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Sonic Hedgehog Control of Size and Shape in Midbrain Pattern Formation

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Science  16 Mar 2001:
Vol. 291, Issue 5511, pp. 2147-2150
DOI: 10.1126/science.1058624

Abstract

Little is known about how patterns of cell types are organized to form brain structures of appropriate size and shape. To study this process, we employed in vivo electroporation during midbrain development to create ectopic sources of Sonic Hedgehog, a signaling molecule previously shown to specify different neuronal cell types in a concentration-dependent manner in vitro. We provide direct evidence that a Sonic Hedgehog source can control pattern at a distance in brain development and demonstrate that the size, shape, and orientation of the cell populations produced depend on the geometry of the morphogen source. Thus, a single regulatory molecule can coordinate tissue size and shape with cell-type identity in brain development.

The determination of cell fate and the spatial organization of differentiated cells are the fundamental processes by which any tissue is organized during development. An attractive mechanism for achieving spatial patterns of different neuronal cell types is through a “positional signal” from a morphogen source that elicits distinct molecular responses in target cells according to their distance from that source (1, 2). Evidence that Sonic Hedgehog (SHH) can serve as a positional signal in vertebrate central nervous system (CNS) development has come mainly from in vitro studies demonstrating that different cell types in spinal cord explant cultures can be produced by different concentrations of recombinant SHH (3–5). We have sought in vivo evidence for positional signaling by SHH in brain development.

To demonstrate SHH effects on brain development, we employed controlled electroporations to express SHH ectopically in chick midbrain at embryonic day 2 (E2), an age when endogenous SHHgene expression is restricted to the ventral midline (Fig. 1A). The embryonic ventral midbrain is an attractive system for studying brain patterning, because it is transiently organized into a regular set of discrete arcuate territories (midbrain arcs) arrayed bilateral to the ventral midline (6). Each of these arcuate territories has a unique molecular identity, based on its expression of specific transcription factors. We assessed the effects of our electroporations at E5, when five molecularly distinct territories can be identified (Fig. 1, B through E). From the ventral midline outward, these territories are arc 1 (marked by PHOX2A + motorneurons), arc 2 (GATA2 +, FOXA2 +), thePAX6 stripe, arc 3 (GATA2 +,FOXA2 ), and the EVX1 stripe. By employing multiple probe wholemount in situ hybridization, we examined the effects of SHH misexpression on midbrain patterning as a whole, rather than on single cell types. Electroporations with alkaline phosphatase cDNA showed that we could make half of the ventral midbrain transgenic, with the other half serving as a control (Fig. 2A). In addition, electroporation itself did not disrupt midbrain arc patterning (Fig. 2B).

Figure 1

SHH gene expression and midbrain arc anatomy. (A and B) Nested expression of SHH (blue) andFOXA2/HNF3β (brown) in E2 brain (A) and E5 midbrain (B). SHH and FOXA2gene expression were used interchangeably to determine extent ofSHH misexpression. In all photomicrographs, dissected brains are presented as flattened wholemounts, with rostral to the top and the ventricular surface facing the viewer. The genes detected with two-color in situ hybridization (20) are noted by color-coded text at the bottom right of each panel. (C to E) Five molecularly distinguished arcuate territories are identified in the E5 ventral midbrain mantle layer (see text). (C) ThePHOX2A+ arc 1 is spatially segregated from theGATA2+ lateral arcs 2 and 3. (D) Spatial relationship of SHH, arcs 1 through 3, and homeobox (Hx) gene expression of PHOX2A (P2),PAX6 (P6), and EVX1 (E1). (E) Cartoon summary of gene expression patterns for SHH (brown),FOXA2 (dark gray) and arc-specific transcription factors at E5. Domains of SHH and FOXA2 gene expression are exaggerated caudally to illustrate the spatial relationships of the markers. The rostral extensions of the SHH andFOXA2 into diencephalon are omitted. III, third ventricle; FB, forebrain; FP, hindbrain floor plate; HB, hindbrain; IS, isthmus; rFP, rostral (or midbrain) floor plate.

Figure 2

SHH misexpression in ventral midbrain. (A and B) Outcome of unilateral control electroporations at E2.5. (A) Alkaline phosphatase (AP) transgene expression at E5 shows AP transfection encompassing one entire side of the ventral midbrain. Electroporated side in this and subsequent panels is the right. (B) E5 arc pattern identified by Hx gene expression is not disrupted by electroporation. PAX3 gene expression marks midbrain tectum, which surrounds the midbrain arcs in flattened whole-mounts. [(C) to (H)] Effects of SHH electroporation at E2. (Cand D) Expanded gene expression of SHH andFOXA2 (C), and PATCHED (PTC) (D) at E5. (E and F) Expanded PHOX2A and GATA2 gene expression at E5. Little overlap is seen between thePHOX2A+ motorneurons in arc 1 and theGATA2+ lateral arcs regardless of the nature of the enlargement of the SHH source. Note that the arc expansions include increases of both length (F) and width (E and F). (G) Increased BrdU labeling (21) over thePHOX2A+ territory at E3. (H) Enlarged right midbrain at E9. The outline of the control left ventral midbrain is imposed (black dots) on the electroporated side to illustrate the increase in midbrain tissue mass. Expanded ISL1+motorneuron population (right) confirms successful SHHelectroporation. TEC, tectum.

We first studied the effects of enlarging the SHH territory in ventromedial midbrain (7). As expected (8–10), SHH overexpression led to the up-regulation of transcriptional targets of SHH, includingFOXA2/HNF3β and the SHH receptor PATCHED (Fig. 2, C and D). In addition, the entire arcuate pattern of the ventral midbrain, including the PHOX2A +motorneurons of arc 1 and the GATA2 +lateral arcs, was expanded (Fig. 2, E and F). Despite these expansions, the relative positions of the arcuate territories to each other and to the SHH source were maintained.

SHH misexpression stimulated cell proliferation (Fig. 2G) and produced frank tissue growth of the ventral midbrain (Fig. 2H). Thus, the observed patterning effects could be due to the expansion of pools of dedicated precursor cells rather than the respecification of cell fates by SHH. Consequently, we used microelectroporation to create small sources of ectopic SHH in dorsal midbrain, where arcs are never seen. These experiments established that an ectopic SHH source is sufficient to elicit a complete set of midbrain arcs (Fig. 3, A and B).

Figure 3

SHH serves as a positional signal in midbrain pattern formation. (A and B) An ectopic SHH source is sufficient to elicit a full set of tegmental arcs. (A) Side view of an E5 brain showing ectopic arcs (arrowhead) in lateral tectum. Arrow indicates position of normal arcs in ventral midbrain. Rostral is to the right. (B) Dorsal view of an E5 brain showing a complete ectopic arc pattern in caudal tectum. Rostral is to the top. Isthmus (blue) identified by WNT1 gene expression. (C and D) Respecification of ventral midbrain cell types as an ectopic source of SHH is moved toward the endogenousSHH source. (C) Ectopic SHHin lateral tegmentum, assayed by FOXA2 gene expression, results in a mirror-image duplication of the normal arc pattern. (D) As the distance between the two sources is reduced, only a partial duplication of the arc pattern results. Arc 3 and theEVX1 territory are present on both sides of the merged pattern, but are lost from the center of this “contour map” of SHH positional signaling. (E) Orientation of the SHHsource determines the orientation of the pattern. An ectopicSHH source concentrated along the isthmus elicits arcs arrayed parallel to the isthmus and at 90° to the axis of the normal arc pattern. (F) The shape of the SHH source determines the shape of the pattern. A spot source of SHHproduces a bull's-eye pattern of gene expression. Tr, trochlear nucleus.

To explore the mechanism by which SHH generates multiple midbrain cell types, we introduced sources of SHH within the ventral midbrain at varying distances from the ventral midline. When placed in lateral tegmentum, the ectopic SHH source produced a complete mirror-image duplication of the arc pattern (Fig. 3C). As the two SHH sources were brought close together, however, cell types normally occupying arcuate territories most distant from theSHH source, such as arc 3 and the EVX1 territory, were lost from the center of the pattern (Fig. 3D), suggesting that the level of the signal provided by the two nearby SHH sources is too high to specify arc 3 or the EVX1 territory. Thus, a SHH source does not simply induce the midbrain arc pattern, but provides a positional signal to specify cell-type identity.

The next set of experiments explored how a positional signal controlling cell-type specification could be deployed to produce brain structures of the required shape and orientation. In hindbrain and spinal cord development, most neuronal cell types are arranged into longitudinal columns bilateral to the ventral midline. A simple account of this patterning is that the SHH in the ventral midline provides a positional signal and that the readouts of this signal are longitudinal columns because the source is a longitudinal stripe. Shifting the orientation of the SHH source by 90° should still produce a parallel array of stripes, but they should lie perpendicular to the normal arc pattern. By microelectroporation, we directed ectopic SHH to the isthmus (midbrain-hindbrain junction), creating a morphogen source orthogonal to the ventral midline. The ectopic arcs ran perpendicular to the normal arcs, forming an L-shaped pattern of midbrain cell types that conformed to the L-shaped SHHsource (Fig. 3E). Thus, the orientation of the SHH source determines the orientation of the arc pattern.

If a line source of SHH elicits stripes, then a spot source should produce a radial pattern. Such patterns have been demonstrated with morphogens of the TGFβ family (11, 12). We found that microelectroporations producing spots of SHH gene expression created bull's-eye patterns of arc-specific rings, which resembled the eyespot of a butterfly's wing (Fig. 3F). Eyespots are known to express hedgehog transcripts (13) and to function as organizers in transplant experiments (14). Our findings establish that ectopic hedgehog is sufficient to organize an eyespot-like pattern in epithelial tissue.

Finally, we found that small SHH sources elicited small patterns of ectopic arcs and large sources produced large patterns (Fig. 4). In essence, the size of the SHH source sets up the size of the field of ectopic arcs and the sizes of the individual arcuate territories.

Figure 4

The size of the ectopic arc pattern is determined by the size of the SHH source. (A and B) Hx gene expression shows that small (A) or large (B) arc patterns can be produced and that the size of the pattern is proportional to the size of the SHH source (B). The right ventral midbrain and the part of tectum containing the ectopic pattern are shown in each flat-mount. Ventral midline is to the left. Bar, 448 μm.

Our findings provide evidence that a SHH positional signal is able to establish a complete pattern of ventral midbrain cell types. We also demonstrate that SHH in brain development, like hh,wg, and dpp in Drosophila limb development (15–17), can coordinate patterning with size control. Our results further point to the critical role that the geometry of a morphogen source plays in patterning vertebrate CNS structures and suggest a reason why the development of the floor plate, the principal source of SHH in spinal cord and brainstem development, is under such extensive developmental regulation (18, 19). To pattern the CNS correctly, not only the location, but also the shape and size, of the SHH source must be precise because the consequences for the patterning of neuronal assemblies are profound. In spinal cord development, SHH is expressed in a tight midline stripe and longitudinal columns are formed. By contrast, in midbrain development, SHH expression fans out from the ventral midline, and arcuate territories in register with the morphogen source are the result.

  • * To whom correspondence should be addressed. E-mail: cliff{at}drugs.bsd.uchicago.edu

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