Her6 regulates the neurogenetic gradient and neuronal identity in the thalamus

Steffen Scholppa,b,1, Alessio Delogua, Jonathan Gilthorpea,2, Daniela Peukerta,b, Simone Schindlerb, and Andrew Lumsdena

aMRC Centre for Developmental Neurobiology, New Hunt’s House, Guy’s Campus, King’s College London, London SE1 1UL, United Kingdom; and bInstitute of Toxicology and , Institute of Technology Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany

Communicated by Thomas M. Jessell, Columbia University College of Physicians and Surgeons, New York, NY, September 30, 2009 (received for review June 10, 2009) During vertebrate brain development, the onset of neuronal dif- expression of Ascl1 in the dorsal forebrain induces ectopic ferentiation is under strict temporal control. In the mammalian differentiation of GABAergic (15). thalamus and other brain regions, neurogenesis is regulated also Another subfamily of bHLH proteins, the hairy-related Hes/ in a spatially progressive manner referred to as a neurogenetic Her proteins, generally function as DNA-binding transcriptional gradient, the underlying mechanism of which is unknown. Here we repressors and antagonize proneural function (16, 17). describe the existence of a neurogenetic gradient in the zebrafish Hairy-related proteins form homodimers through the bHLH thalamus and show that the progression of neurogenesis is con- region and have a conserved WRPW domain at the carboxyl (C) trolled by dynamic expression of the bHLH repressor her6. Mem- terminus, which functions as a repression domain by recruiting bers of the Hes/Her family are known to regulate proneural , co-repressors of the Groucho family (18). Some of these hairy- such as Neurogenin and Ascl. Here we find that Her6 determines related proteins keep cells in a progenitor state, preventing not only the onset of neurogenesis but also the identity of thalamic initiation of the neurogenic program and thereby maintaining neurons, marked by proneural and neurotransmitter gene expres- local organizer populations at signaling boundaries. Such a role sion: loss of Her6 leads to premature Neurogenin1-mediated gen- for Hes/Her proteins has been described in relation to the

esis of glutamatergic (excitatory) neurons, whereas maintenance mid-diencephalic organizer (MDO) at the intrathalamic bound- BIOLOGY ary (zona limitans intrathalamica; ZLI) and isthmic organizer of Her6 leads to Ascl1-mediated production of GABAergic (inhib- DEVELOPMENTAL itory) neurons. Thus, the presence or absence of a single upstream (ISO) at the -hindbrain boundary (19–21). regulator of proneural gene expression, Her6, leads to the estab- Here we decipher the molecular mechanism leading to tem- lishment of discrete neuronal domains in the thalamus. porally controlled thalamic neurogenesis in , and un- cover a underlying mechanism leading to the correct acquisition of thalamic neuronal identity. We describe a function for the diencephalon ͉ Hes1 ͉ mash1 ͉ ngn1 ͉ zona limitans intrathalamica hairy-related gene her6, which is expressed in the entire pre- sumptive thalamic complex at early stages and is subsequently eurogenesis in the developing vertebrate CNS is regulated restricted to ascl1-positive neuronal progenitors within the PTh, Nwith a high degree of temporal and spatial precision, with the rTh and the MDO. The dynamic regression of her6 expres- stereotypic patterns of neuronal differentiation and extensive sion from the cTh is accompanied by the caudal-to-rostral neuronal migration (1, 2). Dynamic patterns of mitotically active progression of the neurog1 expression, making her6 a candidate neuronal precursors, known as ‘neurogenetic gradients’ (3) have regulator of the neurogenic gradient in the glutamatergic thal- been described in several brain regions, including the neocortex amus. We show that Her6 blocks neurog1-mediated neurogenesis (4, 5), the dorsal midbrain colliculi (6), and the dopaminergic cell-autonomously by interaction with the co-factor Groucho1. region of the ventral midbrain (7). In the mammalian dienceph- Furthermore, loss of Her6 leads to ectopic induction of neurog1 alon, in particular the thalamus (formerly known as dorsal in both the rTh and the PTh. Conversely, forced expression of thalamus), two main neurogenetic gradients have been de- Her6 in the cTh switches cells to a GABAergic fate. In an scribed: from posterior to anterior and from lateral to medial (8, epistatic analysis, we demonstrate genetic suppression of neurog1 9). In rodents, all thalamic neurons are generated in about 6 days, by Her6: double knock-down of both genes rescues the single and the orthogonal gradients of glutamatergic neurogenesis knock-down of Her6, such as the maintenance of the sweep across the boundaries of future nuclei. The underlying MDO and the GABAergic population of the rTh. molecular mechanisms responsible for generating the neuroge- In summary, we propose that Her6 determines both the spatial netic gradients of the thalamus are unknown. progression of neurogenesis through the thalamic territory and The major constituent of the thalamus is a population of the decision to become a glutamatergic relay or a excitatory neurons generated in the caudal thalamus (cTh), GABAergic interneuron in this region. whereas a minor population of inhibitory neurons is generated Results in the rostral thalamus (rTh). The latter is thought to give rise to the reticular nucleus and the ventral lateral geniculate nu- The Neurogenetic Gradient in the Thalamus. The term ‘neurogenetic cleus, including the intergeniculate leaflet (10). During devel- gradient’ was coined in the 1970s to describe the process of opment, this rostro-caudal partitioning is seen in the expression domains of proneural basic helix-loop-helix (bHLH) genes: the Author contributions: S. Scholpp, A.D., J.G., and A.L. designed research; S. Scholpp, D.P., achaete-scute-like complex genes (Ascl formerly known as Mash and S. Schindler performed research; S. Scholpp contributed new reagents/analytic tools; S. in mouse and Zash in zebrafish) mark the GABAergic rTh and Scholpp, and A.L. analyzed data; and S. Scholpp and A.L. wrote the paper. the prethalamus (PTh) and the Neurogenin genes (Neurog, The authors declare no conflict of interest. formerly known as Ngn) mark the glutamatergic cTh (2, 11, 12). Freely available online through the PNAS open access option. Several lines of evidence show that these genes function as 1To whom correspondence should be addressed. E-mail: [email protected]. determinants of transmitter phenotype: in the mouse telenceph- 2Present address: Umeå Center for Molecular Medicine, Umeå University, 901 87 Umeå, alon, Neurog1/2 are required to specify the glutamatergic char- Sweden. acter of cortical neurons, while simultaneously repressing an This article contains supporting information online at www.pnas.org/cgi/content/full/ alternative subcortical GABAergic fate (13, 14), whereas forced 0910894106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910894106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 3, 2021 and neuronal gene expression spreading, with time, from pos- terior to anterior. A BC Our observations suggest the presence of a mechanism that regulates both temporal and spatial aspects of thalamic neurogen- esis in vertebrates. Since members of the Hairy/Enhancer of Split (HES) family have been shown to be important regulators of neurogenesis in a number of contexts (17), we focused our attention on this family of bHLH transcription factors. We found that the D E F hairy-related factor her6, a close relative of mammalian HES1, was also dynamically expressed in the developing diencephalon (25). Initially, her6 is broadly expressed in the presumptive telencephalon and mid-diencephalon at the open neural plate stage (Fig. S2 A–C). By the early somitogenesis stage, her6 becomes refined toward the mid-diencephalon and marks the entire thalamic complex including G HI the anteriorly located PTh, the MDO, and the posteriorly located thalamus (Fig. 1B). Interestingly, her6 expression was found to be reciprocal to that of neurog1.At33hpf,her6 expression abuts the expanding expression domain of neurog1 precisely in the cTh. The second major neuronal population in the diencephalon consists of the GABAergic interneurons in the PTh and the rTh and their Fig. 1. The neurogenetic gradient in fish. Glutamatergic neurogenesis precursors, which are marked by the expression of achaete-scute spreads in a wave from posterior to anterior in the developing thalamus. Analysis of the dynamic expression of proneural genes during the develop- complex genes. Therefore, we studied the expression of ascl1a relative to her6 in the developing Th. At the 20-somite stage, ment of the thalamic complex by in vivo imaging of double transgenic ϩ zebrafish and double in situ hybridisations. Upon induction of shh:GFP in the expression of ascl1a is induced within the her6 PTh and at 24 hpf ϩ MDO, neurog1:RFP is induced first in the posterior Th (A, arrowheads). Over ascl1a is further found in the her6 rTh (Fig. 1 C and F). At the 33 time, the caudal thalamus is filled with neurog1 positive cells (D and G). At the hpf, overlapping expression domains of her6 and ascl1a are main- 20-somite stage, neurog1 mRNA can be detected within the thalamic complex tained in the PTh and the rTh (Fig. 1I), whereas neurog1 marks the (B). Over time, the expression increases from posterior to anterior and fills the her6Ϫ cTh (Fig. 1H). entire cTh at 33 hpf (E and H). At 20 somite stage, her6 expression marks the entire thalamic complex (B and C) and is gradually down-regulated in the Knock-Down of Her6 Leads To an Increase of neurog1 Expression. The caudal part over time. her6 expression is maintained in the PTh, MDO, rTh and absent from the cTh at 33 hpf (H and I). The expression domains of her6 and expression of her6 is consistent with its regulating progression of neurog1 are abutting. In contrast to neurog1, the proneural gene ascl1a is the glutamatergic neurogenetic wave by upstream repression of induced from ventral to dorsal within the her6 positive PTh and rTh from the neurog1. To test whether this is the case we used to 20-somite stage (C), to 24 hpf (F) and 36 hpf (I). Embryos are shown laterally, create an antisense knock-down in vivo (26). In her6 morphant white double-headed arrows indicate the increasing width of the cTh. III, third embryos, we found induction of neurog1 in the entire thalamic brain ventricle; cTh, caudal Thalamus; MDO, mid-diencephalic organizer; PTh, complex including the PTh, MDO and thalamus (Fig. 2 A and B). prethalamus; rTh, rostral Thalamus; ss, somite stage. Notably, the timing of neurog1 induction at 24 hpf seems unchanged, suggesting that the inductive signal for neurog1 is unaffected. At 33 hpf in her6 morphant embryos, the PTh, the dynamic neurogenesis in the developing mammalian thalamus. MDO and the entire thalamus becomes neurog1 positive and shh To see whether this is a feature of vertebrates in general, we expression in the MDO is down-regulated (Fig. 2 C and D). analyzed the expression dynamics of genes marking different These results indicate that Her6 regulates the progression of the stages of neuronal development in the embryonic zebrafish neurogenetic gradient by acting as a brake to suppress premature diencephalon: the proneural gene neurogenin1 (neurog1), the or ectopic glutamatergic neurogenesis in the thalamus. In a neuronal precursor marker deltaA, the postmitotic marker of second set of experiments, we analyzed the phenotype of the glutamatergic neurons, the vesicular glutamate transporter 2.1. GABAergic cells in the thalamus of her6 morphants. We ana- To visualize the dynamics of proneural gene expression in the lyzed the expression of markers of the PTh such as the Ascl1 thalamus in vivo, we used a transgenic strategy by generating downstream factor dlx4 (27) by using the dlx4/6:GFP transgenic embryos that express a nuclear-localized form of the red fluo- line (28). For the rTh marker we used the tal1:GFP line (29). tal1 rescent protein (RFP) under the control of the neurog1 promoter (formerly known as scl) is essential for the development of the (22) and green fluorescent protein (GFP) under the control of interneuron population originating from the rTh such as retic- the shh promoter (23). The proneural gene neurog1 has been ular nucleus, and the ventral lateral geniculate nucleus, including shown to mark glutamatergic precursors (15), and shh deter- the intergeniculate leaflet (30). We find that both expression mines the anterior and ventral limit of the thalamus (Fig. 1 and domains – dlx:GFP and tal1:GFP - are strongly reduced in the Movie S1). In the diencephalon at 24 h post-fertilization (hpf), diencephalon (Fig. 2 E–H), whereas vasculature development shh:GFP expression was localized to the MDO and the basal such as of the tal1ϩ anterior cerebral vein (31) is unaltered (Fig. plate (Fig. 1A). Between 24 hpf and 33 hpf, we observed an 2 G and H). As both domains are normally ascl1aϩ (Fig. S1I), we ϩ increase in the number of neurog1:RFP cells in a high-posterior asked whether this determinant of GABAergic fate is also to low-anterior gradient (Fig. 1 D and G). Over the same period, affected. Indeed, we find that ascl1a is strongly down-regulated we also found that the expression of shh and neurog1 is co- in the morphant embryos (Fig. 4 A and B), suggesting that the localized in the ventral cells that have been suggested to partially loss of dlx4/6:GFP and tal1:GFP expression is a consequence of form basal forebrain dopaminergic neurons in fish (24). In the down-regulation of ascl1a. In support of this conclusion, we addition, the progressive activation of neurog1:RFP and neurog1 find that a marker for terminal GABAergic neurons, glutamic mRNA are accompanied by the expression of dla mRNA, which acid decarboxylase 65/67 (GAD 65/67), is strongly down- marks cells undergoing active neurogenesis (Fig. S1 A, D, and G). regulated in her6 morphant embryos (Fig. 4 E and F). After 33 hpf, the region begins to express the differentiated It is possible that a global knock-down of Her6 could have an neuronal marker vglut2.1 (Fig. S1 B, E, and H). Thus, as in the early non-specific effect on thalamic patterning. To assess this we mammalian thalamus, we find a wave of proneural/neurogenic performed a temporally and spatially regulated knock-down

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II‘ JJ‘ Fig. 3. Maintenance of Her6 is required of fate of the rTh. Mis-expression of her6 represses neurog1 expression and activates rostral thalamic fate. Acti- vation of a heat-shock inducible construct driving her6 and the lineage tracer GFP leads to the cell-autonomous down-regulation of neurog1:RFP at 33 hpf (9/15; B and BЈ; arrowheads). If Her6 lacks the Groucho interaction motif WRPW, the construct does not alter neurog1:RFP expression in a control experiment and shows a co-localization of GFP and nuclear-localized RFP Fig. 2. Her6 is required to repress the proneural gene neurogenin1. In vivo (8/12; A and AЈ; arrowheads). Knock-down of grg1 resembles the her6 mor- analysis of the loss of Her6 function in double transgenic zebrafish embryos by phant phenotype (23/43; D and E) and leads to an increase in the caudal confocal microscopy. Knock-down of her6 (her6MO) leads to an increase of expression of neurog1:RFP (white arrowhead) as well as a decrease of shh:GFP neurog1 expression in the thalamic complex at 27 hpf (62/86; A and B). At 33 in the MDO (yellow arrowhead) at 30 hpf. Clonal overexpression of her6 and hpf, lack of Her6 function leads to a massive up-regulation of neurog1 in the the membrane-bound red lineage marker mCherry in the cTh leads to the entire thalamic complex including PTh and MDO (72/91; C and D). The Shh activation of the GABAergic rTh marker tal1:GFP (14/21; I; arrowhead). Nota- positive MDO is fragmented. Up-regulation of neurog1 leads to a down- BIOLOGY bly, dividing cells at the ventricular surface are only positive for the mCherry

regulation of markers, such as dlx4/6:GFP in the PTh at 36 hpf (53/78; E and F). DEVELOPMENTAL lineage marker (asterisk) but become tal1:GFP positive after radial migration Similarly, the expression of the rTh marker tal1:GFP is completely lacking in (arrowhead). Cells containing the lineage tracer only do not express Tal1 (H). her6 morphant embryos 42 hpf (23/28; G and H). By delivering the her6 MO III, third brain ventricle; cTh, caudal thalamus; PTec, pretectum; PTh, prethala- antisense oligomers into a few cells of the rTh by electroporation, neurog1 mus; MDO, mid-diencephalic organizer; rTh, rostral thalamus. expression was induced cell-autonomously, as shown by the expression of RFP tagged with a nuclear localisation sequence (5/11; J, higher magnification of the electroporated cell clone marked by arrowheads in JЈ). Electroporation of a control MO showed no effect (I and IЈ). III, third brain ventricle; ACeV, followed by an up-regulation of ascl1a expression at 30 hpf (Fig. S3 anterior cerebral vein; cTh, caudal thalamus; PTh, prethalamus; PTec, pretec- C–F). We then analyzed the fate of small groups of clonally related tum; rTh, rostral thalamus. cells maintaining Her6 in the cTh by injecting her6 mRNA in a single blastomere at the 32-cell stage. These cells express the rTh marker tal1:GFP ectopically in the cTh (Fig. 3 H and I). These experiment using an in vivo electroporation assay (32). Fluores- experiments suggest that Her6 is able to repress neurog1 expression cently tagged her6 Morpholinos were delivered into a small via a Groucho-dependent mechanism and that the down-regulation group of cells in the rTh of neurog1:RFP embryos at 24 hpf (Fig. of Her6 is required for the formation of the cTh. If Her6 is not 2 I–JЈ). Expression of neurog1:RFP was analyzed 12 h later. We down-regulated, cells acquire the identity of the rTh. found that neurog1:RFP was induced in a cell-autonomous manner in rostral thalamic cells that had taken up the antisense Genetic Suppression of neurog1 by her6. We then performed a her6 MO (Fig. 2 I and JЈ). knock-down experiment for her6 and neurog1. If Her6 acts solely via the repression of neurog1, it should be possible to rescue the Maintenance of Her6 Leads To the Acquisition of rTh Cell Fate. In a her6 morphant phenotype by the double knock-down of both further set of experiments, we investigated the Her6 maintenance her6 and neurog1. phenotype in the cTh. Therefore, we used an inducible vector her6 morphant embryos display ectopic expression of neurog1 consisting of heat-shock responsive elements that drive bidirec- throughout the thalamus, down-regulation of ascl1 in the rTh and tional expression of her6 and gfp (33) (Fig. 3 A–BЈ). Injection of the PTh, leading to an decrease in GABAergic neurons, and frag- plasmid DNA into the neurog1:RFP line at the one-cell stage leads mented shh expression in the MDO (Fig. 4 B, F, and J). In contrast, to mosaic distribution of the plasmid. Embryos were heat-shocked neurog1 morphants display posterior expansion of the ascl1a ex- at the 10-somite stage, before the endogenous down-regulation of pression domain resulting in an increase in the domain of GABAer- her6 expression in the thalamus. This experimental setting allowed gic neurons of the rTh (Fig. 4 C and G), while shh expression at the us to analyze the phenotype resulting from maintained Her6 MDO appears normal (Fig. 4K). In her6/neurog1 double morphant expression with high temporal precision. Although located in the embryos, both the ascl1a expression domains in the PTh and in the normally neurog1ϩ cTh, cells that ectopically expressed her6 were rTh and the corresponding domains of GABAergic neurons, as well unable to activate neurog1:RFP expression (Fig. 3 B and BЈ). In a as the shh expression in the MDO were rescued (Fig. 4 D, H, and control setting, we found that cells expressing a her6 construct that L). Furthermore, the rTh showed an expansion of ascl1a expression lacks the interaction domain for the Groucho co-repressor co- comparable to the neurog1 morphant embryos, suggesting genetic expressed neurog1:RFP (26) (Fig. 3 A and AЈ). To assess the suppression of neurog1 by her6/neurog1. To further test our hypoth- requirement for Groucho in more detail, we performed a MO- esis for this epistatic relationship, we performed an over-expression mediated knock-down experiment for Groucho1. This led to the analysis of neurog1. Forced expression of neurog1 mRNA led to a ectopic induction of neurog1:RFP as well as to down-regulation of down-regulation of ascl1a and gad1 expression in the fore- and shh:GFP at the MDO, a similar phenotype to that of her6 morphant midbrain area (Fig. S4 A–D), consistent with the observation in embryos (Fig. 3 D and E). We then injected embryos with 50 pg her6 mouse (13, 14) and a cell autonomous down-regulation of shh in the mRNA. This led to an down-regulation of neurog1 at 24 hpf MDO (Fig. S4 E and F) and of tal1 in the rTh (Fig. S4 G and H).

Scholpp et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 3, 2021 cell fate and (iii) it maintains the mid-diencephalic organizer as a non-neurogenic signaling boundary (Fig. S5). A BCD Hes/her Genes and the Regulation of Neurogenesis. In the develop- ing CNS, Hes or Hes-related (Her) proteins have been shown to be involved in the regulation of neuronal differentiation. In Hes1 and Hes5 knockout mice, neural stem cells cannot be maintained EFGH and neurons differentiate prematurely (34, 35), whereas over- expression of Hes1 prevents neuronal differentiation in the brain (36). Furthermore, in the absence of Hes1 and its related genes, Hes3 and Hes5, proneural bHLH genes are ectopically expressed in boundaries, resulting in ectopic neurogenesis and disruption IJKL of their local organizing properties; for example, the MDO is disrupted in Hes1 mutant mice (21). These data suggest that Hes genes are critical for the proper spatial control of neuronal differentiation and for the maintenance of local organizing boundaries in the CNS. Following analysis of the Hes1/3/5 triple knockout mouse, a model was suggested in which the Hes genes Fig. 4. Her6 acts to genetically suppress neurog1. Analysis of ascl1a and irx1b, a pan-thalamic marker, at 30 hpf (A–D) and vglut2.1 and GAD65/67 at repress all proneural bHLH factors, including Neurog, Ascl1 or 48 hpf (E–H)inher6 and neurog1 morphant embryos. Knock-down of her6 Atonal (21). Similarly in the zebrafish thalamus, we show that leads to the down-regulation of ascl1a (45/61; A and B) as well as GAD65/67 neurog1 and coe2 (a member of the Collier/Olf1/EBF family of (22/35; E and F) in the rTh (arrowheads) and in the PTh. Knock-down of transcription factors and the ortholog of mouse Ebf2) are neurog1 leads to an unaltered PTh but an increase in width in ascl1a expres- repressed by Her6, the orthologue of mouse Hes1, consistent sion (22/34; C) as well as GAD65/67 expression (12/21; G) in the rTh (yellow with the presence of a bHLH binding motif (E-box) in the arrows). Compared to the her6 single knock-down, double knock-down of neurog1 promoter (20, 37). In contrast to mouse, however, we her6 and neurog1 rescues the prethalamic as well as the rostral thalamic observe no repression of ascl1a by Her6. Rather, we find that the expression domain (ascl1a: 17/22; GAD: 8/17; D and H). Analysis of the MDO in mRNAs of ascl1a and her6 are co-expressed, a phenomenon also shh:GFP transgenic animals co-expressing ubiquitously the membrane-bound fluorophore mCherry (I–L): Down-regulation of her6 leads to disintegration of seen in the figures of (21) but not discussed therein. the Shh positive MDO (20/26; I and J; arrowheads). In embryos knocked-down As well as disruption of the thalamic complex, the midbrain- for neurog1, the MDO seems unaltered in extension and width compared to hindbrain boundary (MHB) area including the ISO is missing in the control (8/10; K, arrowhead). Double knock-down of her6 and neurog1 Hes1 mutant mouse embryos. From this perspective, Hes1 seems rescues the extend of the shh:GFP expression within the MDO (6/8; L, arrow- to be functionally equivalent to another bHLH factor, her5, in head). III, third ventricle; cTh, caudal thalamus; MDO, mid-diencephalic orga- zebrafish (19, 20). her5 morphant embryos show premature nizer; PTh, prethalamus; rTh, rostral thalamus. up-regulation of the proneural genes neurog1 and coe2 at the ISO and, as with her6, her5 does not repress either ascl1a nor ascl1b (20). No orthologue of her5 has yet been identified in mammals, These outcomes mimic the her6 morphant phenotype. Thus, the suggesting that Hes1 has adopted the functions of both her6 at the main function of Her6 is to repress neurog1 and Her6 is necessary MDO/Th and her5 at the ISO in zebrafish. but not sufficient to induce a subsequent GABAergic fate. The direct upstream signal regulating the activity of Her6 is Discussion unknown. Transcription of her6 seems to be dependent on Hdac1 (38) but independent of Notch signaling (39). However, recent data We have explored the function of the bHLH from the mouse retina suggest that Shh is able to stabilize Hes1 Her6 during development of the zebrafish thalamic complex. We protein (40), suggesting one possible way in which the progressive find that her6, the expression of which is initially widespread, retreat of Hes/her genes from the cTh could be controlled. represses the expression of neurog1 cell-autonomously through- out the thalamus. The subsequent posterior-to-anterior regres- Proneural Genes in the Thalamic Complex. Following the ectopic sion of her6 expression is accompanied by up-regulation of activation of her6 we saw first the down-regulation of neurog1, and neurog1 in cells that formerly expressed her6. The progression of subsequently the up-regulation of ascl1a. Thus, we suggest that this gene expression transition presents as a neurogenetic gra- Her6 is required to repress neurog1 expression and does not dient, a phenomenon that was observed, but not explained, in the ϩ regulate ascl1a in the thalamus. This is consistent with the finding mammalian thalamus. In addition, we find that the ascl1 that Neurog1 is able to repress the transcription of ascl1, whereas domains in the thalamic complex, the PTh and the rTh, require Ascl1 is able to direct neuronal precursors toward a GABAergic the maintenance of her6 expression (and her6-mediated repres- fate (15). Interestingly, both genes are induced by the same signal, sion of neurog1) for proper GABAergic specification. In ze- Shh (41–43) (Fig. S6). In the thalamus, concentration dependent brafish, two orthologues of the mammalian ascl1 gene exist, Shh activity has been suggested to be involved in regionalization of ascl1a and ascl1b, which show a similar expression pattern in the thalamic nuclei: high Shh concentration defines rTh fate proximal thalamic complex, suggesting a similar function (Fig. S2I). In to the source and lower concentration determines cTh fate distal to parallel, her6 is required to maintain the MDO in a non- the source (44, 45). However, one single gradient cannot define a neurogenic state to fulfil its function as a signaling boundary. sharp border between abutting expression domains (46) such as the Lack of Her6 leads to ectopic de-repression of neurog1 in the rTh and the cTh. Therefore, we propose that a different mechanism PTh and the rTh, the repression of ascl1a, and the ectopic to assign correct neuronal fate - before the induction of proneural acquisition of glutamatergic fate. Additionally, the MDO starts genes by Shh and the mutual repressive interaction of neurog1 and to ectopically express neurog1, leading to the down-regulation of ascl1: Her6 perform this function by acting as a neurog1 repressor. shh expression at the organizer. In contrast, cells forced to When her6 function is abrogated, neurog1 expression is induced express her6 ectopically in the cTh adopt a GABAergic fate. prematurely in normally glutamatergic domains of the thalamus, Thus, we propose a triple role for Her6 during thalamic devel- and is induced ectopically in normally GABAergic domains, bring- opment: (i) Her6 determines the spatial activation pattern of ing about a switch of fate. Spatial knock-down of Her6 by electro- neurog1,(ii) it is required for the development of GABAergic poration in an area exposed to high Shh concentration leads to the

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910894106 Scholpp et al. Downloaded by guest on October 3, 2021 induction of Neurog1, supporting the hypothesis that Shh acts as Methods an concentration-independent but general upstream inducer of Maintenance of Fish. Breeding zebrafish (Danio rerio) were maintained at proneural genes expression. Furthermore, rescue of the her6 knock- 28 °C on a 14-h light/10-h dark cycle (55, 56). To prevent pigment formation, down phenotype by simultaneous knock-down of neurog1, suggest- some embryos were raised in 0.2 mM 1-phenyl-2-thiourea (PTU, Sigma). The ing that the down-regulation of ascl1 expression is a secondary data we present in this study were obtained from analysis of King’s College effect of the her6 knock-down and represents the genetic suppres- wild-type (KWT) fish, the neurog1:RFP transgenic line (22), and the shh:GFP sion of ascl1 by neurog1. In this light, it would be interesting to transgenic line (23). examine the thalamic phenotype in Hes1Ϫ/Ϫ NeurogϪ/Ϫ mice. In Injections. Transient knock-down of gene expression was performed as de- support of our findings, it has been shown in a different context that scribed in (54), The following -antisense oligomers (MO, Gene- other bHLH factors are required for GABAergic cell fate deter- tools LLC) were used at a concentration of 0.5 mM. her6MO (26), neurog1MO mination: Helt1 seems to have a function only in the mouse (57), gro1MO (5Ј- CGGCCCTGCGGATACATCTTGAATG-3Ј), and 5-bp mismatch midbrain and acts in a different way as it lacks the Groucho binding control gro1MO (5Ј-CGcCCCTaCGGATAgATgTTcAATG-3Ј). domain (47), and Ptf1a is expressed only in the spinal cord (48). For mis-expression experiments mRNA was synthesized in vitro (Message The MDO is fragmented in the her6 morphant embryos, with Machine Kit, Amersham) from full-length pCS2ϩ-her6 (26) and pCS2ϩ- glutamatergic proneural genes being expressed in place of shh. neurog1 (24). Together with rhodamine dextran as lineage tracer. 120pg We suggest that the abrogation of Her6 function converts the mRNA was injected into one out of 64 cells (MiniRuby, Invitrogen). For temporally controlled over-expression studies, 60 ng of the heat-shock organizer cells into neurons and, therefore, the characteristics of (hs)-inducible pSGH2-her6 and her6-trunc DNA constructs were used per blas- an organizing boundary (i.e., expression of the principal signal- tomere. At 10-somite stage, embryos were heat-shocked for 30 min at 42 °C. ing molecule Shh) are lost, similar to the loss of Shh expression in Hes1 knock-out mouse (21). Thus, Her6 can be seen as Single-Cell Electroporation. The single-cell electroporation technique (32) was essential for the maintenance of this local organizer. adapted to efficiently transfer MOs to a small group of cells in the rostral The activity of Her6 appears to be Groucho-dependent. thalamus of the neurog1:RFP line. Electroporation was performed on the right Indeed, it has been reported that Hairy related factors contain side of the thalamus at 22 hpf. Control embryos were electroporated with 0.5 a Groucho interaction domain (49). It is unlikely that the ␮g/␮L fluorescein-labeled control MO and experimental embryos with 0.5 down-regulation of shh expression at the MDO in the gro1 ␮g/␮L fluorescein-tagged her6 MO. To ensure stable concentration levels in morphant embryos is due to Gro function directly, as it has been the capillary, continuous but low outflow was used, causing green fluorescent staining of brain ventricular fluid. The following stimulation parameters were shown that Shh expression is unaltered when Groucho function used as described for electroporation of Morpholinos in embryos BIOLOGY

is abrogated in the mouse spinal cord (50). (58): 1-s long trains of 7-ms square pulses at 200 Hz and a voltage of 45 V. Trains DEVELOPMENTAL were delivered three times with a 1-s interval between trains. Pulses were Temporal Control of neurog1 Induction. Although we find ectopic generated with a Grass SD9 stimulator (Grass-Telefactor). neurog1 expression in her6 morphant embryos, the onset of neurog1 expression, at 24 hpf in the thalamus, is unaltered. The timing of Confocal Microscopy and Live Imaging. For live imaging, larvae were embed- induction of neurog1 expression as well as ascl1a strongly correlates ded in 1.5% low-melting-point agarose dissolved in Ringer medium contain- with the induction of Shh in the MDO and, indeed, Shh has been ing 0.016% tricaine at 22 hpf. Confocal image stacks were acquired using a demonstrated as a crucial factor for the regulation of proneural Nikon C1 confocal laser-scanning microscope. To reconstruct the imaged area, we collected a series of optical planes (z-stacks) spanning one body-half of the gene expression in a variety of neural tissues (41–43) and to be larvae and projected them into a single image (maximum intensity projection) essential for diencephalic maturation (51–54). We show that block- or rendered the volume in three dimensions to provide views of the image ade of Shh before its expression in the MDO is sufficient to block stack at different angles. The step size for each z-stack was chosen upon expression of the proneural genes neurog1 and ascl1a in the calculation of the theoretical z-resolution of the objective used (typically thalamus (Fig. S6). In support of these data we find that in embryos 2-␮m) or for multiday imaging, typically 15-␮m. The data sets were decon- carrying a in the nodal co-receptor one-eyed pinhead,in volved by AutoDeblur X Gold-Edition (AutoQuant) and further processed which shh expression is expressed in the MDO but is absent from using Imaris 4.1.3 (Bitplane AG). the basal plate, neurog1 expression is unaltered within the thalamus (54), suggesting that Shh expression from the MDO alone is Whole-Mount In Situ Hybridization. Whole-mount mRNA in situ hybridizations sufficient to induce neurog1 in the cTh. Compared to the phenotype (ISH) were performed as in ref. 59. Expression patterns have been described for ascl1a (originally described as zash1a) (60), coe2 (61), irx1b (originally de- in wild-type embryos, expression of her6 is independent of Shh scribed as Ziro1) (62), neurog1 (also known as ngn1) (41), shha (originally signaling, as in smu mutant embryos her6 expression is still detect- described as shh) (63), and wnt8b (64). For GAD staining post ISH, the poly- able. Reduction of the expression domain in smu mutant embryos clonal GAD65/67 antibody (Abcam) was used. may be explained by the overall reduced size of the diencephalon in the mutant (Fig. S6 F and I). ACKNOWLEDGMENTS. We thank Corinne Houart, Jon Clarke (MRC CDN) for We therefore propose a model in which Shh from the MDO experimental suggestions and comments, and Pia Aanstad (KIT) for help with defines the timing of induction of proneural genes in the thalamus. the purmorphamine treatment. Patrick Blader (Centre de Biologie du Dével- oppement, Toulouse) supplied the neurog1:RFP fish line; Jon Clarke advised Subsequently, Her6 determines the spatial dynamics of neurog1 on the electroporation technique; Thomas Czerny (Institute of Animal Breed- induction and neuronal identity. Thus, Hairy genes are not only ing and Genetics, Vienna) supplied the heat-shock inducible pSGH2 vector; required for the spatial control of neurogenesis but are also Andrea Pasini (University of Padua) and David Wilkinson (NIMR, London) supplied the her6 probe; Steve Wilson (UCL) supplied the ascl1 probe. This important for the determination of a fundamental neuronal cell work was funded primarily by a grant from the Medical Research Council fate decision between excitatory projection neuron and inhibitory (G0601064) and also by a Deutsche Forschungsgemeinschaft Emmy-Noether interneuron. Fellowship (SCHO 847/2-1).

1. Salzberg A, Bellen HJ (1996) Invertebrate versus vertebrate neurogenesis: Variations 6. Cooper ML, Rakic P (1981) Neurogenetic gradients in the superior and inferior colliculi on the same theme? Dev Genet 18:1–10. of the rhesus monkey. J Comp Neurol 202:309–334. 2. Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of 7. Bayer SA, Wills KV, Triarhou LC, Ghetti B (1995) Time of neuron origin and gradients neural cell types. Nat Rev Neurosci 3:517–530. of neurogenesis in midbrain dopaminergic neurons in the mouse. Exp Brain Res 3. Bayer SA, Altman J (1987) Directions in neurogenetic gradients and patterns of 105:191–199. anatomical connections in the telencephalon. Prog Neurobiol 29:57–106. 8. Angevine JB, Jr (1970) Time of neuron origin in the diencephalon of the mouse. An 4. Angevine JB, Jr, Sidman RL (1961) Autoradiographic study of cell migration during autoradiographic study. J Comp Neurol 139:129–187. histogenesis of cerebral cortex in the mouse. Nature 192:766–768. 9. McAllister JP, II, Das GD (1977) Neurogenesis in the epithalamus, dorsal thalamus and 5. Aboitiz F, Morales D, Montiel J (2001) The inverted neurogenetic gradient of the mam- ventral thalamus of the rat: An autoradiographic and cytological study. J Comp Neurol malian isocortex: Development and evolution. Brain Res Brain Res Rev 38:129–139. 172:647–686.

Scholpp et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on October 3, 2021 10. Jones EG (2007) The Thalamus (Cambridge University Press, New York) 2nd Ed. 38. Cunliffe VT (2004) Histone deacetylase 1 is required to repress Notch target gene 11. Vue TY, et al. (2007) Characterization of progenitor domains in the developing mouse expression during zebrafish neurogenesis and to maintain the production of motoneu- thalamus. J Comp Neurol 505:73–91. rones in response to hedgehog signalling. Development 131:2983–2995. 12. Parras CM, et al. (2004) Mash1 specifies neurons and oligodendrocytes in the postnatal 39. Hans S, et al. (2004) her3, a zebrafish member of the hairy-E(spl) family, is repressed by brain. EMBO J 23:4495–4505. Notch signaling. Development 131:2957–2969. 13. Schuurmans C, et al. (2004) Sequential phases of cortical specification involve Neuro- 40. Wall DS, et al. (2009) Progenitor cell proliferation in the retina is dependent on genin-dependent and -independent pathways. EMBO J 23:2892–2902. Notch-independent Sonic hedgehog/Hes1 activity. J Cell Biol 184:101–112. 14. Britz O, et al. (2006) A role for proneural genes in the maturation of cortical progenitor 41. Blader P, Fischer N, Gradwohl G, Guillemot F, Strahle U (1997) The activity of neuro- cells. Cereb Cortex 16:i138–151. genin1 is controlled by local cues in the zebrafish embryo. Development 124:4557– 15. Fode C, et al. (2000) A role for neural determination genes in specifying the dorso- 4569. ventral identity of telencephalic neurons. Genes Dev 14:67–80. 42. Ota M, Ito K (2003) Induction of neurogenin-1 expression by sonic hedgehog: Its role 16. Bally-Cuif L, Hammerschmidt M (2003) Induction and patterning of neuronal devel- in development of trigeminal sensory neurons. Dev Dyn 227:544–551. opment, and its connection to cell cycle control. Curr Opin Neurobiol 13:16–25. 43. Ma YC, et al. (2008) Regulation of motor neuron specification by phosphorylation of 17. Kageyama R, Ohtsuka T, Kobayashi T (2007) The Hes gene family: Repressors and neurogenin 2. Neuron 58:65–77. oscillators that orchestrate embryogenesis. Development 134:1243–1251. 18. Paroush Z, et al. (1994) Groucho is required for Drosophila neurogenesis, segmenta- 44. Szabo NE, Zhao T, Zhou X, Alvarez-Bolado G (2009) The role of Sonic hedgehog of tion, and sex determination and interacts directly with hairy-related bHLH proteins. neural origin in thalamic differentiation in the mouse. J Neurosci 29:2453–2466. Cell 79:805–815. 45. Vue TY, et al. (2009) Sonic hedgehog signaling controls thalamic progenitor identity 19. Geling A, et al. (2003) bHLH transcription factor Her5 links patterning to regional and nuclei specification in mice. J Neurosci 29:4484–4497. inhibition of neurogenesis at the midbrain-hindbrain boundary. Development 46. Ashe HL, Briscoe J (2006) The interpretation of morphogen gradients. Development 130:1591–1604. 133:385–394. 20. Geling A, Plessy C, Rastegar S, Strahle U, Bally-Cuif L (2004) Her5 acts as a prepattern 47. Nakatani T, Minaki Y, Kumai M, Ono Y (2007) Helt determines GABAergic over factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit glutamatergic neuronal fate by repressing Ngn genes in the developing mesenceph- neurogenesis at the midbrain-hindbrain boundary. Development 131:1993–2006. alon. Development 134:2783–2793. 21. Baek JH, Hatakeyama J, Sakamoto S, Ohtsuka T, Kageyama R (2006) Persistent and high 48. Glasgow SM, Henke RM, Macdonald RJ, Wright CV, Johnson JE (2005) Ptf1a determines levels of Hes1 expression regulate boundary formation in the developing central GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn. nervous system. Development 133:2467–2476. Development 132:5461–5469. 22. Blader P, Plessy C, Strahle U (2003) Multiple regulatory elements with spatially and 49. Fisher AL, Ohsako S, Caudy M (1996) The WRPW motif of the hairy-related basic temporally distinct activities control neurogenin1 expression in primary neurons of the helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and zebrafish embryo. Mech Dev 120:211–218. protein-protein interaction domain. Mol Cell Biol 16:2670–2677. 23. Neumann CJ, Nuesslein-Volhard C (2000) Patterning of the zebrafish retina by a wave 50. Muhr J, Andersson E, Persson M, Jessell TM, Ericson J (2001) Groucho-mediated of sonic hedgehog activity. Science 289:2137–2139. transcriptional repression establishes progenitor cell pattern and neuronal fate in the 24. Jeong JY, et al. (2006) Neurogenin1 is a determinant of zebrafish basal forebrain ventral . Cell 104:861–873. dopaminergic neurons and is regulated by the conserved zinc finger protein Tof/Fezl. 51. Hashimoto-Torii K, et al. (2003) Differential activities of Sonic hedgehog mediated by Proc Natl Acad Sci USA 103:5143–5148. Gli transcription factors define distinct neuronal subtypes in the dorsal thalamus. Mech 25. Pasini A, Henrique D, Wilkinson DG (2001) The zebrafish Hairy/Enhancer-of-split- Dev 120:1097–1111. related gene her6 is segmentally expressed during the early development of hindbrain 52. Kiecker C, Lumsden A (2004) Hedgehog signaling from the ZLI regulates diencephalic and somites. Mech Dev 100:317–321. regional identity. Nat Neurosci 7:1242–1249. 26. Pasini A, Jiang YJ, Wilkinson DG (2004) Two zebrafish Notch-dependent hairy/ Enhancer-of-split-related genes, her6 and her4, are required to maintain the coordi- 53. Vieira C, Garda AL, Shimamura K, Martinez S (2005) Thalamic development induced by nation of cyclic gene expression in the presomitic mesoderm. Development 131:1529– Shh in the chick embryo. Dev Biol 284:351–363. 1541. 54. Scholpp S, Wolf O, Brand M, Lumsden A (2006) Hedgehog signaling from the zona 27. Petryniak MA, Potter GB, Rowitch DH, Rubenstein JL (2007) Dlx1 and Dlx2 control limitans intrathalamica orchestrates patterning of the zebrafish diencephalon. Devel- neuronal versus oligodendroglial cell fate acquisition in the developing forebrain. opment 133:855–864. Neuron 55:417–433. 55. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic 28. Zerucha T, et al. (2000) A highly conserved enhancer in the Dlx5/Dlx6 intergenic region development of the zebrafish. Dev Dyn 203:253–310. is the site of cross-regulatory interactions between Dlx genes in the embryonic fore- 56. Brand M, Granato M, Nuesslein-Volhard C (2002) Zebrafish, Keeping and Raising brain. J Neurosci 20:709–721. Zebrafish (Oxford Univ Press, Oxford), 1st Ed. 29. Zhang XY, Rodaway AR (2007) SCL-GFP transgenic zebrafish: In vivo imaging of blood 57. Cornell RA, Eisen JS (2002) Delta/Notch signaling promotes formation of zebrafish and endothelial development and identification of the initial site of definitive hema- neural crest by repressing Neurogenin 1 function. Development 129:2639–2648. topoiesis. Dev Biol 307:179–194. 58. Bestman JE, Ewald RC, Chiu SL, Cline HT (2006) In vivo single-cell electroporation for 30. Bradley CK, et al. (2006) The essential haematopoietic transcription factor Scl is also transfer of DNA and macromolecules. Nat Protoc 1:1267–1272. critical for neuronal development. Eur J Neurosci 23:1677–1689. 59. Scholpp S, Brand M (2003) Integrity of the midbrain region is required to maintain the 31. Isogai S, Horiguchi M, Weinstein BM (2001) The vascular anatomy of the developing diencephalic-mesencephalic boundary in zebrafish no isthmus/.1 mutants. Dev zebrafish: An atlas of embryonic and early larval development. Dev Biol 230:278–301. Dyn 228:313–322. 32. Tawk M, Bianco IH, Clarke JD (2009) Focal electroporation in zebrafish embryos and 60. Allende ML, Weinberg ES (1994) The expression pattern of two zebrafish achaete-scute larvae. Methods Mol Biol 546:145–151. homolog (ash) genes is altered in the embryonic brain of the cyclops mutant. Dev Biol 33. Bajoghli B, Aghaallaei N, Heimbucher T, Czerny T (2004) An artificial promoter con- 166:509–530. struct for heat-inducible misexpression during fish embryogenesis. Dev Biol 271:416– 61. Bally-Cuif L, Dubois L, Vincent A (1998) Molecular cloning of Zcoe2, the zebrafish 430. homolog of Xenopus Xcoe2 and mouse EBF-2, and its expression during primary 34. Ishibashi M, et al. (1995) Targeted disruption of mammalian hairy and Enhancer of split neurogenesis. Mech Dev 77:85–90. homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature 62. Wang X, Emelyanov A, Sleptsova-Friedrich I, Korzh V, Gong Z (2001) Expression of two neurogenesis, and severe neural tube defects. Genes Dev 9:3136–3148. 35. Ohtsuka T, Sakamoto M, Guillemot F, Kageyama R (2001) Roles of the basic helix-loop- novel zebrafish iroquois homologues (ziro1 and ziro5) during early development of helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain. axial structures and . Mech Dev 105:191–195. J Biol Chem 276:30467–30474. 63. Krauss S, Concordet JP, Ingham PW (1993) A functionally conserved homolog of the 36. Ishibashi M, et al. (1994) Persistent expression of helix-loop-helix factor HES-1 prevents Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in mammalian neural differentiation in the central nervous system. EMBO J 13:1799– zebrafish embryos. Cell 75:1431–1444. 1805. 64. Kelly GM, Greenstein P, Erezyilmaz DF, Moon RT (1995) Zebrafish wnt8 and wnt8b 37. Blader P, et al. (2004) Conserved and acquired features of neurogenin1 regulation. share a common activity but are involved in distinct developmental pathways. Devel- Development 131:5627–5637. opment 121:1787–1799.

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