Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning

Abstract

Telencephalic patterning centers, defined by the discrete expression domains of distinct morphogens, Fgfs in the commissural plate (CoP), Wnts and Bmps in the cortical hem, and a ventral domain of Sonic hedgehog (Shh), are postulated to establish during development the initial patterning of the telencepahlon, including the neocortex. We show that the expression patterns of Sp5, Sp8, and Sp9, members of the Sp8-like family that are homologues of Drosophila buttonhead, correlate during early embryonic development with these three telencephalic patterning centers. To study potential functional relationships, we focused on Sp8, because it is transiently expressed in the CoP coincident with the expression of Fgf8, a morphogen implicated in area patterning of the neocortex. We also show that Sp8 is expressed in cortical progenitors in a high to low anterior-medial to posterior-lateral gradient across the ventricular zone. We used in utero electroporation of full-length and chimeric expression constructs to perform gain-of-function and loss-of-function studies of interactions between Sp8 and Fgf8 and their roles in cortical area patterning. We show that Fgf8 and Sp8 exhibit reciprocal induction in vivo in the embryonic telencephalon. Sp8 also induces downstream targets of Fgf8, including ETS transcription factors. In vitro assays show that Sp8 binds Fgf8 regulatory elements and is a direct transcriptional activator of Fgf8. We also show that Sp8 induction of Fgf8 is repressed by Emx2 in vitro, suggesting a mechanism to limit Fgf8 expression to the CoP. In vivo expression of a dominant negative Sp8 in the CoP indicates that Sp8 maintains expression of Fgf8 and also its effect on area patterning. Ectopic expression of Sp8 in anterior or posterior cortical poles induces significant anterior or posterior shifts in area patterning, respectively, paralleled by changes in expression of gene markers of positional identity. These effects of Sp8 on area patterning oppose those induced by ectopic expression of Fgf8, suggesting that in parallel to regulating Fgf8 expression, Sp8 also activates a distinct signaling pathway for cortical area patterning. In summary, Sp8 and Fgf8 robustly induce one another, and may act to balance the anterior-posterior area patterning of the cortex.

Figure 1

Expression patterns of Sp5 in embryonic mouse forebrain and relationship with cortical hem and choroid plexus. (a-i) Sp5 expression relates to the cortical hem, a source of Wnts and Bmps. Shown are in situ hybridizations using DIG-labeled riboprobes for Sp5 on sagittal (a, d, g) or coronal (b, e, h) sections and Wnt2b (c, f, i) of mouse forebrain at E10.5 to E13.5. Sp5 expression is observed around the cortical hem and Cpe as being highest in medial and posterior parts of dorsal telencephalon (dTel; arrowhead), and quickly downregulated at E13.5. Unlike the expression of Sp8, Sp5 expression domains overlap those of Wnts throughout E10.5 to 13.5. Ctx, cortex; GE, ganglionic eminence; Hp, hippocampus. Scale bars: 0.5 mm

Figure 2

Expression patterns of Sp9 in embryonic mouse forebrain and relationship with the Shh expression domain in ventral telencephalon. (a-i) Comparison of Sp9 and Shh expression. Sp9 expression is not detected in cortex, but it is expressed highly in the SVZ of the MGE, the source of GABAergic interneurons that populate the cortex (arrowheads), and weakly in the LGE that populates interneurons migrating to olfactory bulbs. Sp9 expression domains are observed in the vicinity of the Shh expression domain, but does not overlap them. Ctx, cortex. Scale bars: 0.5 mm.

Figure 3

Expression patterns of Sp8 in embryonic mouse forebrain and relationships with expression domains of Fgf8 in the CoP. (a-i) Shown are in situ hybridizations of sections of mouse forebrain at E10.5 to E13.5 using DIG-labeled riboprobes for Sp8 on sagittal (a, d, g) or coronal (b, e, h) sections, and for Fgf8 (c, f, i). Sp8 is expressed in the dTel VZ, with highest levels in the medial and anterior parts. Sp8 is also expressed robustly in the LGE and at lower levels in the MGE. Sp8 expression overlaps the Fgf8 expression domain in the CoP at E10.5 (arrowhead in (b, c)), but gradually become excluded at E11.5 (arrowhead in (e, f)). (j, k) Whole mount in situ hybridizations done on E9.5 embryos with DIG-labeled riboprobes for Sp8 (j), and Fgf8 (k), respectively. The Sp8 A/P gradient is evident at this stage (j). Arrowheads indicate expression domains. Ctx, cortex; GE, ganglionic eminence; Hp, hippocampus. Scale bars: 0.5 mm (a-f); 0.5 mm (g-i); 0.5 mm (j, k).

Figure 4

Reciprocal induction between Sp8 and Fgf8 in vivo. (a-h) Induction of Sp8 by ectopic expression of Fgf8. Coronal sections of E13.5 forebrains that were electroporated in utero at E11.5 with Fgf8b (a-d) or a control vector (e-h) mixed with EGFP and processed for in situ hybridization using S³⁵ riboprobes for Sp5 (d, h), Sp8 (b, f) and Sp9 (c, g), respectively. Induction of Sp8 was detected in the electroporated domain marked by EGFP (a, e) (arrowheads). Sp5 and Sp9 were not induced by ectopic expression of Fgf8 (c, d). (i-p) Induction of Fgf8 by ectopic expression of Sp8 or the dominant active form of Sp8 (Sp8-VP16). E13.5 brains were electroporated with Sp8 (i, j), Sp9 (k, l), Sp5 (m, n), or Sp8-VP16 (o, p), mixed with EGFP, and processed for in situ hybridization using Fgf8 riboprobes. Fgf8 induction was detected in Sp8 (j) and Sp8-VP16 (p) electroporated brains. Weak induction of Fgf8 was also observed by the electroporation of Sp9 (l), which shares identical btd and Zn-finger domains with Sp8. Fgf8 induction was not detected in brains electroporated with Sp5 (n). Scale bars: 0.5 mm (a-p).

Figure 5

Sp8 induces Fgf8 downstream targets. (a-j) Induction of ectopic ETS TFs, Pea3, Erm and Er81 by the ectopic expression of Sp8. Coronal sections of E13.5 forebrains that were electroporated at E11.5 with Sp8 (a-e) or a control vector (f-j), mixed with EGFP, and processed for in situ hybridization using S³⁵ riboprobes for Sp8 (b, g), Pea3 (c, h), Erm (d, i) and Er81 (e, j), respectively. EGFP (a, f) marks the electroporation domains (arrowheads). Pea3 (c), Er81 (d) and Erm (e) were induced by ectopic expression of Sp8. Scale bars: 0.5 mm.

Figure 6

Sp8 directly binds the Fgf8 promoter and Emx2 represses Sp8 induction of Fgf8. (a) Sequence of a 585 bp fragment, containing the 555 bp immediate upstream region of the mouse Fgf8 transcription start site and the 30 bp 5' UTR of Fgf8 having six putative Sp8 binding sites. Putative Sp8 binding sites predicted from Sp1-binding motifs (GGGGCGG or CCCCGCC) are underlined. (b) Gel retardation assay of Sp8 and Fgf8 promoter fragments. P³²-labeled Fgf8 promoter fragments show slow mobility after incubation with the Sp8-expressed lysate (black arrow) compared to the unbound DNA (gray arrow). These bands are not detected in the control lysate sample and are diminished in the presence of an equal (×1) or five-fold (×5) amount of unlabeled DNA compared to labeled probes. (c) Sp8 binding to the oligonucleotide of the Fgf8 promoter region. HA-Sp8 was co-precipitated with biotinylated non-mutated oligonucleotide corresponding to a putative Sp8 binding site, but not with oligonucleotides with a mutated core recognition sequence. (d) Luciferase reporter assay for induction of Fgf8. Control or test vectors (Sp8, Sp9, Emx2) were transfected independently or in combinations into C3H10T1/2 cells also transfected with an Fgf8 reporter construct consisting of a promoter element for Fgf8 and a firefly luciferase reporter vector. The relative effectiveness of Fgf8 induction was assessed by measuring luciferase activity. Sp8 robustly induces expression of the Fgf8 reporter construct; this Sp8 induction is suppressed by co-expression of Emx2. Sp9 modestly induces expression of the Fgf8 reporter construct; again, this is suppressed by Emx2. Emx2 alone has no effect on Fgf8 induction, similar to a control empty vector.

Figure 7

Anterior electroporation of a dominant negative form of Sp8 inhibits endogenous Fgf8 expression in the commissural plate and results in an anterior shift in area patterning. Anterior electroporation of a dominant negative form of Sp8 inhibits endogenous Fgf8 expression in the commissural plate and results in an anterior shift in area patterning. (a-h) Anterior electroporation of a dominant negative form of Sp8 inhibits endogenous Fgf8 expression in the CoP. Electroporation of the Sp8 dominant negative chimeric construct, Sp8-engrailed, suppresses endogenous Fgf8 expression in the CoP (b), as well as expression of Pea3, a downstream target of Fgf8 (f) at E14.5. Fgf8 and Pea3 expression in brains with similar electroporation of the control engrailed construct (a, e) is indistinguishable from non-transfected brains (not shown). Arrowheads indicate Fgf8 (a, b) and Pea3 expression domains (e, f). An EGFP expression construct co-electroporated with Sp8-engrailed and engrailed control constructs are shown in parallel and define the transfection domain (c, d, g, h). (i, j) Anterior ectopic expression of Sp8-engrailed results in an anterior shift of cortical areas. Tangential cortical sections through layer 4 of a P7 brain electroporated at E11.5 with control vector (i) or Sp8-engrailed (j) and processed for serotonin immunostaining to reveal the primary sensory areas: somatosensory (S1), auditory (A1) and visual (V1). In cortex of brains with anterior electroporation of Sp8-engrailed, S1 shifts far anteriorly, resulting in a very small amount of cortex remaining for motor (M) areas. The arrow in (j) indicates putative visual areas. Scale bars: 0.5 mm (a-h), 1.0 mm (i, j). (k) Quantification of motor area size in Sp8-engrailed electroporated brains. The length of motor areas is measured as a ratio of the length from the edge of anterior flattened brains to the staining of S1 regions (M_L) and the total length (T_L) of serotonin staining of flattened brains. Compared to engrailed control vector cases (28.89 ± 1.35% standard error of the mean (SEM, n = 5), brains electroporated with Sp8-engrailed show shrunken motor areas (13.66 ± 1.34% SEM, n = 6), as indicated in the middle panel. The right panel shows a scatter plot of individual cases.

Figure 8

Cortical area shifts by ectopic expression of Fgf8 at anterior or posterior sites. Electroporation of a CAG-Fgf8 vector was done at E11.5 and later, at P7, the brains were processed and tangential sections of their cortical areas were visualized by serotonin staining as described in Methods. (a) Ectopic expression of Fgf8 at an anterior site in E11.5 brains causes posterior area shifts with motor (M) area expansion (n = 2 of 2 electroporated cases; this finding replicates that of Fukuchi-Shigomori and Grove [18], therefore we did not perform additional cases) compared to control electroporated brains. (b) In contrast, a posterior electroporation of Fgf8 induces anterior area shifts accompanied by an expansion of V1 (n = 4 of 6 electroporated cases). We also performed electroporations using medial and lateral approaches at mid-posterior levels in an attempt to target the presumptive barrelfield of S1 (posteromedial barrel subfield, PMBSF). (c, d) Medial-posterior electroporation of Fgf8 typically causes an 'elongated' S1 (c) (n = 7 of 10 electroporated cases), whereas lateral-posterior electroporation within the cortical field that would develop as S1 can result in a 'split barrel field' (d) (n = 2 of 9 electroporated cases). The middle of the 'PMBSF' in S1 is pointed to by the line. We did not observe 'duplicate' barrels as reported by Fukuchi-Shigomori and Grove [18]. We assume that duplicated barrels are produced only in a unique situation with an appropriate combination of timing, size and position of the ectopic Fgf8 expression domain, and level of Fgf8 expression, required to partially duplicate the barrel pattern. Arrows indicate targeted locations of the electroporation sites. M, motor areas. Scale bar: 1.0 mm.

Figure 9

Ectopic expression of Sp8 and a dominant active form of Sp8 induce area shifts that oppose Fgf8. (a, b) Ectopic expression of Sp8 (b) at an anterior site of E11.5 brains causes anterior area shifts with a substantial reduction of motor (M) areas compared to control electroporated brains (a) that are indistinguishable from wild type (not shown). Areas of tangential section at P7 cortices are visualized by serotonin immunostaining. (c, d) Anterior ectopic expression of a dominant active form of Sp8, Sp8-VP16 (d), but not theVP16 domain alone (c), induces anterior area shifts similar to Sp8. The dashed line is at the E1 barrel position of control brain for the A-P comparison. (e) Quantification of motor areas in Sp8-, Sp8-VP16, or control-transfected brains. Anterior electroporation of either Sp8 or Sp8-VP16 causes reduction of the motor areas ratio (Sp8, 18.07 ± 1.52% SEM, n = 10; Sp8-VP16, 19.12 ± 2.18% SEM, n = 9), compared to the control cases (control vector; 29.05 ± 0.691%, SEM, n = 5; VP16, 26.88 ± 0.853%, SEM, n = 5). The right panel shows a scatter plot of individual cases. (f-i) Posterior ectopic expression of Sp8 and Sp8-VP16 induces posterior cortical area shifts. Sp8 (g) and Sp8-VP16 (i) ectopic expression at posterior sites causes a posterior shift in area patterning with a reduction in size of V1 areas, whereas expression of an empty vector (F) or the VP16 domain alone (H) has no effect on area patterning. The dashed line is at the A1 barrel position of control brain. Scale bar: 1.0 mm. (l) Quantification of caudal areas in Sp8-, Sp8-VP16, or control-transfected brains. Posterior electroporation of either Sp8 or Sp8-VP16 causes reduction of caudal area ratio (Sp8, 24.42 ± 2.03%, SEM, n = 6; Sp8-VP16, 24.89 ± 0.700% SEM, n = 18), compared to the control cases (control vector, 33.02 ± 0.581%, SEM, n = 5; VP16, 31.16 ± 0.430%, SEM, n = 5). The right panel shows a scatter plot of individual cases. EP, electroporation; M_L, motor length; C_L, caudal length;T_L, total length.

Figure 10

Sp8 changes region-specific gene expression in the cortex. (a, d, g, j) Dorsal views of P1 brains processed for whole mount in situ analysis with digoxigenin labeled-cad8 riboprobes following electroporation at an anterior (a, d), or posterior (g, j) location of empty control (a, g) or Sp8 (d, j) vectors at E11.5. Arrowheads indicate the posterior border of the cad8 expression domain in anterior cortex. Anterior electroporation of Sp8 results in an anterior contraction of the cad8 domain (d), whereas posterior electroporation of Sp8 results in a posterior expansion of the cad8 domain (j). Electroporation of an empty vector has no effect on the cad8 domain (e, m). (b-f, h-l) Sagittal sections of P1 brains stained with an anterior marker gene, RORb probes (b, e, h, k), or a S1 marker gene, ephrin-A5 probes (c, f, I, l), following electroporation at an anterior (b, c, e, f) or posterior (h, I, k, l) domain of empty vector (b, c, h, i) or Sp8 (e, f, k, l). Arrowheads indicate the posterior border of the RORb expression domain or the putative M1/S1 and S1/V1 borders of ephrin-A5 expression. Coincident to the area shifts detected by cad8 expression, RORb and ephrin-A5 expressions are shifted anteriorly in anterior Sp8-overexpressed brains, and shifted posteriorly in posterior Sp8-electoporated brains. Scale bars: 0.5 mm (e, f, m, l); 0.5 mm (b, c, e, f, h, I, k, l). (m) Quantification of cad8 expression domains in Sp8- or control transfected brains. Anterior electroporation of Sp8 reduces motor area (M_A) size detected as cad8 domain (8.2 ± 1.12%, SEM, n = 13) compared to the control cases (15.2 ± 0.297%, SEM, n = 9). In contrast, posterior electroporation of Sp8 enlarges motor areas (19.97 ± 0.771%, SEM, n = 6) compared to the control cases (14.37 ± 0.241). The bottom panel shows a scatter plot of individual cases. EP, electroporation.

Figure 11

Summary of the Sp8-like family expression patterns related to telencephalic patterning centers. Position of patterning centers in relationship to Sp expression at E10.5. Sp5 expression is observed around the cortical hem, which expresses Wnts and Bmps, as being highest in medial and posterior parts of dTel. Sp8 is expressed in a high to low anterior-medial to posterior-lateral gradient across the entire cortical ventricular zone, and transiently overlaps with Fgf8 expression in the commissural plate. Sp9 is highly expressed in the mantle zone of the MGE, coincident with the domain of Shh expression. See the text for details.

Figure 12

Summary of the Sp8 function related to telencephalic patterning centers and cortical area patterning. (a) Schematic diagram of time-dependent expression domains of Sp8 and Fgf8. At E10.5, Sp8 expression (green) is expressed in both progenitor cells in the cortical VZ (cortex) and within the CoP, where it overlaps with the Fgf8 expression domain (overlapping Sp8 and Fgf8 expression in the CoP is colored yellow). Sp8 expression is gradually excluded from the Fgf8 expression domain (red) at E11.5 and later. (b) Domain-dependent regulation of Sp8 TF activity by Emx2. Sp8 forms a reciprocal induction loop with Fgf8 in the CoP. Emx2 (blue) is expressed in cortical progenitors, but not in the CoP. Although Sp8 is expressed in cortex, Emx2 represses the ability of Sp8 to induce Fgf8, thereby restricting Fgf8 expression to the CoP. (c) Electroporation (EP) of Fgf8 and Sp8 results in opposing shifts in cortical area patterning. In early stages of cortical patterning, Sp8 maintains Fgf8 expression in CoP and the Fgf8 signaling pathway, which imposes anterior identity to cortical progenitors. Anterior EP of Fgf8 expression constructs results in enhanced anterior area identities and a corresponding posterior shift in cortical areas. Anterior EP of Sp8 expression constructs has an opposing effect on area patterning to that of Fgf8, and results in an anterior shift in cortical areas. Posterior EP of either Fgf8 or Sp8 have the opposing effect on area patterning compared to their anterior EP. These opposing effects of ectopic expression of Sp8 on area patterning compared to Fgf8 indicate that Sp8 activates a signaling pathway(s) that can overcome the effect of Fgf8 signaling, perhaps by interfering with Fgf8 signaling or dominating it. Furthermore, Sp8 expression in cortical progenitors may trigger distinct signaling pathways that function to facilitate posterior area identity.