Subdivisions of the adult zebrafish pallium based on molecular marker analysis

Abstract

Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species.

Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish ( Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain.

Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases.

Keywords: Actinopterygii; amygdala; evolution; hippocampus; homology; neuroanatomy; neurogenesis; telencephalon; teleost; vertebrate brain.

Figure 1.. Expression of eomesa in the pallium.

A. In the rostral telencephalic, eomesa expression is found in the ventricular zone (vz) of Dm (arrowheads) and in the vz and neuronal layer (nl) of Dc and Dlv. B. At mid-telencephalic levels, eomesa expression is present in the vz of Dm (arrowheads), in the nl of Dc and in the vz and nl of Dld and Dlv. C. At the anterior commissure (Cant), eomesa expression is present in the vz of Dm (arrowhead) and in the vz and nl of Dld and Dp. Inset shows close-up of vz of Dm D. Caudal to Cant, eomesa expression is present in the vz of Dm (arrowhead), in the vz and nl of Dp and in BNSM. Inset shows close-up of vz of Dm E.– H. Summary of the expression pattern of eomesa at rostral ( E.), mid-telencephalic ( F.), commissural ( G.) and postcommissural levels ( H.). A.– D. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A– D.

Figure 2.. Expression of emx1, emx2 and emx3 in the pallium.

A. At the anterior commissure (Cant), emx1 expression is found in neuronal layer (nl) and ventricular zone (vz) of Dp. B. Caudal to Cant, emx1 expression is present in the vz and nl of Dp and in EN. C. Caudal to Cant, emx2 expression is present in part of Dc. D.– F. Summary of the expression pattern of emx1 and emx2 at commissural ( D.) and postcommissural levels ( E., F.). G. In the rostral telencephalon, emx3 expression is present in the vz and nl of Dm, weakly in scattered cells of the nl od Dc and in the vz of Dlv (arrowheads). H. At mid-telencephalic levels, emx3 expression is found in the vz and nl of Dm, in scattered cells in Dc, and in the vz and nl of Dlv. I. At Cant, emx3 expression is present in the vz and nl of Dm, in scattered cells in Dc and in the vz and nl of Dp. J.– L. Summary of the expression pattern of emx3 at rostral ( J.), mid-telencephalic ( K.) and commissural levels ( L.). A.– F. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A– D.

Figure 3.. Expression of Prox1 in the pallium.

A. At mid-telencephalic levels, Prox1 positive cells are found in the neuronal layer (nl) of Dld shortly before the anterior commissure (Cant). B. At Cant, Prox1 positive cells are found in the nl of Dld. C.– D. Summary of expression pattern of Prox1. A.– B. Confocal images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The yellow dashed line indicates the boundary between D and V. Scale bars = 50µm in A– B.

Figure 4.. Expression of ascl1a in the pallium.

A.– B. In the rostral telencephalon, ascl1a expression is present in scattered cells in the ventricular zone (vz) of Dm ( A, arrowheads) and Dlv ( B, arrowheads). C.– D. At mid-telencephalic levels, ascl1a expression is present in scattered cells in the vz of Dm ( C, arrowheads) and Dlv ( D, arrowheads). E.– F. At Cant, ascl1a expression is present in scattered cells in the vz of Dm ( E, arrowheads) and Dp ( F, arrowheads). G.– H. Posterior to Cant, ascl1a expression is present in scattered cells in the vz of Dm ( G, arrowheads) and Dp ( H, arrowheads). I.– L. Summary of the expression pattern of ascl1a rostral ( I.), mid-telencephalic ( J.), commissural ( K.) and postcommissural levels ( L.). A.- H. Brightfield images of cross-sections at the levels indicated through the telencephalon. Red dashed line indicates subdivisions based on the current marker. White dashed line indicates subdivisions based on other markers. The black dashed line indicates the boundary between D and V. Scale bars = 50µm in A– C.

Figure 5.. Summary of the gene expression patterns in the adult zebrafish pallium.

A.– D. Cross-sections at the levels indicated through the telencephalon. E. Schematic diagram of a cross section through the mouse telencephalon for comparison (modified after ), note that the amygdaloid complex (AC, brown) is derived both from subpallium (grey) and pallium. Light grey areas (Th/Hyp) are part of the diencephalon. Indicated is also a model of the putative homology of the subdivisions in the adult zebrafish pallium to regions in the tetrapod pallium taking the data presented in this paper into account. Additional marker analysis, lineage tracing experiments and functional analyses are necessary to substantiate the proposed homology to pallial nuclei in tetrapods. The different shades of green (Dc) and yellow (Dld) indicate gene expression changes along the rostro-caudal axis. * in scattered cells, ** in Dlv in the ventricular zone, moving caudally expression in the neuronal layer and ventricular zone, note that Prox1 positive cells are present in Dld shortly before the anterior commissure, n/a = not applicable. The black dashed line indicates the boundary between D and V. The red dashed lines indicate the boundaries between different nuclei in D. AC amygdaloid complex, ACo anterior cortical amygdalar area, BC basal amygdalar complex, BNSM bed nucleus of the stria medullaris, Ce central amygdala, CP Caudateputamen, DG dentate gyrus, EN entopeduncular nucleus, HF hippocampal formation, Hyp hypothalamus, Me medial amygdala, NCx neocortex, pCx piriform cortex, Po preoptic region, Th thalamus, V area ventralis telencephali, Vp postcommissural nucleus of the area ventralis telencephali, Vs supracommisural nucleus of the area ventralis telencephali.

Figure S1.. Teleost eomes genes.

A. The topology of the Ensembl Gene Tree suggest that the teleost eomes co-orthologs, eomesa and eomesb, were duplicated at the base of the teleost lineage and thus most likely during the teleost genome duplication (TGD). B. Dotplot from the Synteny Database shows that the human EOMES gene region on chromosome Hsa3 shows extensive double conserved synteny to zebrafish chromosomes Dre19 and Dre16 which contain eomesa and eomesb, respectively, providing strong evidence for the TGD origin of teleost eomes co-orthologs.

Figure S2.. Expression of eomesb in the embryonic zebrafish brain.

A. eomesb expression is not found in the telencephalon (T), but present in the midbrain (M), arrowhead, shown is a lateral view. Di Diencephalon, H Hindbrain.

Figure S3.. Expression of emx1, emx2 and emx3 in the zebrafish larval brain at 7dpf.

A. emx1 expression is present in the lateral part of the dorsal telencephalon ( D, arrowhead), shown is a ventral view. B. emx2 expression is present in the lateral part of D (arrowhead), shown is a parasaggital view. C. emx3 expression is present throughout D, shown is a parasaggital view. D. Summary of the distribution of emx1 and emx2 expression in D in a sagittal view. E. Summary of the distribution of emx3 expression in D in a sagittal view. A.– C. Brightfield whole-mount images, A. ventral, B.– C. sagittal views. Scale bars = 50µm A– C.