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A mitogen gradient of dorsal midline Wnts organizes growth in the CNS

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

View larger version (78K): [in a new window]   Fig. 1. Wnt1 promotes proliferation and inhibits differentiation. (a-c) Transfection of forelimb level chick spinal neural precursors by in ovo electroporation with a plasmid containing an internal ribosome entry sequence followed by nuclear GFP (a), gives rapid, ubiquitous, unilateral expression with cellular resolution and does not perturb normal development (b,c). (d,e) Embryos transfected with Wnt1 show an increase in BrdU incorporation (4 hour labeling) at ventral levels (marked by bracket) and a decrease in post-mitotic neurons marked with N-tubulin. (f-h) Rates of proliferation and differentiation were quantitated using BrdU cumulative labeling and N-tubulin staining in stage 22 embryos in both the transfected (green) and untransfected sides (red). Ectopic Wnt1 expression increased the BrdU labeling index to a progressively greater degree ventrally (f). BrdU cumulative labeling shows that ectopic Wnt1 increases both the proliferation rate and the fraction of cells in S-phase in the ventral three fifths of the ventricular zone (g). Wnt1 transfected precursors have a 55% reduction in the rate of differentiation (h).

View larger version (70K): [in a new window]   Fig. 2. Mitogenic Wnt signaling is transduced through the ß-catenin/TCF pathway in neural precursors across the DV axis. (a) Dominant-active ß-catenin causes a large expansion in the ventricular zone and a reduction in post-mitotic neurons. (b-d) Dominant-active ß-catenin reduces D1, D2, D3, V1 and V2 interneurons and ventral motor neurons. (e) Triple staining for GFP, TUNEL and Isl1 shows that dominant-active ß-catenin reduces differentiation of motorneurons prior to and independent of inducing apoptosis. (f-h) Dominant-active ß-catenin causes precursor expansion at dorsal (f), intermediate (g), and ventral (h) levels without primarily changing positional information. (i,j) Dominant negative TCF4 cell-autonomously blocks precursors from entering S-phase of the cell cycle across the DV axis. Arrows mark cells on the transfected side that were not transfected (i) and are BrdU positive (j). All images are representative of at least 5 transfected embryos.

View larger version (100K): [in a new window]   Fig. 3. Only dorsal midline Wnts have mitogenic activity. (a,c) Wnt1 and Wnt3a have similar expression patterns in the dorsal midline. (b,d) Neural precursors transfected with Wnt1 or Wnt3a have increased BrdU labeling index (4 hour incorporation) and a decreased number of post-mitotic neurons. (e,g) Wnt3 and Wnt4 are expressed in broad dorsal domains. Wnt4 is also expressed in the floor plate (g). (i,k) Wnt7a and Wnt7b are expressed in broad ventral domains. (f,h,j,l) Neural precursors transfected with the non-dorsal midline Wnts, Wnt3, Wnt4, Wnt7a or Wnt7b, have normal BrdU incorporation and numbers of post-mitotic neurons. (m,n) The BrdU labeling index in the ventral three fifths of the VZ and the number of post-mitotic neurons were quantitated for these and other embryos. All sections were visualized for GFP to ensure good transfection before staining.

View larger version (108K): [in a new window]   Fig. 4. Regulation of D-type cyclins by Wnts. (a-d) cyclin D1 is expressed in a dorsal to ventral gradient. In situ hybridization at E8.5, E9.5 and E11.5 (a,b,c) shows cyclin D1 is expressed in neural precursors in a DV gradient. cyclin D1 is also transiently expressed in some differentiating neurons as marked by arrows in b and c. (d) Normalized quantitation of cyclin D1 expression levels shows that the gradient extends completely across the DV axis when the spinal cord is small and becomes more dorsally restricted as the spinal cord grows. (e-q) Sections of transfected embryos were first visualized for GFP (e,g,i,k,m,o,q) and then stained by in situ hybridization (f,h,j,l,n,p). Dominant active ß-catenin upregulates cyclin D1 and cyclin D2 (e-h). cyclin D1 can be upregulated at all dorsal-ventral levels but cyclin D2 can only be upregulated dorsally (upregulation marked by bracket). Dominant active ß-catenin does not upregulate the expression of the G2/M cyclins cyclin A1 (i,j) or cyclin B3 (k,l). Wnt1 upregulates cyclin D1 expression at ventral levels but not at dorsal levels (upregulation marked by bracket) (m,n). Dominant negative TCF4 downregulates cyclin D1 at all levels of high transfection efficiency as marked by bracket (o,p). Dominant negative cyclin D1 reduces precursor expansion (q).

View larger version (82K): [in a new window]   Fig. 5. An endogenous dorsal to ventral gradient of Wnt signaling. Cotransfection pTOPRed, a reporter construct containing synthetic TCF binding sites driving red fluorescent protein, with pCIG was used to investigate the range of ß-catenin signaling in the neural tube. (a) GFP expression from pCIG shows the entire DV axis was transfected. (b) RFP from pTOPRed shows that the reporter is active across the dorsal three quarters of the neural tube and is more active in the dorsal-most quarter. The dorsal to ventral gradient of RFP expression is jagged, likely due to the variable levels of transfection between cells. By comparing the level of transfection of each cell as marked by GFP with the level of ß-catenin signaling as marked by RFP, a gradient of ß-catenin signaling across much of the DV axis is apparent in the merged image (c). (d-f) Transfection of pFOPRed in which the TCF binding sites are mutated shows no activity (e) despite high levels of cotransfection of pCIG (d). (f) Merged pFOPRed and pCIG images.

View larger version (57K): [in a new window]   Fig. 6. An endogenous growth gradient in the spinal cord. The rates of proliferation and differentiation were determined by BrdU cumulative labeling (1, 2, 3, 4, 5 and 6 hours) and N-tubulin staining for 5 bins across the DV axis at HH stages 11, 14, 17, 21 and 25. (a-e) Representative images from each stage, labeled for 4 hours with BrdU and immunostained for BrdU and N-tubulin. A dorsal to ventral gradient of proliferation and a ventral to dorsal gradient of differentiation are apparent. The rates across space and time are represented by color intensity for (f) proliferation (green) and (g) differentiation (red). (h) The pattern of Wnt signaling determined by medial cyclin D1 expression is shown in blue. (i) The proliferation rate, differentiation rate, and amount of Wnt signaling are merged to show the positive correlation between the proliferation rate and Wnt signaling and the inverse correlation between the differentiation rate and Wnt signaling. There is a growth gradient from early to late and dorsal to ventral that correlates with the pattern of Wnt signaling.

View larger version (29K): [in a new window]   Fig. 7. Computer modeling demonstrates the mitogen gradient model can robustly pattern growth. (a) Diagram of the computer simulation of the mitogen gradient model. The proliferation rates and differentiation rates of a one-dimensional row of precursors are regulated by their distance from the growth organizer according to a linear function set by the user. Precursors are pushed ventrally away from the growth organizer when they divide. (b) To show the emergence of the growth zone, the state of cells in the model after every 30 iterations for the first 1020 iterations is shown. The squiggly line indicates where additional cells in the simulation are not shown. The growth zone is a robust and dynamically stable region whose size and shape is controlled by the proliferation and differentiation rate gradients and is independent of the starting number of precursors. (c) The mitogen gradient model can pattern cell cycle divisions and exits across a field of cells as seen in a chart of the total number of divisions and exits at each distance from the growth organizer after 1500 iterations of the simulation. The mitogen gradient model can also generate a constant rate of outgrowth and a constant supply of differentiated cells along an outgrowing structure. (d) The total length of the field of cells, including precursors and post-mitotic cells, at the end of every 30 iterations is shown. The total length of the field of cells increase roughly linearly with time.

View larger version (30K): [in a new window]   Fig. 8. Mitogen gradient model of how growth is patterned across the DV axis of spinal cord to determine its size and shape. (a) At the cellular level, Wnt1 and Wnt3a act as mitogens on neural precursors through the ß-catenin pathway to promote cell cycle progression and inhibit cell cycle exit. (b) Diagram of cross section of the developing spinal cord. Mitotically active neural precursors comprise the medially located ventricular zone. Neural precursors exit the cell cycle and differentiate to form laterally located neurons. The mitogens Wnt1 and Wnt3a are expressed in the dorsal midline and form a dorsal to ventral concentration gradient across the field of neural precursors. (c) The potential size and shape of the mitogen gradient defines the potential rates of proliferation and differentiation across the DV axis. The proliferation and differentiation rate gradients level off dorsally because mitogenic Wnts are saturating at dorsal levels. The net effects of proliferation and differentiation form a gradient termed ‘growth potential’ (d). The proliferation rate gradient and the differentiation rate gradient cross about midway along the DV axis in this diagram. At this crossing point, the proliferation rate is equal to the differentiation rate meaning the population of precursors should be steady in number. Dorsal to this point the number of precursors is continually increasing, and ventral to this point the number of precursors is continually decreasing. The ventral extent of the growth potential gradient corresponds to the point at which the area between the curves above the crossing point equals the area between the curves below the crossing point such that the field of cells should not grow any further ventrally. (e) Different stages of the development of the spinal cord showing how it ‘grows into’ the size and shape specified by the growth potential gradient. The neural tube is initially small and round in cross section. At this early stage, Wnt1 and Wnt3a can diffuse across the entire DV axis to cause high rates of proliferation and low rates of differentiation across the entire DV axis. These high rates of proliferation and low rates of differentiation cause the neural tube to expand. As the neural tube expands, Wnt1 and Wnt3a become limiting at ventral levels causing higher rates of differentiation and lower rates of proliferation at ventral levels. This difference in growth potential across the DV axis causes the neural tube to become asymmetric; the ventricular zone becomes thicker dorsally than it is ventrally, and the mantle zone becomes thicker ventrally than it is dorsally. The neural tube continues to grow across its DV axis until it reaches a limit defined by the growth potential. Once this limit in DV size is reached, the developing spinal cord only grows across the medial-lateral and anterior-posterior axes by adding more neurons to the mantle zone. The arrows show the treadmilling movement of neural precursors ventrally away from the growth organizer caused by proliferation and the movement of precursors laterally to form neurons as they differentiate.