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doi: 10.1242/10.1242/dev.00179

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A role for midbrain arcs in nucleogenesis

Seema Agarwala and Clifton W. Ragsdale*

Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, IL 60637, USA



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  		Fig. 1. The medial arc contains three molecularly distinct territories. In each row, pairs of E5 embryos are shown in whole-mount preparations (left column) and in cross-sections (right column). In the left panel, rostral is towards the top and the ventricular surface faces the viewer. In the right panel, the ventricular surface lining the midbrain vesicle is towards the top. Markers are shown in color-coded text at the bottom of each panel. (A,B) ACHE gene expression (purple) distinguishes arcs (labeled 1-3, medial to lateral). Two-color demonstration of ACHE, PHOX2A and ISL1 gene expression establishes that the PHOX2A+ and ISL1+ cells are restricted the medial arc (arc 1), but do not fill it completely. Arrowhead in A indicates the rostral limit of PHOX2A labeling. Arrows in B indicate ISL1-negative territories within the ACHE-rich medial arc (see below). (C) TH (arrow) and PHOX2A gene expression demonstrates that some midbrain DA neurons are also found in the medial arc. (D) The PHOX2A+ and the TH+ neurons occupy discrete ventricular-pial positions within the medial arc. (E,F) A population of BRN3A+ cells is detected in the medial arc rostrally. (E) These cells overlap the PHOX2A labeling caudally and conform to the ACHE+ medial arc territory that lies rostral to the arrowhead in A. (F) When viewed in cross-section, the BRN3A+ cells lie pial to the PHOX2A+ territory. (G,H) EMX2 gene expression also identifies this rostral medial arc territory. Relationship of medial arc EMX2 labeling to the ISL1+ OMC is illustrated. Within the medial arc, BRN3A and EMX2 are expressed exclusively within this rostral territory. BRN3A and EMX2 expression is not, however, restricted to the medial arc in midbrain development. Both genes are expressed in developing tectum (E, and data not shown), EMX2 identifies arcs 2-4 (G,H) and by E7 BRN3A marks out other ventral midbrain structures as well (not illustrated) (Fedtsova and Turner, 2001Go; Fedtsova and Turner, 1995Go). 1,2,3, arcs 1, 2 and 3; III, third ventricle; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; tec, tectum.

 


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  		Fig. 2. Axonal projections distinguish red nucleus and oculomotor complex territories within the medial arc. (A,B) Tracer deposits delivered to the third cranial nerve establish that OMC neurons lie within the PHOX2A-rich territory of the medial arc. Retrogradely labeled neurons identified by anti-fluorescein alkaline phosphatase histochemistry appear as dark purple cells in E5 midbrain whole-mount (A) and cross-section (B) preparations. (C,D) Whole-mount preparations of E6 explants injected with fluoresceinated dextrans and cultured for 4 hours. (C) Anterograde labeling from a deposit in ventromedial midbrain (asterisk) identifies a crossed projection from the medial arc to the contralateral hindbrain (arrow). (D) Retrograde labeling from a hindbrain tracer deposit (asterisk) identifies red nucleus (RN) neurons in contralateral midbrain. Arrowheads in C,D indicate the midbrain-hindbrain junction. Control deposits placed in more caudal and lateral hindbrain (n=54) failed to label contralateral ventral midbrain, suggesting that at E6 RN axons have not reached the spinal cord. (E,F) Cross-sections of E6 explants where PHOX2A (E) and BRN3A (F) gene expression were combined with retrograde labeling from tracer deposits in contralateral (left) hindbrain. The retrogradely labeled RN neurons lie pial to the PHOX2A+ OMC (E) and are contained within the BRN3A+ territory of the medial arc (F). III, third ventricle; FP, floor plate of hindbrain; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus.

 


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  		Fig. 3. The pRN originates within the ventral midbrain. Explant cultures of ventral midbrain prepared at E3, a day before a BRN3A+ pRN can be identified, and collected 2-3 days later. (A,B) BRN3A+ pRN is detected in ventral midbrain explants grown in the presence (A) or absence (B) of adjoining dorsal midbrain tissue (PAX3+/BRN3A+). (C,D) Expression of BRN3A (blue) in the ventral midbrain overlaps the rostral half of the PHOX2A (brown) gene expression territory, showing that an organotypic medial arc with appropriate three-dimensional organization is formed in midbrain explants grown with (C) or without (D) tectal tissue. Tectal tissue in cultured midbrain explants is distinguished by two markers: PAX3, which is expressed in dorsal midbrain at the time of culturing; and BRN3A, which is detected in dorsal midbrain by E5. OMC, oculomotor complex; RN, red nucleus; tec, tectum; Tr, trochlear nucleus.

 


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  		Fig. 4. The mouse ventral midbrain has a medial arc expressing molecular markers of the OMC and the pRN. (A) Emx2 labeling identifies arcs in mouse E12.5 midbrain wholemounts, oriented as in Fig. 1. (B,C) Isl1 (B) and Brn3a (C) labeling of the OMC and the pRN in the mouse medial arc. (D) Combined Isl1 and Emx2 labeling confirms that the organization of the mouse medial arc is similar to that of the chick. IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus.

 


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  		Fig. 5. Emx2 is required for the formation of the RN in mice. (A,B) Brn3a+ cells are present in midbrain wholemounts of E14.5 heterozygous (A) and homozygous (B) mutant littermates, demonstrating that pRN neurons are initially specified in Emx2 mutants. (C-F) Brn3a (C,D) and class III ß-tubulin (E,F) gene expression fail to identify a RN at E18.5 in homozygous Emx2 mutant mice. (G,H) Normal Isl1 (G) and Th (H) gene expression in Emx2 homozygous mice at E18.5 suggests that among medial arc derivatives, the RN is selectively disrupted by mutations in the Emx2 gene. Aq, cerebral aqueduct.

 


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  		Fig. 6. Relationship of SHH gene expression to the developing chick OMC and pRN. (A-C) PHOX2A and SHH gene expression at E 2.5 (A) and E5 (B,C), shown in whole-mount preparations (A,B) and in transverse section (C). The SHH source is confined to the midline at E2 (A), but from E3 expands within the ventricular layer (asterisk in C) to overlie the OMC (B,C). (D) E5 midbrain wholemount demonstrating that ventricular SHH expression also overlies the BRN3A+ pRN. Midbrain BRN3A labeling is first detected at E4 (data not shown). FP, floor plate of hindbrain; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus; tec, tectum.

 


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  		Fig. 7. Coordinate regulation of medial arc pronuclei by SHH manipulations. (A,B) Expanded gene expression for SHH (A) and its downstream transcriptional targets FOXA2 and ISL1 (B) is seen at E5, after electroporation of pXeX-SHH into chick E2 ventral midbrain. In this example, the electroporated side of the brain is the right side; the left side serves as an internal control. In the hindbrain the expanded ISL1 gene expression identifies the trochlear (Tr) nucleus. (C-F) An enlarged SHH source elicits a strictly coordinate expansion of the OMC and the pRN, as demonstrated by two-color gene expression for PHOX2A and BRN3A (C,D), and PHOX2A and EMX2 (E,F). The E5 wholemounts shown in C and E were cut at the level of the asterisks to prepare the sections illustrated in D and F. The OMC and pRN territories, although expanded, maintain their spatial relationship in all three dimensions: co-extensive along the mediolateral axis (D,F), partially overlapping along the anteroposterior axis (C,E) and completely segregated along the ventricular-pial axis (D,F). (G) E5 wholemount of EMX2 and PHOX2A gene expression shows that SHH in lateral tegmentum elicits a properly patterned ectopic medial arc (1*). Arrows indicate EMX2 labeling of the ectopic pRN, which partially overlaps the PHOX2A labeling of the ectopic OMC and lies pial to it. (H) Wholemount of E5 midbrain hemitegmentum (below dotted line) and tectum (above dotted line) demonstrating that SHH misexpression in dorsal midbrain also produces an ectopic medial arc (1*) containing an ectopic PHOX2A+ OMC and an ectopic BRN3A+ pRN. Note that SHH misexpression in dorsal midbrain results in the loss of BRN3A labeling of the tectum, which is found in the inner part of the mantle layer and is thereby readily distinguished from pRN-like BRN3A labeling, which is found more pially. (H, inset) Cross-section through an ectopic medial arc in dorsal midbrain documents the ventricular-pial segregation of the PHOX2A+ (brown) and BRN3A+ (blue) territories. The insert is oriented with the tectal ventricular surface marked by an asterisk. 1 and 2, arcs 1 and 2; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus; tec, tectum; Tr, trochlear nucleus.

 


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  		Fig. 8. The OMC and the pRN maintain their anteroposterior relationship, even when the isthmus is shifted rostrally and the midbrain is reduced in size. Rostral movement of the isthmus is induced by mouse Fgf8 misexpression in chick midbrain, which converts midbrain into hindbrain and thereby creates a more rostral midbrain-hindbrain junction. (A) Relationship of the isthmus (IS), identified by WNT1 labeling (blue), to midbrain arc patterning, identified with probes for the homeobox genes PHOX2A (arc 1, blue), PAX6 (P6, brown) and EVX1 (E1, blue). (B) FGF8 gene expression at E5 after unilateral mouse Fgf8 misexpression at E2 documents the rostral shift of the isthmus on the electroporated (right) side, with the left side serving as a control. Asterisk indicates expression of the mouse Fgf8 transgene. Chick FGF8 gene expression, detected by cross-hybridization of the mouse Fgf8 riboprobe, identifies the normal (left) and ectopic (right) isthmi (IS). (C,D) Side views of unelectroporated (C, Control) and Fgf8-electroporated (D, Fgf8 EP) E5 brainstems show that the rostral shift of the isthmus elicits a transformation of the dorsal midbrain from the globose shape characteristic of the tectum into a tubular shape similar to that of the hindbrain. Dorsal is towards the top, and rostral is towards the lower right. Red lines in C and D mark the extent of rhombomere 1. Arrow in D indicates location of the ectopic isthmus induced by Fgf8 misexpression. (E) Elongated trochlear nucleus (PHOX2A+) and compressed midbrain arcs (Hx indicates homeobox genes PHOX2A, PAX6 and EVX1) illustrate the expansion of rhombomere 1 at the expense of ventral midbrain tissue after Fgf8 misexpression. (F) BRN3A gene expression demonstrates rostral shift of the pRN territory after Fgf8 misexpression. Expanded GBX2 labeling demonstrates the concomitant rostral shift in the isthmus (IS) and a transformation of midbrain to hindbrain. (G,H) The PHOX2A+ and BRN3A+ territories of the medial arc maintain their relative anteroposterior positions whether the rostral shift of the isthmus, as identified by WNT1 gene expression (blue in G, brown in H), is moderate (G) or substantial (H). To demonstrate the rostral shift of BRN3A labeling in these wholemounts, the right brainstem has been kinked (G) or the left forebrain has cropped (H). 1, arc 1; III, third ventricle; IS, isthmus; OMC, oculomotor complex; rFP, rostral, or midbrain, floor plate; RN, red nucleus; Tr, trochlear nucleus.

 

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