Figure 1.
Comparison of axon guidance defects in mid, slit and robo mutants.
Embryos from wild type (panels A–C), mid H15 deficiency (panels D–F), slit (panels G–I) and robo (panels J, K) were stained with an antibody against Fas II. Arrow indicates projection from the pCC neuron as well as the medial tract (in older embryos) or its abnormal crossing at the midline; arrowheads in panel F indicate breaks, and star indicates groups of stalled longitudinal axon tracts. These mid-specific defects are not seen in either slit or robo mutant embryos. Anterior end is up, midline is marked by vertical lines. Note that to define age of the embryo we prefer “hours” of development (at room temperature) to the traditional “stages” of development. We think that this is particularly important for describing events at the molecular level. Assigning different “stages” was done based on gross morphological milestones during embryogenesis, and each stage can be 10 minutes short or 2–3 hours long. For defining age or development at the molecular level this approach is not meaningful and can be misleading.
Table 1.
Penetrance of the axon tracts defects in mid mutants.
Figure 2.
Patterns of aberrant axon projections in mid mutant embryos.
Anterior end is up, the midline is marked by vertical lines. Embryos in panels A–E were stained with Mab 22C10 to visualize vMP2 and dMP2 neurons and their projections. Arrow indicates a normal projection in wild type (A, B), arrow with star indicates an abnormal/stalled projection in the mutant from dMP2 and vMP2 neurons (panels C–E). Panel F, G: Embryos were double-stained for GFP (green) and Odd-skipped (Odd, red). Arrow indicates normal projection from vMP2 in wild type (F), arrow with star indicates projection from vMP2 abnormally exiting the nerve cord in the mutant (G). Panels H, I, J: Embryos were double-stained for GFP (green) and Eve (red). Arrow indicates normal projections from MP1 in wild type (H), arrow with star indicates abnormal MP1 projection in the mutant (I, J). Panels K, L: Embryos were stained for LacZ (green). Arrows indicate normal projections from pCC in wild type (K), arrow with star indicates abnormal projection from pCC in the mutant (L). Panels M, N: Embryos were stained for LacZ (green). Smaller arrow indicates normal motor intersegmental nerve bundle from aCC and RP2 neurons in wild type (M) and in the mutant (N).
Figure 3.
Expression of Slit protein and slit transcription in different mid mutants.
Embryos from wild type (panels A, C and E), two mid mutant alleles (mid1 and los1; panels B and D) and mid H15 deficiency (panel F) were stained with an antibody against Slit. In Panel G, Western blotting analysis of Slit expression in wild type, mid H15 deficiency and sli2 mutant embryos is shown using an antibody raised against the N-terminal portion of Slit [see ref. 7]. The levels of the unprocessed (UP) and processed (P) N-terminal fragment of Slit were quantified using the AlphaEase FC software. Levels of the β-Tubulin (∼55 kDa band), determined by probing the same blot with an antibody against β-Tubulin, was used as a loading control. In panels H–L, transcription of slit was examined in wild type (panels H and I) and in mid H15 deficiency embryos with whole mount RNA in situ using a probe against slit. Anterior end is up, midline is marked by vertical lines. Arrows in panels K and L indicate occasional disorganization or displacement of the slit-expressing midline glial cells in mid mutants.
Figure 4.
Expression of Robo is not affected in mid mutant embryos.
Wild type (panels A and E) and mid H15 deficiency (panel B), mid1 (panel C), mid H15df/mid1 transheterozygous (panels D and F) embryos were stained with an antibody against Robo. Anterior end is up, midline is marked by vertical lines. Severe axon tract disruptions/breaks were observed in mutant embryos but the Robo levels were similar to wild type. In panel G, Western blotting analysis of Robo in wild type, mid H15 deficiency and robo deficiency embryos is shown. Note that the Robo protein band is not seen in robo-deficiency embryos indicating that the antibody is specific to Robo. β-Tubulin was used as a loading control and the levels were quantified using the AlphaEase FC software.
Figure 5.
Real time quantitative pCR for slit, robo and frazzled.
The qRT-PCR was done for samples from three seperate embryo collections for each genotype and in triplicates for each collection-sample. Standard errors (SE) were calculated from the pooled data for each genotype.
Figure 6.
Wingless and Gooseberry are inappropriately expressed in row 2 cells in mid mutants.
Panel A–H: Wild type (A, B) and mutant embryos (C–H) were double-stained with antibodies against Wg (Green) and Gsb (Red). Anterior end is up, midline is marked by vertical lines. Rows of NBs are numbered in both 5 hours old and 6 hours old embryos. Arrow-head in mutant embryo panels indicates ectopic Wg (and less frequent ectopic Gsb) expression. Note that in some hemisegments, ectopic Wg expression is extensive (panels G and H). Panels I and J: Slp expression in wild type and mutant. Note the ectopic Slp in mutant embryos, indicating transformation of row 2 into row 5 NBs (arrowheads). Panel K: Expression of some of the major segment polarity genes in the neuroectoderm (NE) and neuroblasts (NB) is shown (saggital view). Two rows of NE cells correspond to one row of NBs (each NB is twice the size of a NE cell); thus, for example, Wg is expressed in two rows of NE cells whereas it is expressed only in one row of NB. Expression of Gsb in the most posterior row of NE cells is restricted to only the cells closest to the midline, from which NB7-1 is derived. Panels L–O: Expression of Mid in early embryonic neurogenesis in wild type. A strong Mid expression is seen in rows 7 and 1 and remains so throughout neurogenesis, whereas weak and varying expression of Mid can be observed in other rows of NBs and this expression pattern changes with time.
Figure 7.
Longitudinal axon tracts stall just before the region of ectopic Wg and Gsb expression in mid mutant embryos.
Wild type (panels A, B, I) and mutant (panels C–H, J) embryos were stained with BP102 and Eve (panel A), BP102 (panels B–H), and Robo (panels I and J). Anterior end is up, midline is marked by vertical lines. aCC/pCC and RP2 are Eve-positive neurons. The numbers 2, 3, and 4 indicates approximate location of NB rows 2, 3 and 4 formed earlier during neurogenesis. Neurons formed from a NB generally stay in the same location as the parent NB [see ref. 28]. For example, aCC/pCC pairs are formed from NB1-1 in row 1; RP2 is formed from NB4-2 in row 4. AC, anterior commissure; PC, posterior commissure; LC, longitudinal connectives; star indicates stalled blobs of axon tracts. Arrowhead indicates missing longitudinal axon tracts; black arrow indicates outward projection of longitudinal tracts and star indicates stalled axon tracts.