Expression of Zash-1a in the postembryonic zebrafish brain allows comparison to mouse Mash1 domains

Gene Expression Patterns 1 (2002) 187–192 www.gxp-interactive.com

Report

Expression of Zash-1 a in the postembryonic zebrafish brain allows comparison to mouse Mash1 domains Mario F. Wullimann*, Thomas Mueller Brain Research Institute, FB 2, University of Bremen, P.O. Box 330440, 2834 Bremen, Germany Accepted 3 September 2002

Abstract Four areas in the late embryonic murine forebrain, i.e. the subpallium (striatum), the preoptic region, the ventral thalamus, and the hypothalamus, have been described to express the basic helix–loop–helix (bHLH) gene mammalian achaete-scute homolog Mash1 (Ascl1, Mouse Genome Informatics) in a complementary fashion to another bHLH gene, neurogenin1 (ngn1 ) (Neurod3, Mouse Genome Informatics), which is expressed in directly adjacent forebrain regions. We report here that the four regions previously identified as subpallium, preoptic region, ventral thalamus and hypothalamus (i.e. ventral inferior lobe) in the postembryonic zebrafish brain show Zash-1 a expression at 3 days postfertilization (dpf), whereas none of those areas express the bHLH gene neuroD (nrd) between 2 and 5 dpf. This indicates that two well established alternative genetic pathways involved in neurogenesis in the amniote (mammalian) brain are present in homologous phenotypic locations in the anamniote (zebrafish) brain as well and that these pathways possibly act similarly in the generation of different neuronal phenotypes (e.g. subpallial GABAergic interneurons versus pallial glutamatergic projection neurons, or dopaminergic neurons versus other neurotransmitter phenotypes). Furthermore, previous initial identification of early postembryonic brain subdivisions in the zebrafish is strongly corroborated by these expression patterns.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Pattern formation, compartments, and boundaries Keywords: Zebrafish; Hu protein; PCNA; neuroD; nrd; Forebrain; Diencephalon; Posterior tuberculum; Telencephalon; Cerebellum; External granular layer; bHLH transcription factor; Neurogenesis; Mitosis; Proliferation; Zash-1 a; Mash1 ; Ascl1 ; neurogenin1 ; ngn1 ; Tyrosine hydroxylase; GABA; Dopamine

1. Background and results Basic helix–loop–helix (bHLH) genes involved in neurogenesis (such as NeuroD and Neurogenin1 ) are dynamically expressed and show some spatiotemporal changes in early expression domains in the zebrafish [3,10] and in the mouse central nervous system (CNS) [7,11,20]. For example in murine mammals, neurogenin1 (Neurod3, Mouse Genome Informatics) and another bHLH gene, the mammalian achaete-scute homolog Mash1 (Ascl1, Mouse Genome Informatics); display complementary expression

*Corresponding author. Tel.: 149-421-218-4702; fax: 149-421-2184549. E-mail addresses: [email protected] (M.F. Wullimann), [email protected] (T. Mueller).

domains with sharp boundaries in the spinal cord and forebrain [7,8,12,21] at a particular developmental stage (i.e. mouse: E12.5, rat: E14.5). More specifically, four areas in the late embryonic murine forebrain, i.e. the subpallium (striatum), the preoptic region, the ventral thalamus and the hypothalamus, have been described to express Mash1 in a complementary fashion to Neurogenin1 which is expressed in directly adjacent forebrain regions. Meanwhile, it is well accepted that Neurogenin1 /NeuroD and Mash1 are part of two alternative genetic pathways involved in the generation of two different main classes of neuronal phenotypes in the telencephalon, e.g. glutamatergic neurons in the pallium versus GABAergic neurons in the subpallium [5,16,19]. Here, we identify the 3 days postfertilization (dpf) zebrafish brain as the corresponding developmental time point of complementarity of expression domains of the orthologous

1567-133X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S1567-133X( 02 )00016-9

188

M.F. Wullimann, T. Mueller / Gene Expression Patterns 1 (2002) 187–192

Table 1 Complementarity of bHLH gene expression in 3 dpf zebrafish brain neuroD

Zash-1 a

Huprotein

Tyrosine hydroxylase a

1 1

1 2b

1 1

1 2

2 2

1 1

1 1

1 1

1 1 1 2 1 2 1 1

(1) 2 (1) 1 1 1 1 2

1 1 1 2 1 1 1 1

2 2 2 1 1 1 1 2

2 2 2 1

1 1 1 1

1 1 1 2

1 1 1 2

Mesencephalon Optic tectum Torus semicircularis Tegmentum

1 1 1

1 1 2

1 1 1

2 2 2

Rhombencephalon Cerebellar plate Valvula cerebelli Medulla oblongata

1 1 1

1 1 1

1 1 1

2 2 1

Telencephalon Olfactory bulb Pallium Subpallium Precommissural Supra- / postcommissural Diencephalon Epiphysis Habenula Dorsal thalamus Ventral thalamus Pretectum Preoptic region Ventral posterior tuberculum M1 / M2 Hypothalamus Rostral Intermediate Caudal Pituitary

(1) Zash-1 a expression very restricted within area indicated (see text). a Data on 5 dpf zebrafish brain described in Rink and Wullimann [18]. b Except for most caudomedial pallial ventricular zone which is Zash-1 a-positive.

genes (i.e. neuroD identified by Blader et al. [3], and Zash-1 a identified by Allende and Weinberg [1]). The expression patterns of bHLH genes neuroD (nrd) and Zash-1 a, the Hu-protein distribution in the zebrafish brain at 3 dpf, as well as tyrosine hydroxylase-containing cell groups at 5 dpf are summarized in Table 1. Huproteins form a family of mRNA binding proteins serving as a marker for early (postmitotic) differentiating neurons [2,13,15] and their immunohistological description helps in identifying the morphogenetic maturation state of a given central nervous region. Previously described data on tyrosine hydroxylase [17,18] are added in the table because they reveal correlation with bHLH gene expression.

In the telencephalon, Zash-1 a is expressed in the subpallium, but not in the rostral two thirds of the pallium (Fig. 1A2–C2), although there is some expression in the most caudal pallial ventricular zone (not shown). In the alar plate diencephalon, Zash-1 a has prominent expression domains in the ventral thalamus and preoptic region, but not in the habenula, and only minor ones, if any, in the epiphysis and dorsal thalamus (Fig. 1D2). The ventral thalamus may be further identified by the absence of differentiating Hu-positive (Fig. 1D3) and presence of proliferating, PCNA-positive cells (Fig. 1D4), similar to what has been described before at 5 dpf [23]. In the basal plate diencephalon, Zash-1 a is strongly expressed in all

Fig. 1. Neuronal differentiation (Hu-protein) and complementarity of bHLH gene expression (neuroD5nrd and Zash-1 a) in the 3-dpf zebrafish brain. Levels shown are: olfactory bulb (A1–3), precommissural telencephalon (B1–3), supracommissural telencephalon (C1–3), ventral thalamus / preoptic region (D1–4); note that PCNA-positive cells are additionally shown for this level in D4 and that these proliferative cells are perfectly complementary to differentiating Hu-positive cells shown in D3), hypothalamus (E1–2,F1–3), cerebellar plate (G1–2). Abbreviations: ac, anterior commissure; CeP, cerebellar plate; DT, dorsal thalamus; E, epiphysis; EGL, external granular layer; EPi, eye pigment; Ha, habenula; Hi, Hr intermediate, rostral hypothalamic zones; Hy; hypophysis (pituitary); MO, medulla oblongata; M1, migrated pretectal region; M2, migrated posterior tubercular (preglomerular) region; OB, olfactory bulb; OE, olfactory epithelium; P, pallium; Pi, meningeal pigment; Po, preoptic area; PTv, ventral part of posterior tubercle; S, subpallium; Sd, dorsal subpallium; Sv, ventral subpallium; TeO, optic tectum; VCP, ventral cerebellar proliferation; VT, ventral thalamus; ZI, zona limitans intrathalamica.

M.F. Wullimann, T. Mueller / Gene Expression Patterns 1 (2002) 187–192

189

190

M.F. Wullimann, T. Mueller / Gene Expression Patterns 1 (2002) 187–192

three, i.e. rostral, intermediate and caudal, divisions of the hypothalamus (Fig. 1E2–F2). In contrast, neuroD is expressed in a complementary fashion (compare with Table 1) in the forebrain since its transcripts are massively present in the pallium (Fig. 1B1), habenula (Fig. 1C1), epiphysis (Fig. 1C1), and dorsal thalamus (Fig. 1D1), but neuroD transcripts are absent in the subpallium (Fig. 1B1,C1), ventral thalamus (Fig. 1D1), preoptic region (Fig. 1D1) and entire hypothalamus (Fig. 1E1,F1). Huproteins are present in Zash-1 a-positive as well as in neuroD-positive forebrain regions. Two forebrain areas are characterized by strong proliferation at 3 dpf which is upheld at least into 5 dpf [15,23], the preoptic region and the ventral thalamus (Fig. 1D4). However, the anterior as well as posterior poles of the preoptic region clearly are Hu-positive (Fig. 1D3) and there are also some differentiating cells in the ventral thalamus (Fig. 1D3,D4). This suggests that—irrespective of Zash-1 a or neuroD expression—differentiating neurons exist essentially in the entire zebrafish forebrain at 3 dpf. Furthermore, the four Zash-1 a-positive zebrafish forebrain regions identified above (i.e. subpallium, ventral thalamus, preoptic region and hypothalamus) do not express neuroD at any later point in time during brain development between 2 and 5 dpf [15]. By 5 dpf, neuroDexpression is massively down-regulated already [15]. Expression of neurogenin1 at 2 dpf in the zebrafish brain parallels that of neuroD [14] in that it too is absent in the subpallium, the ventral thalamus and the hypothalamus. However, there is a lateral blob of ngn1 -positive cells in the zebrafish preoptic region at 2 dpf (TM and MFW, unpublished observations). In summary, this indicates that two well established alternative genetic pathways (i.e. one using neurogenin1 / neuroD, the other one using Mash1; reviewed in Schuurmans and Guillemot [19]) involved in neurogenesis in the amniote (mammalian) brain are present in homologous phenotypic locations in the anamniote (zebrafish, 3 dpf) brain and that they possibly act similarly in the generation of different neuronal phenotypes, e.g. subpallial GABAergic interneurons versus pallial glutamatergic projection neurons [5,16,19]. It is furthermore intriguing that the presence of very many dopaminergic neurons in the early [18] and adult [17] zebrafish brain in the subpallium, ventral thalamus, preoptic region and hypothalamus, and also in the olfactory bulb, pretectum and ventral posterior tuberculum (see below) correlates with early Zash-1 a expression in these locations (Table 1). This offers an additional hypothesis about the involvement of Zash1 -1 a with neuronal phenotype development (i.e. dopaminergic neurons). In any case, the previous initial identification of early postembryonic brain subdivisions in the zebrafish [23,25,26] is strongly corroborated interspecifically by selective bHLH expression patterns reported here. In addition to the complementary expression patterns

reported above, there are regions where both neuroD and Zash-1 a are expressed, i.e. the olfactory bulb (Fig. 1A1,A2), the most caudal pallium, the pretectum (not shown), the ventral posterior tuberculum (Fig. 1E1,E2,F1,F2), the optic tectum (Fig. 1E1,E2,F1,F2), the torus semicircularis (not shown), the medulla oblongata (Fig. 1F1,F2), the valvula (Fig. 1F1,F2), the cerebellar plate (Fig. 1G1,G2), and the pituitary (Fig. 1F1,F2). Except for the valvula, cerebellar plate and pituitary, where neurogenin1 expression is absent, ngn1 is expressed similarly as Zash-1 a in the regions just mentioned at 2 dpf (TM and MFW, unpublished observations). In some of these regions, Zash-1 a and neuroD expression domains are clearly segregated. For instance, whereas Zash-1 a (and ngn1 -) cells lie within the proliferative zone of the olfactory bulb, neuroD cells lie clearly dorsal to it (Fig. 1A1,A2). Although this supports that Zash-1 a is involved in neurogenesis directly within the early zebrafish olfactory bulb, the Zash-1 a positive cells noted there could alternatively have migrated into the olfactory bulb from a more posterior ventricular telencephalic position. This was observed in late postembryonic to adult mice where neurons are added to the olfactory bulbs via the rostral migratory stream and where Mash1 mutants showed a considerable decrease of GABAergic cells in the olfactory bulbs compared to the wild type [5]. While the presence of a rostral migratory stream has not been demonstrated in the zebrafish brain, the telencephalic ventricle remains proliferative into adulthood and neuronal cell numbers increase in the zebrafish olfactory bulb [4]. A segregation of (more medially lying) neuroD-positive and (laterally lying) Zash1 a-positive cells is also seen in the ventral posterior tubercular area (Fig. 1F1,F2). Although in other regions (e.g. torus semicircularis, not shown) strongly Zash-1 apositive and neuroD-positive cell clusters overlap, they still may be separate on the cellular level as in the remaining cases. With the exception of the pituitary (which remains Hu-negative until 5 dpf; TM and MFW, unpublished observations), Hu-proteins and, thus, differentiating neurons, are again present at least partially within all these regions just mentioned. Even in the optic tectum, which is mostly free of Hu-positive cells, some of the latter are present in the periphery of its most rostral aspect. With respect to the distribution of neuroD versus Zash-1 a expression, the cerebellar plate represents a particularly interesting case because neuroD expression is present in two locations here, one close to the proliferative ventral layer and one towards the subpial external granular layer (Fig. 1G1). In contrast, Zash-1 a is only expressed close to the proliferative ventral layer, and not towards the pial periphery (Fig. 1G2). Also, Zash-1 a is expressed in repetitive blobs at the basal cerebellar plate and does not form an uninterrupted band of expression as neuroD does in this location. An attractive explanation is that GABAer-

M.F. Wullimann, T. Mueller / Gene Expression Patterns 1 (2002) 187–192

gic cerebellar neurons (such as Purkinje cells) may only be generated in the proliferative ventral layer, while other neuronal types are produced in the proliferative ventral as well as in the external granular layer. Similarly, in the olfactory bulb, pretectum and ventral posterior tuberculum, dopaminergic and additional neuronal phenotypes may be produced in accordance with expression of both neuroD and Zash-1 a. Thus, these two bHLH genes may generally be involved with alternative development of different neurochemical phenotypes of neurons.

2. Material and methods Zebrafish were kept and bred according to standard techniques [22]. Fertilized zebrafish eggs were raised at 28.5 8C and staged according to Kimmel et al. [9]. For the in situ hybridizations, Hu-protein- and PCNA-immunohistochemistry performed here, we used 2, 3, 4 and 5 dpf zebrafish larvae (three specimens each per gene / protein marker and age). The animals used for in situ hybridization were paraformaldehyde-fixed overnight (4%, in phosphate buffer, pH 7.2) and embedded in paraffin. The zebrafish neurogenin1 and neuroD (nrd) plasmids were received as ¨ a gift from Drs Uwe Strahle, Patrick Blader and the IGBMC-184-ULP, Strasbourg, France and the zebrafish Zash-1 a plasmid was given to us by Dr Eric Weinberg (Pennsylvania, PA, USA). The antisense RNA-probes were generated by in vitro transcription with digoxigenin-11UTP (Boehringer, Mannheim, Germany). The neuroD probe is against the complete cds (1053 bp) of nrd (GenBank accession number AF017302). The neurogenin probe is against the complete cds (939 bp) of ngn1 (GenBank accession number AF017301). The Zash-1 a probe is against the complete cds (1561 bp) of Zash-1 a (GenBank accession number U14587). In situ hybridization with digoxigenin-labeled antisense probes of neurogenin1, neuroD or Zash-1 a were performed on paraffin sections (thickness: 8–10 mm) according to Dorsky et al. [6]. The hybridization step was carried out overnight at 55 8C, and the color reaction was allowed to proceed for 1–3 days at room temperature. For immunohistochemical detection of Hu-proteins, the primary anti-Hu monoclonal antibody 16A11 (Monoclonal Antibody Facility, Eugene, OR) was used. Details of the procedure were described elsewhere [15]. The immunohistochemical PCNA-material was prepared as described previously [23,24]. Details of tyrosine hydroxylase immunohistochemistry were reported elsewhere [17,18]. The planned number of animals to be used in this study were reported prior to starting the investigations to the local authorities (Senate of the City of Bremen, Germany). Thus, treatment of all animals used herein is according to the ‘Deutsche Tierschutzgesetz’ and is furthermore in

191

¨ agreement with the guidelines of the ‘Veterinaramt’ of the Senate of the City of Bremen (Germany).

Acknowledgements We thank Professor Dr Gerhard Roth, Director of Brain Research Institute (University of Bremen), for furthering our work in various ways, Professor Dr Dietmar Blohm for access to his molecular biological facility (Department of Biotechnology and Molecular Genetics, UFT, University of Bremen), Katja Ahrens for help with the in situ hybridization method, Andrea Schaffrath for technical ¨ help, Drs Uwe Strahle, Patrick Blader and IGBMC-184ULP (Strasbourg, France) for plasmids of nrd and ngn1, Dr Eric Weinberg (Pennsylvania, Philadelphia, USA) for plasmids of Zash-1 a and Zash-1 b, and the Deutsche Forschungsgemeinschaft (Bonn, Projekt Wu-211 / 1-3) as well as the E.A.C. Lange-Stiftung (Bremen) for financial support.

References [1] M.L. Allende, E.S. Weinberg, The expression pattern of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant, Dev. Biol. 166 (1994) 509–530. [2] K. Barami, K. Iversen, H. Furneaux, S.A. Goldman, Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain, J. Neurobiol. 28 (1995) 82–101. ¨ [3] P. Blader, N. Fischer, G. Gradwohl, F. Guillemot, U. Strahle, The activity of neurogenin1 is controlled by local cues in the zebrafish, Development 124 (1997) 4557–4569. [4] C.A. Byrd, P.C. Brunjes, Neurogenesis in the olfactory bulb of adult zebrafish, Neuroscience 105 (2001) 793–801. [5] S. Casarosa, C. Fode, F. Guillemot, Mash1 regulates neurogenesis in the ventral telencephalon, Development 126 (1999) 525–534. [6] R.I. Dorsky, D.H. Rapaport, W.A. Harris, Xotch inhibits cell differentiation in the Xenopus retina, Neuron 14 (1995) 487–496. [7] C. Fode, Q. Ma, S. Casarosa, S.-L. Ang, D.J. Anderson, F. Guillemot, A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons, Genes Dev. 14 (2000) 67–80. [8] S. Horton, A. Meredith, J.A. Richardson, J.E. Johnson, Correct coordination of neuronal differentiation events in ventral forebrain requires the bHLH factor Mash1, Mol. Cell Neurosci. 14 (1999) 355–369. [9] C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253–310. [10] V. Korzh, I. Sleptsova, J. Liao, J. He, Z. Gong, Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation, Dev. Dyn. 213 (1998) 92–104. [11] Q. Ma, C. Kintner, D.J. Anderson, Identification of neurogenin, a vertebrate neuronal determination gene, Cell 87 (1996) 43–52. [12] Q. Ma, L. Sommer, P. Cserjesi, D.J. Anderson, Mash1 and neurogenin1 expression patterns define complementary domains of neuroepithelium in the developing CNS and are correlated with

192

[13]

[14]

[15]

[16] [17]

[18]

[19]

[20]

M.F. Wullimann, T. Mueller / Gene Expression Patterns 1 (2002) 187–192 regions expressing Notch ligands, J. Neurosci. 17 (1997) 3644– 3652. M.F. Marusich, H.M. Furneaux, P.D. Henion, J.A. Weston, Hu neuronal proteins are expressed in proliferating neurogenic cells, J. Neurobiol. 25 (1994) 143–155. T. Mueller, M.F. Wullimann, Expression domains of neuroD (nrd) in the early postembryonic zebrafish brain, Brain Res. Bull. 57 (2002) 377–379. T. Mueller, M.F. Wullimann, BrdU-, neuroD-(nrd) and Hu-studies show unusual non-ventricular neurogenesis in the postembryonic zebrafish forebrain, Mech. Dev. 117 (2002) 123–135. J.G. Parnavelas, The origin and migration of cortical neurones: new vistas, Trends Neurosci. 23 (2000) 126–131. E. Rink, M.F. Wullimann, The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum), Brain Res. 889 (2001) 316– 330. E. Rink, M.F. Wullimann, Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study, Dev. Brain Res. 137 (2002) 89–100. C. Schuurmans, F. Guillemot, Molecular mechanisms underlying cell fate specification in the developing telencephalon, Curr. Opin. Neurobiol. 12 (2002) 26–34. L. Sommer, Q. Ma, D.J. Anderson, Neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian

[21]

[22] [23]

[24]

[25]

[26]

neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS, Mol. Cell Neurosci. 8 (1996) 221–241. M.-A. Torii, F. Matsuzaki, N. Osumi, K. Kaibuchi, S. Nakamura, S. Casarosa, F. Guillemot, M. Nakafuku, Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system, Development 126 (1999) 443–456. M. Westerfield, The Zebrafish Book, University of Oregon Press, Eugene, OR, 1995. M.F. Wullimann, L. Puelles, Postembryonic neural proliferation in the zebrafish forebrain and its relationship to prosomeric domains, Anat. Embryol. 329 (1999) 329–348. M.F. Wullimann, S. Knipp, Proliferation pattern changes in the zebrafish brain from embryonic through early postembryonic stages, Anat. Embryol. 202 (2000) 385–400. M.F. Wullimann, E. Rink, Detailed immunohistology of Pax6 protein and tyrosine hydroxylase in the early zebrafish brain suggests role of Pax6 gene in development of dopaminergic diencephalic neurons, Dev. Brain Res. 131 (2001) 173–191. M.F. Wullimann, E. Rink, The teleostean forebrain: A comparative and developmental view based on early proliferation, Pax6 activity and catecholaminergic organization, Brain Res. Bull. 57 (2002) 363–370.