Analysis of Po mRNA at early developmental stages of the rat using in situ hybridization at the trunk level

Whole mount embryos at ElO, 11 and 12 were chosen to analyze the spatial correlation o f the expression o f the Po gene and the migrating crest population The blue/purple color precipitation o f the Po ISH in the whole mount embryos was seen in the ElO rat embryo in the cranial area (Figure 4-2). E l l , 12 embryos were hybridized with Po probe and immunostained with TUJ-1 antibody, which stains growing neurites and immature sensory neurons (Memberg and Hall, 1994) (Figure 4- 3 A,B and Figure 4-4A,B). The Po signal was visualized by a mixture o f NBT/X-P solution, and the TUJ-1 was visualized by incubation in DAB solution, as described in chapter 2.

At E l l there is an active spectrum o f migrating neural crest ranging from premigratory stage to mid migration stages (Erickson et al, 1989; Serbedzija et al., 1990). The blue/purple color precipitation o f the Po ISH in the whole mount embryos was seen as repeating wedged or squared streams o f cells, spreading from the edge o f the tube (Figure 4-3A). Each stream was contained within one somite. In the more caudal somites, the Po positivity was seen as slim stripes running dorsal-ventrally 1-2 mm lateral from the tube midline. These cells were not restricted to the anterior half o f the somite when they first emerged from the tube, but soon after they migrated out o f the near vicinity o f the dorsal midline o f the neural tube, the positive cells were seen to be arranged in 2 to 3 finer streams, and the migration was restricted to the anterior half o f the somite. In the more rostral, and hence more mature somites, the positive cells formed a wedge, with the exception o f the first 3 somites, due to the lack o f neural crest derivatives in this area. The ventral most, thus supposedly front o f the migrating crest was always restricted to the anterior part o f the somite (Figure 4- 3 A, also Figure 4-4C), while nearer to the dorsal edge of the neural tube, the positive cells spanned nearly the whole length o f the somite and formed the fat end o f the

wedge. This is typical o f neural crest populations in that the restriction to the anterior part o f the somite only happens after the cells migrate some short but significant length ventrally. This is often less addressed than the phenomenon o f restriction to the anterior somite, as it is not always seen in classical longitudinal sections.

A longitudinal line o f dark ISH precipitation was seen running across two thirds o f the length from the top o f the wedged form beginning from the first somite and fading away at around the 10th to 12th somite (Figure 4-3A, arrow). Results from segments partially digested with collagenase to expose the somites showed that this line consisted o f cells situated at the area where the motor axons first meet the migrating crest cells. At the 3rd to 6th somites, the blue/purple color was also seen much more ventrally in the area where future sympathetic ganglia would locate. The level o f Po expression went down gradually towards the tail o f the embryo. There were no Po signals in the last 5 to 6 somites.

The migration o f the neural crest continues on the next day ( E l2). Neural crest migration into the periphery is initiated in an anterior to posterior wave (Erickson et al, 1989). The anterior to posterior progression is most obvious when forelimb and himdlimb levels are compared in the same embryos. The correlation o f Po expression to the migrating crest population was studied with serial cross sections o f the embryo. The ventral extent o f Po expression forms a repetitive pattern, repeating itself every 200 p M on average, which is the length o f a somite. This pattern is also seen in a longitudinal section o f E l 2 embryo hybridized with Po probe (Figure 4-4C), where the Po signal o f wedged blue precipitation was seen only in the anterior half o f the somite, and not in the posterior. In cross sections at the hind limb level Po signal was present in a location typically described as the ventral-lateral pathway o f neural crest, as shown in the cross section o f the anterior half o f the somite (Figure 4-4F). Note that the Po is not present in cells o f the migration pathway as soon as they emerged from the neural tube, and it is not expressed in the posterior half o f the somite (Figure 4-4G), apart from two points which are probably the ventral exit zone for motor axons and in the notochord. A cross section o f the whole mount embryo confirmed that these Po signals in the ventral exit zone run parallel to the neural tube to form a

longitudinal line which is only interrupted between somites. Sections from the same rostral-caudal level o f same age o f embryo hybridized with Krox-20 were compared. Po was expressed at the ventral exit zone when it is still Krox-20 negative(Figure 4- 4J). This showed that the Po is expressed earlier than Krox-20 at this location. However, cells in the entry/exit zone o f the cranial nerves o f the same embryo were already Krox-20 positive (Figure 4-4K), indicating the temporal proximity o f Po and Krox-20 expression.

In the front limb level the derivatives o f neural crest have aggregated to form the DRG, as shown by the presence o f TUJ-1 antigen. The Po signal was present in the now substantial structure o f the DRG, as well as along the length o f the spinal nerves (Figure 4-4H, I). The crest-derived cells that coalesce to form the young DRG were observed as early as E12, as shown by the presence o f TUJ-1 antigen (Figure 4-4B). A t this stage the Po positive cells were seen in the entire area o f these condensations (Figure 4-1). At the later stage o f E14 the Po signal was down-regulated in the DRG itself and was only seen in the roots and spinal nerves (Figure 4-5), and this was similar in the mature adult DRG. At a more rostral level the Po was seen in the anlage o f the sympathetic chain (Figure 4-4A).

E l 2 rat embryos were hybridized with ErbB3 probe and cryosectioned as described for Po. Cross sections o f the hind limb level were compared to those hybridized with Po probe, and sections chosen for comparison were from similar anterior-posterior positions within the somite. This was done by counting a similar number o f sections from the boundary o f the somite. The ErbB3 signal was very similar to the Po signal, with small but significant differences. In the anterior half o f the somite the ErbB3 was seen in the cells between the ectoderm and the neural tube and in the wedge between the neural tube, dermamyotome, and the overlying ectoderm (Figure 4-4D). These bands o f expression on the two sides o f the neural tube were, or were nearly, continuous with the top o f the neural tube. This continuity stretched ventrally, first between the dermatome and neural tube and, as the cells migrate more ventrally, near to the neural tube. It then extends laterally when reaching the exit point o f the motor axons (Figure 4-4D). In the posterior half o f the somite, the ErbB3 was expressed in

a group o f aggregated cells sitting in a wedge between the neural tube and dermamyotome but above the sclerotome. The location o f these grouped cells was nearer to the dermatome than to the neural tube, and the expression was highest on the ventral extreme (Figure 4-4E). Some cells in the myotome were ErbB3 positive, in agreement with results reported by Meyer and Birchmeier in mouse (1995). However, this signal in myotome is only significant in the anterior half o f the somite (Figure 4-4D)

4.2.3 Analysis of Po mRNA at early developmental stages of the rat using in situ