Expression of Barhl2 and its relationship with Pax6 expression in the forebrain of the mouse embryo

Abstract

Background: The transcription factor Barhl2 is an antiproneural transcription factor with roles in neuronal differentiation. The functions of its homologue in Drosophila development are better understood than its functions in mammalian brain development. Existing evidence suggests that its expression in the embryonic forebrain of the mouse is regional and may complement that of another transcription factor that is important for forebrain development, Pax6. The aim of this study is to provide a more detailed description of the Barhl2 expression pattern in the embryonic forebrain than is currently available, to relate its expression domains to those of Pax6 and to examine the effects of Pax6 loss on Barhl2 expression.

Results: We found that Barhl2 is expressed in the developing diencephalon from the time of anterior neural tube closure. Its expression initially overlaps that of Pax6 in a central region of the alar diencephalon but over the following days their domains of expression become complementary in most forebrain regions. The exceptions are the thalamus and pretectum, where countergradients of Pax6 and Barhl2 expression are established by embryonic day 12.5, before overall Pax6 levels in these regions decline greatly while Barhl2 levels remain relatively high. We found that Barhl2 expression becomes upregulated in specifically the thalamus and pretectum in Pax6-null mice.

Conclusions: The region-specific expression pattern of Barhl2 makes it likely to be an important player in the development of region-specific differences in embryonic mouse forebrain. Repression of its expression in the thalamus and pretectum by Pax6 may be crucial for allowing proneural factors to promote normal neuronal differentiation in this region.

Keywords: Barhl2; Development; Forebrain; Gene expression; Mouse; Pax6; Thalamus; Zona limitans intrathalamica.

Fig. 1

A–L In situ hybridization data for Pax6 and Barhl2 mRNA in adjacent sections cut from embryos at E8.5 and E9.5. M, N Schematics to illustrate the planes of the sections in A–L and to summarize the results. Scale bar for A–F 200 μm, G–L 500 μm. Tel telencephalon, Di diencephalon, BP basal plate

Fig. 2

A–L In situ hybridization data for Pax6 and Barhl2 mRNA in adjacent sections cut from embryos at E10.5 and E11.5. M Schematics to illustrate the planes of the sections and to summarize results at E10.5. Scale bars 500 μm. PT pretectum, Th thalamus, pTh prethalamus, ZLI zona limitans intrathalamica, EmT eminentia thalami, Hyp hypothalamus, PSPB pallial–subpallial boundary, Tel telencephalon, vTel ventral telencephalon, dTel dorsal telencephalon

Fig. 3

A–L In situ hybridization data for Pax6 and Barhl2 mRNA in adjacent sections cut from embryos at E12.5 and E13.5. M Schematic to illustrate the planes of the sections. Scale bar for A–F 500 μm, G–L 250 μm. PT pretectum, Th thalamus, pTh prethalamus, Hyp hypothalamus, ZLI zona limitans intrathalamica, EmT eminentia thalami, PSPB pallial–subpallial boundary, Tel telencephalon

Fig. 4

A–O Sagittal sections of embryos treated with immunostaining for Pax6 protein and in situ hybridization for Barhl2 mRNA. P Schematic to illustrate the approximate plane of section. Scale bars for A–I 250 μm, J–0 500 μm. Tel telencephalon, Di diencephalon, Mes mesencephalon, BP basal plate, AP alar plate, PT pretectum, Th thalamus, pTh prethalamus, ZLI zona limitans intrathalamica

Fig. 5

A Sagittal section of E9.5 embryo immunostained for Pax6 protein and in situ hybridization for Barhl2 mRNA. Scale bar 200 μm. B Detail of area outlined in A. Scale bar 25 μm. C, D DAPI staining and triple-staining for DAPI, Pax6 and Barhl2 within the prethalamus in the area outlined in B. Scale bar 10 μm

Fig. 6

A Coronal section of E12.5 embryo treated with immunostaining for Pax6 protein and in situ hybridization for Barhl2 mRNA. B–E, F–I Higher magnification of tissue outlined in A. J–M Detail of the eminentia thalami. Scale bars for A 100 μm, B–M 25 μm. PT pretectum, Th thalamus, pTh prethalamus

Fig. 7

A–L Coronal sections of embryos treated with immunostaining for Pax6 protein and in situ hybridization for Barhl2 mRNA. M Schematic to illustrate the approximate plane of section. Scale bars 500 μm. PT pretectum, Th thalamus, pTh prethalamus, ZLI zona limitans intrathalamica, EmT eminentia thalami, Tel telencephalon

Fig. 8

A Schematic to illustrate the area of the ventricular zone across which the intensity plots were calculated. Scale bar 100 μm. B Intensity plots for 12 embryos, three at each of four ages from E10.5 to E13.5, with linear regression trend lines. C Table of the gradients of the trend lines for each developmental stage, and the mean gradient for each set of three embryos. D Bar chart of the mean values of the gradients recorded for each group of three embryos, with standard errors. PT pretectum, ZLI zona limitans intrathalamica, dpc days post conception

Fig. 9

A–F In situ hybridization for Barhl2 mRNA on comparable sections from Pax6 ^+/+ and Pax6 ^Sey/Sey embryos at E11.5. G–L In situ hybridization for Barhl2 mRNA on comparable sections from Pax6 ^+/+ and Pax6 ^Sey/Sey embryos at E12.5. PT pretectum, Th thalamus, pTh prethalamus, ZLI zona limitans intrathalamica, EmT eminentia thalami, Tel telencephalon, Hyp hypothalamus. Scale bars 500 μm

Fig. 10

Immunohistochemistry for Nestin on sections of the E13.5 Pax6 ^+/+ and Pax6 ^Sey/Sey diencephalon. Th thalamus, pTH prethalamus. Scale bars 500 µm in A, C and 250 µm in B, D

Fig. 11

A–L In situ hybridization for Barhl2 and Ngn2 mRNA on coronal sections from Pax6 ^+/+ embryos at E11.5–E13.5. Scale bars for A–I 500 μm, J–L 50 μm. M–O In situ hybridization for Barhl2 and Shh mRNA, high magnification image showing detail of the ZLI. Scale bar 50 μm. PT pretectum, Th thalamus, pTh prethalamus, ZLI zona limitans intrathalamica, EmT eminentia thalami, Tel telencephalon, EmT eminentia thalami