The embryonic nervous system of homozygous Ror 4 embryos displays a mild

During embryonic and larval development Drosophila Ror is primarily expressed in the nervous system. Within the central nervous system, the protein is found in all neuronal cells and in all axonal projections. In the peripheral nervous system it can be found at the membrane of all neurons including the sensory axons (see 3.1). Although the expression pattern of Ror is not identical to Otk and Otk2, they can both also be found in the larval central nervous system as well as in the larval brain. However, the morphology of the

viable (Linnemannstöns et al., 2014). To analyze if the loss of Ror alone or of Ror, otk and otk2 together has any effect on the development of the embryonic CNS, I have stained homozygous embryos of the respective mutant lines for the CNS axon marker BP102, for Fasciclin II which marks a subset of CNS axons and for Repo to visualize glial cells. After staining, fillet preparations of the CNS of stage 17 embryos were prepared.

The CNS axon tracts visualized by BP102 in all analyzed mutant nervous systems resembled the wild type. The neuronal processes forming the longitudinal connectives are intact and the anterior and posterior commissures were separated from each other (Figure 23 A-D’).

In stage 16/17 embryos, Fasciclin II labels three longitudinal axon bundles, termed fascicles. In wild type and homozygous Ror⁴, Df(otk,otk2)D72 embryos, all three fascicles were well formed and intact (Figure 23 A/A’,H/H’). In homozygous Ror⁴ embryos every now and then discontinuities in the outermost lateral fascicle were visible (Figure 23 F’, arrowhead). However, a detailed analysis of the number of segments in which disrupted fascicles were observed showed that the difference to wild type embryos is not statistically relevant (Figure 24). A closer look at the three fascicles in the Ror⁴ embryos revealed that some axons appear wavy and it seems as if not all axons are tighly fasciculated into the bundle (Figure 23 F’). Many Df(otk,otk2)D72 nervous systems display a similar phenotype. While axons not incorporated into the fascicle are not so frequent, many fascicles are wavy (Figure 23 G’). Surprisingly, most homozygous Ror⁴, Df(otk,otk2)D72 embryos exhibit no axons outside of the bundles and the fascicles themselves are straightly formed (Figure 23 H’). However, some homozygous triple mutant embryos display a more severe CNS phenotype than the Ror single mutant. In these samples, the outermost lateral fascicle is disrupted in every segment (Figure 23 M, arrowheads). The remaining fascicles also appear somewhat wavy and unorganized.

The differentiation and maintenance of glial cells is not disturbed in all analyzed mutants. I have not observed any lack or misplacement of glial cells and the pattern in the mutants is comparable to the wild type.

Taken altogether it appears as if Ror⁴ mutant embryos and homozygous Df(otk,otk2)D72 embryos both display a mild axon guidance or fasciculation phenotype.

Figure 23: The morphology of the ventral nerve cord in wild type embryos compared to Ror, otk and otk2 mutants. (A-D) Axon tracts of the CNS are visualized using the BP102 antibody in wt (A), Ror⁴ (B), Df(otk,otk2)D72 (C) and Ror⁴,Df(otk,otk2)D72 (D) embryos. All mutant embryos resemble the wild type. (E-H) Fasciclin II labels the axons of a subset of neurons within the CNS. In Ror⁴ embryos the fascicles have a wavy appearance, and some axons are not tightly incorporated into the fascicles. Some disruptions in the lateral fascicle are also visible (arrowhead); in otk, otk2 double mutants the fascicles appear wavy as well and in Ror, otk, otk2 triple mutants all fascicles are intact. (I-L) Glial cells visualized with the anti-Repo antibody. The pattern is not disturbed in any of the investigated mutants. Images A’-L’ are magnifications of sections in the images A-L. All images show three abdominal segments of late stage embryos; anterior is up. (M) Some Ror, otk, otk2 triple mutant embryos exhibit a stronger CNS phenotype. The lateral fascicle display many breaks (arrowheads).

As mentioned above, the nervous systems of a small amount of homozygous Ror⁴, Df(otk,otk2)D72 triple mutant embryos appear to have an increased number of disrupted fascicles, while the morphology of the other nervous systems was comparable to wild type nervous systems (Figure 23 M). This fact is visible as the high standard error bar in Figure 24. It is possible that these noticeable nervous systems are from embryos mistakenly dissected at an earlier developmental stage or that they are the nervous systems of fully developed but unhatched embryos (mentioned above). The latter would indicate that at least a small percentage of Ror, otk, otk2 triple mutant embryos display a lethal phenotype associated with defects in CNS development.

Figure 24: The number of CNS segments with disrupted fascicles in Ror and otk,otk2 mutant embryos is not statistically relevant. In stage 17 Ror⁴ mutant embryos the percentage of segments with disrupted fascicles is comparable to the number observed in white^- and in homozygous Df(otk,otk2)D72 mutant embryos. In homozygous Ror⁴,Df(otk,otk2)D72 triple mutant embryos, some nervous systems display an increased number of disrupted fascicles. Number of analyzed segments: white^-: n = 105; Ror⁴: n = 101;

Df(otk,otk2)D72: n = 48; Ror⁴,Df(otk,otk2)D72: n = 47. *: p-value < 0.05.

I have also analyzed the peripheral nervous system of Ror⁴ mutants for any defects. For this reason I stained homozygous Ror⁴ embryos for the PNS marker 22C10 (Futsch). This marker visualizes all PNS neurons. There are three clusters on the lateral side of the embryo, the dorsal cluster, the lateral cluster and the ventral cluster. The PNS of Ror mutant embryos is normally developed. All clusters of neurons are present and axons grow into the CNS as usual (Figure 25).

Figure 25: The PNS in Ror⁴ mutant embryos is not affected. Stage 17 homozygous Ror⁴ embryo stained with 22C10 (Futsch) to reveal the peripheral nervous system. The dorsal cluster (d) and the lateral cluster (l) are shown in a higher magnification. Anterior is to the left, Scale bars = 50 µm.

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segments with disrupted fascicles (%) *