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 Genetics, 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 neurons (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 gene 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 genes, 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 midbrain-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 zebrafish, 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 phenotype 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 neuron 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