal sections through the epithalamus of wild type host embryos containing transplanted cells from wild type (A) or smo^hi229 donors (B). Donors carried the Tg(huC:GFP), which appears in white, and transplanted cells appear in red; red cells that are not white in B are epiphysial photoreceptors (white arrow). Host embryos were immunolabelled with an anti-HuC/D antibody (in blue) that highlights the habenular neurons (Hb) and projection neurons in the epiphysis (ep). Embryos are viewed dorsally with anterior up. A histogram indicates the number of embryos incorporating cells from wild type or smo^hi229 mutant donors into either the habenulae or epiphysis (C). Unlike cells transplanted from wild type donors (white arrowheads in A), those from homozygous smo^hi229 mutants are systematically excluded from becoming habenular neurons; transplanted cells from either donor can become epiphysial projection neurons.

Expression of pax6a and Tg(neurog1:GFP) co-localises in dorsal habenulae.

Confocal sections (A-C) of the head of a Tg(-8.4neurog1:GFP)^sb1 embryo at 36 hpf after whole-mount immunostaining against GFP (A, green), in situ hybridization against pax6a (B, red) and Immunostaining against HuC/D (D, magenta); cell nuclei staining (in blue) makes brain structures visible in A, B, D, and merges are shown in C (A+B) and E (A+D). The Tg(-8.4neurog1:GFP) transgene is expressed in the epithalamus, both in epiphyseal (ep) and habenular neurons (Hb, white brackets), as well as other brain structures such as the telencephalon (Tel) and the tectum (Tc). The expression of Tg(-8.4neurog1:GFP)^sb1 recapitulates endogenous neurog1 expression in habenular progenitors (described previously in [25]), although it can also be detected in newly-born HuC+ habenular neurons, probably due to persistence of the fluorescent reporter which acts as a short term lineage label (E). The expression of pax6a overlaps broadly with most of the Neurog1:GFP+ neurons in both the left and right habenulae (n = 15/15; C).

Sonic Hedgehog ligand (Shha) is required for habenular development.

Whole-mount in situ hybridization against cxcr4b at 36 hpf (A,A’,B,B’) or brn3a at 48 hpf (C,C’,D,D’) showing heads (A-D) or the epithalamus (A’-D’) of wild type (A,A’,C,C’) or shha^tbx392 embryos (B,B’,D,D’). While the expression of cxcr4b and brn3a is detected in the habenulae of all the wild type siblings (n = 8 and n = 14 respectively), the expression of both genes is strongly reduced in the epithalamus of shha^tbx392 mutant embryos (B,B’, n = 12/13 and D,D’, n = 6/6). Embryos are viewed dorsally with anterior up.

Hh signalling is required for the expression of pax6a, neurog1 and neurod4 in the epithalamus.

Whole-mount in situ hybridization against pax6a (A,B), neurog1 (C,D) and neurod4 (E,F) showing the head of control treated embryos (A, n = 10; C, n = 12; E, n = 8) or those treated from 16 hpf with cyclopamine (B, n = 10; D, n = 13; F, n = 9) in a lateral view of embryonic heads; all embryos are at 36 hpf and shown with anterior to the left. The expression of pax6a, neurog1 and neurod4 appears perturbed in the most dorsal part of the diencephalon subdivision (black brackets), while their expression does not appear significantly changed more ventrally or in other brain regions, such as the telencephalon (show as a *).

Hedgehog signalling is not required for cxcr4b expression in epiphysial projection neurons.

Whole-mount in situ hybridization against cxcr4b at 28 hpf showing heads (A,B; lateral view) or the epithalamus (A’,B’; dorsal view with anterior up) of wild type (A,A’) or smo^b641 embryos (B,B’). At 28 hpf, cxcr4b is expressed on both side of the epithalamic midline (*) in epiphysial projection neurons in both wild type siblings (n = 9/9) and smo^b641 mutant embryos (n = 8/8). At this stage, cxcr4b is either only expressed in few left habenular neurons or not expressed yet in the habenulae. This expression could be detected in some wild type siblings (A’, black brackets, n = 3/9) but never in the habenulae of smo^b641 mutant embryos (B’, n = 8/8).