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First published online 16 March 2005
doi: 10.1242/dev.01731

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Transcriptional control of early tract formation in the embryonic chick midbrain

Frank R. Schubert^*, and Andrew Lumsden

MRC Centre for Developmental Neurobiology, King's College London, 4th Floor New Hunt's House, Guy's Campus, London SE1 1UL, UK

View larger version (115K): [in a new window]   Fig. 3. Alterations to the mlf following ectopic expression of Sax1. (A-I) Lateral view the ventral mesencephalon of embryos, stained for neurofilament protein (brown) and eGFP mRNA (A-F, blue), analysed 1 day (A-C, HH18) or 2 days (D-F, HH21; G-I, HH19) after electroporation. The line marks the MFB. (A,D,G) After electroporation of the GFP-expressing control construct, the axon tracts appear normal. (B,E,H) Following ectopic expression of pCAß-Sax1-IRES-GFP, the mlf is enlarged (arrows in B,E; also compare brackets in G and H), and the pc is no longer detectable (arrowhead in E). (C,F,I) Ectopic expression of the VP16Sax1 constructs results in a diminished mlf. (J,K) The nucleus of the mlf revealed by retrograde labelling with DiI from the hindbrain, demonstrating enlargement of the mlf after ectopic Sax1 expression (K) compared with an embryo expressing the control construct (J). The caudodorsal and rostroventral subnuclei of the mlf (arrowheads in J) are not distinct in Sax1-expressing embryos (K). (L) Map of the pCAß-LINK-IRESeGFPm5-ClaI expression vector, outlining the CMV enhancer/chick ß-actin promoter (CAß), the multiple cloning site (MCS) into which genes of interest can be inserted and the coding region for GFP linked by the ECMV internal ribosome entry site (IRES). (M,N) Lateral view of the ventral mesencephalon stained for phospho-Histone H3 as proliferation marker. Embryos expressing the control construct (M) or the Sax1 construct (N) show no obvious difference. (O) Schematic representation of the pCAß-Sax1-IRES-GFP and pCAß-VP16Sax1-IRES-GFP expression vectors that are based on pCAß-LINK-IRESeGFPm5-ClaI. Also depicted is the domain swap where the eh1-like transrepression domain of Sax1 (red) is replaced by the transactivation domain of Herpes simplex VP16 (green) to yield the constitutive transcriptional activator VP16Sax1. mlf, medial longitudinal fascicle; nIII, oculomotor nucleus; pc, posterior commissure.

View larger version (56K): [in a new window]   Fig. 1. Differential gene expression at the ventral MFB. (B-E) Cross-section through the mesencephalon of HH20 chick embryos, showing the area depicted in the schematic view (A). (F) Schematic lateral view of a HH25 chick brain, indicating the location of major tracts. Highlighted are the mlf in red and the pc in green. Black circles at the ventral MFB represent the respective nuclei. (G-J) Lateral view of the ventral mesencephalon and pretectum of HH25 chick embryos, focussing on the ventral MFB (see boxed area in F). The position of the MFB is marked by a line. Red staining in B-E,G-I indicates the expression of Isl1 (arrowhead in B,G; ventral stripe, marking the oculomotor nucleus) and Pax6 (arrow in B,G; dorsal stripe). (B,G) Blue staining indicates the expression of Sax1 in a broad domain dorsal and rostral to the oculomotor nucleus. Sax1 and Pax6 signals overlap. (C,H) Expression of Six3 around the ventral MFB. Six3 staining in the ventral mesencephalon (blue) is split into two longitudinal domains by the intervening Pax6 stripe (arrow). (D,I) The mRNA for Emx2 (blue) is also largely localised in two longitudinal stripes in the ventral mesencephalon, divided by the Pax6 signals (arrow). Emx2 and Isl1 signals partially overlap at the MFB (arrowhead). (E,J) Pax6 is expressed in a single longitudinal stripe in the ventral mesencephalon (arrow in J), dorsal to the oculomotor nucleus (red Isl1 signal in E). Pax6 is also expressed in the pretectum, in a ventral patch of cells (arrowhead in J), and dorsally. (K-O) Schematic representation of nuclei position (K) and expression domains for Sax1 (L), Six3 (M), Emx2 (N) and Pax6 (O) in the ventral midbrain and pretectum. Numbers in K represent the midbrain arcs 1-3. III, oculomotor nerve; INC, interstitial nucleus of Cajal; llf, lateral longitudinal fascicle; mes, mesencephalon; mlf, medial longitudinal fascicle; nIII, oculomotor nucleus; nPC, nucleus of the posterior commissure (ventral part); pc, posterior commissure; pt, pretectum; RN, red nucleus; tpoc, tract of the postoptic commissure.

View larger version (63K): [in a new window]   Fig. 2. Expression of Sax1 in the nucleus of the mlf. (A) Schematic lateral view of the embryonic mesencephalon and pretectum, showing the expression domains of Sax1 in blue, and indicating major axon tracts and the location of their cell bodies. The fully developed expression pattern of Sax1 is depicted as seen in embryos from HH21. (B) Lateral view of the MFB of an HH27 chick embryo, with retrograde labelling of the mlf (DiI, red) from the rostral hindbrain and the pc (DiO, green) from the dorsal pretectum (see red and green asterisks, respectively, in A), showing the partial overlap of the corresponding nuclei for these axon tracts originating from the ventral MFB. (C) Lateral view of chick head at HH17, with the Sax1 expression domains in rhombencephalon (arrowhead), mesencephalon (arrow) and pretectum in blue. (D) Lateral view of the ventral mesencephalon of an HH23 embryo. Sax1 mRNA signals are in blue, and the red staining shows the mlf, retrograde-labelled with fluorescein-labelled dextran from the rostral hindbrain (see red asterisk in A). The nucleus of the mlf (arrows) overlaps with the Sax1 expression domain. (E-H) Double-labelling for Sax1 mRNA in blue and neurofilament protein in brown. Lateral views of the ventral mesencephalon of an HH17 (E) and an HH21 (F) embryo show that the Sax1 domain overlaps with the cell bodies for the mlf (arrow in E). At HH21, Sax1 staining is weakly visible in the dorsal pretectum. (G) A horizontal section through the ventral mesencephalon of an HH22 embryo (boxed area magnified in H) shows Sax1-expressing neurons projecting into the mlf (arrows). The MFB is marked by a line in D,F,G. INC, interstitial nucleus of Cajal; mes, mesencephalon; mlf, medial longitudinal fascicle; nIII, oculomotor nucleus; pc, posterior commissure; pt, pretectum; r1, rhombomere 1.

View larger version (137K): [in a new window]   Fig. 4. Regulation of other homeobox genes by Sax1. (A-C) Lateral view of brains at HH18, stained for Emx2 mRNA in blue and GFP mRNA in red. After electroporation of the control construct (A), the single Emx2 stripe in the ventral mesencephalon (the dorsal stripe only appears at HH20) is evident. Ectopic expression of Sax1 represses Emx2 expression in the mesencephalon (B, arrow), while expression of VP16Sax1 leads to an enlargement of the ventral Emx2 stripe (C, arrow), and also induces ectopic spots of Emx2 in the dorsal mesencephalon and diencephalon (arrowheads). (D-F) Expression of Six3 in blue and GFP in red, following electroporation. The pattern of Six3 is normal after electroporation of the control construct (D), while ectopic expression of Sax1 abolishes the expression of Six3 in the mesencephalon (E, arrow). VP16Sax1 has no effect on Six3 expression (F, arrow). (G-I) Phox2a expression in an HH21 embryo, 2 days after electroporation. In a control embryo, Phox2a signals label the trochlear and oculomotor nuclei (G). Unilateral ectopic expression of Sax1 leads to a rostral expansion of the Phox2a expression in the oculomotor nucleus (H, arrow) compared with the control side (arrowheads marks the normal rostral limit of Phox2a expression), while VP16Sax1 represses Phox2a expression in the oculomotor and trochlear nuclei (I, arrows). (J-L) Pax6 expression in an HH21 embryo, 2 days after electroporation. The Pax6 stripe in the ventral mesencephalon is largely unaffected by Sax1 (K, arrowhead) or VP16Sax1 (L) expression. By contrast, the ventral Pax6 domain in the pretectum, prominent in embryos expressing the control construct (J), is lost following ectopic expression of Sax1 (K, arrow). mes, mesencephalon; nIII, oculomotor nucleus; nIV, trochlear nucleus; pt, pretectum.