homozygous embryos exhibit severe axon guidance defects in the longitudinal connective as early as 9h post-fertilization - e.g. growth cones of posterior corner cell neurons tangentially projected towards the midline -, as compared to controls.
The adult brain of sli2
transheterozygotes exhibits major defects in neuropil organization in several central brain areas including the central complex, mushroom body lobes, antennal lobes and Robo2/Robo3-expressing axons, as compared to either heterozygous controls; their adult sLNv neurons exhibit significantly longer axon projections compared to controls.
The adult sLNv neurons of sli2
heterozygotes that also express sliJF01229
under the control of Scer\GAL4ey-OK107
(in combination with Dicer-2, for efficient RNAi) exhibit significantly longer axon projections compared to controls.
homozygous embryonic heart cardioblasts show a rounded morphology, show significant decreases in migration velocity, as well as in filopodial and lamellopodial extensions and activities, as compared to controls; these are associated with gaps in the leading edge (and lost adhesions with ipsilateral partners), cell clumping, improper linear alignment of the cardioblasts, and embryonic heart lumen formation defects, as compared to controls. sli2
heterozygous cardioblasts also show a significant decrease in filopodial activity, but not in lamellopodial activity or migration velocity, as compared to controls.
/+ embryos, the normally ipsilateral-projecting ap
-positive neurons display occasional defects.
Tracheal dorsal branch fusion occurs normally in the tracheal system of sli2
/+ third instar larvae
stage 13 embryos have gonads with unfused somatic gonadal precursor (SGP) clusters. By stage 15, SGP clusters fuse but gonads then fail to compact properly. Mutants also show germ cell ensheathment defects.
Mild ectopic ap neuron crossing defects are seen in heterozygous sli2
stage 16 embryos.
mutant embryos the ventral longitudinal muscles cross dorsally over the CNS, meeting those from the other side to form ectopic muscle attachments along the ventral midline.
mutant embryos, the entire axon scaffold collapses at the midline. The Scer\GAL4ap-md544
-expressing axons project directly to the midline and do not leave.
Many of the dorsal abdominal clusters in sli2
mutant animals have branches that exceed the normal level of extension at approximately 21 and 22 hours after egg laying. The maximal dendrite length with respect to the most dorsally positioned neuronal cell body is significantly altered in mutants.
Class IV sli2
mutant neurons display dendrite over-elongation and reduced branching. Class IV neurons show less high order branches and form longer dendrite process compared to control embryos. The number of branches of class IV neurons is significantly decreased in robo1
mutants at 22 hours after egg laying. The dorsal elongation and the average branch length are significantly increased.
/+ embryos show a mild axon guidance defect of the Apterous neurons; less than one thick bundle of Ap neurons cross the midline on average per sli2
/+ embryo, while these neurons never cross in wild-type embryos.
embryos, the pCC axons aberrantly cross the midline.
mutant embryos present a very severe fused commissure phenotype.
embryos show defective heart development. These embryos have breaks in the continuity of the adherent cardial cells during migration, which can lead to lesions in the final heart vessel. Sometimes, a few cardial cells are not incorporated into the heart, nuclei sometimes cross the midline and irregular bulges in cardial alignment are produced. The hearts of sli2
embryos have either no lumen, or a very small one, and an expanded basolateral zone. Dorsal closure is not affected in these mutants.
/+ mutants have a normal heart.
Although the appropriate number of cardioblasts are specified in sli2
embryos, cell adhesion between cardioblasts is lost. Thus, in 97% of sli2
embryos, the cardioblasts fail to form two continuous rows of cells, showing frequent gaps and inappropriate cell clustering and intermingling with the flanking pericardial cells: approximately 104 Mef2
-positive cardioblast nuclei form two rows at the dorsal midline in wild type embryos, whereas there are only approximately 87.9 Mef2
-positive cells at this position in sli2
mutant embryos. The rows of pericardial cells that flank the rows of cardioblasts are also misaligned in sli2
embryos, and often show gaps that frequently correspond with the gaps in the rows of cardioblasts.
The shape of cardioblasts in sli2
embryos is abnormal.
90% of sli2
embryos have salivary glands that curve medially towards the CNS midline, instead of lying parallel to the midline in the wild-type position.
When the bilateral rows of myocardial progenitors have reached the dorsal midline, they fail to align properly in sli2
mutants compared to wild-type. There are two types of myocardial cell misalignments: type I consist of irregularities in the myocardial cell rows and type II, in addition, has gaps and triple lines of myocardial cells. The apical-lateral polarity of the heart tube is disrupted in sli2
Normal separation of primordial germ cells into lateral clusters on each side of the embryo is observed in homozygous and heterozygous sli2
33% of thoracic segments show transformation of the dch3 chordotonal organs to a morphology resembling that of abdominal lch5 chordotonal organs in mutant embryos; the transformed "dch3" organs are positioned laterally and have dendrites pointing dorsally.
The vp1-4a external sensory organs in the abdomen are correctly specified and positioned in stage 16 mutant embryos. However, the ventral multidendritic (vmd) neuron cluster contains a variable number of md neurons, ranging from 4 (one md neuron missing in 29% of hemisegments) to 6 (one extra md neuron in 24% of hemisegments). The variation in number of neurons in the vmd cluster is due to variation in the number of vmd1a neurons in the cluster (the wild-type vmd cluster contains 1 vmd1a neuron). By comparing the vmd cluster on each side of the midline in a segment, it is seen that whenever two vmd1a neurons are seen in one hemisegment, no vmd1a neuron is seen in the contralateral hemisegment, strongly suggesting that the vmd1a neuron can cross the midline in these embryos. In 3 segments, the loss of the vmd1a neuron did not correlate with its duplication in the contralateral hemisegment. In these cases, the missing vmd1a neuron has probably remained at the midline. Analysis of the position of the vmd1a neuron and its precursor cells in sli2
mutant embryos at different stages indicates directly that the vmd1a neuron can cross the midline in these embryos.
Most of ventral muscles cross the midline in homozygous embryos. The Fas2
-positive fascicles are collapsed into the ventral midline at stages 16 and 17. 0.6% of inner Fas2
-positive fascicles cross the ventral midline in heterozygous embryos.
third instar larvae show defects in photoreceptor axon targeting, with gaps in the lamina and increased numbers of axons entering the medulla. slik04807b
animals show disruption of the lamina glia layer; clumps of glia and gaps in the glial layers are seen. The regions of photoreceptor axon mistargeting correlate with areas of lamin glial disruption. Many distal cell neurons enter the base of the lamina in slik04807b
third instar larvae and some distal cell neurons invade the lamina neuropil, disrupting photoreceptor innervation.
Heterozygous mutants do not exhibit significant longitudinal axon ectopic midline crossing defects.
lch5 axons stall before turning point TP1 in 10% of hemisegments or misproject to the v'ch1 pathway in 12% of hemisegments of mutant embryos. Dorsal cluster axons stall in the lateral region of the embryo in 10% of hemisegments or project in abnormal directions (usually after reaching the lateral region) in 8% of hemisegments. Mutant embryos have defects in the tracheal system, including the spiracular branch and transverse connective. Motor axon projections are often disrupted in mutant embryos. In mutant hemisegments which have a normal location of the spiracular branch, normal position and orientation of the dorsal and lateral sensory neuron cell bodies and normal motor axon trajectories, 14% of the hemisegments have lch5 axons projecting to the v' pathway and 28% of hemisegments have lesA/ldaA axons projecting to the v' pathway, while the dorsal cluster axons follow a normal trajectory to the lateral region in all the hemisegments.
Homozygous embryos show three Fas2
fascicles collapsed on the midline. The ch neurons project across the midline and either stall or reach as far as the outer far edge of the CNS. Either way they fail to branch.
Heterozygous embryos only extremely rarely show Fas2
-expressing axons crossing the midline.
One or two Fas2
-positive axon bundles cross the midline in about 36% of heterozygous embryos.
heterozygotes exhibit 3.4% midline errors (as assayed with Fas2
Heterozygotes show 4% ectopic crossing of the midline by axons in the embryonic central nervous system.
1.0% of expected mutant embryos hatch as first instar larvae. The latest observed stage of mutants is L1. Nerve cord length is 88% of embryo length. Midline guidance failure of repellence phenotype, evidenced by midline crossing, is severe. Mutant embryos show complete fusion of the longitudinal and commissural nerve trunks - few intersegmental connections are maintained. 6.8 ventral oblique muscles per segment remain on the dorsal surface of the ventral nerve cord, instead of having migrated to the edges and inserted into the ectoderm.
All CNS axons converge on the midline which is displaced ventrally.
embryos in which the midline has been rescued by expressing sliScer\UAS.cKa
under the control of Scer\GAL4sim.P3.7
often show misinsertion of dorsal muscles 1,2, 9 and 10. However, the overall position of these muscles is not dramatically affected in these embryos. Muscles 21 to 23 are largely unaffected although occasionally an extra muscle is seen. 1% of segments show crossing of the midline by muscles 6/7, 54% show abnormal insertion of muscle 5 and 36% show abnormal insertion of muscles 6/7 (either failing to reach their specific muscle insertion sites in the epidermis - 12%, or reaching the epidermis but making abnormal connections - 24%). In sli2
embryos expressing sliScer\UAS.cKa
under the control of Scer\GAL4en-e16E
, 49% of segments show muscle cells aligned with sites of ectopic sli
expression. In sli2
embryos expressing sliScer\UAS.cKa
under the control of Scer\GAL4ptc-559.1
, 20% of segments show muscle cells aligned with sites of ectopic sli
expression. Ventral longitudinal muscles 6 and 7 cross the midline in 90% of segments in mutant embryos. Muscle 13 also occasionally crosses the midline. Muscle 5 is present, although it is frequently attached to the wrong site in the epidermis.
The ventral muscles fail to migrate away from the midline in mutant embryos, resulting in muscles that extend over the dorsal surface of the CNS. The initial migration defect seen in the ventral muscle precursors of sli2
embryos is rescued by expression of sliScer\UAS.cKa
under the control of Scer\GAL4sim.P3.7
. However, striking defects are seen during the second phase, as muscles extend towards their (muscle attachment sites) MASs. Many muscles that normally attach at sli
-positive MASs are instead attached to the wrong places in the epidermis. Muscles 6 and 7 either do not reach their MASs or make abnormal connections with the epidermis. Muscle 5 is often missing or is not properly attached at one or both ends. Muscle 4 is also often not properly attached.
The GMC-1 in mutants frequently (about 10%) divide symmetrically to generate two RP2s instead of an RP2 and a sib.
Approximately 24% of heterozygous embryos show periodic crossovers of Fas2
-positive axons across the midline.
All central nervous system axons converge on the midline in sli1
All axons in the longitudinal connectives are collapsed towards a single midline tract. MP pioneers are medially displaced in stage 12/0 mutant embryos. Contralaterally projecting axons persist in sli
mutants despite fusion of longitudinal tracts. Midline glia are displaced to the ventral limit of the neuropil, and maintain contact with commissural axons. Anterior and posterior to the commissural axons the neuropil of sli
mutant nerve cords contains many more growth cone filopodia, and fewer axons than wild type. The longitudinal connectives appear to recross the midline as they project anteriorly or posteriorly, similarly to robo
mutants. Most mesectodermal cells line the ventral midline. The ventral oblique muscles do not insert below the cord, but cross the dorsal surface of the cord and insert contralaterally with the ventral longitudinal muscles. When the mutant phenotype is partially rescued by the sliScer\UAS.cBa
combination, midline structures are partially restored, but errant axons continue to cross the midline. This phenoytpe is comparable to that of sli532
or a robo
hypomorph. Mesectodermal cytoarchitecture is not restored. Embryos heterozygous for either robo1
show deviation of longitudinal fascicles towards the midline in less than 5% of segments.
Axons enter the midline, but fail to leave it and instead run along it in one longitudinal tract in the central nervous system (CNS) of sli2
embryos. The pCC axon aberrantly enters the midline in homozygous embryos, where it fasciculates with its contralateral homologue. The pCC homologues then extend anteriorly along the midline. In some segments the aCC axon crosses the midline and fasciculates with the axon of its contralateral homologue (in contrast to wild-type where it normally extends ipsilaterally away from the midline). Commissural axons such as SP1 do not leave the midline. The ventral muscles extend over the dorsal surface of the CNS.
In stage 13 embryos the dorsal median cells are mislocalised to lateral positions away from the midline. By stage 16 the cells are absent from most segments.
Mutant phenotype as assayed by Ecol\lacZrp
staining: midline missing. Mutant phenotype of lateral chordotonal axons includes: shorter axons, defasciculated axons or dorsally routed axons.
In stage 12/3 homozygous embryos the commissures are pioneered but are closer together than wild type. By stage 14 the longitudinal tracts have collapsed at the midline. Midline glia reach the dorsal midline by stage 12/5, become ventrally displaced during stage 13 and by stage 14 are spread from the dorsal to ventral surface. VUM neurons are present at the proper position during stage 11 by are misplaced ventrally by stage 13. These neurons are still present at the ventral midline at stage 14. In stage 13 embryos the wild-type number of MP1 neurons are present but displaced ventrally, by stage 14 they are fused at the midline. en+
neurons are present at stage 14 but are displaced.
Longitudinal tracts fuse at the ventral midline, though the glial scaffold is normal.
Absence of commissures and the collapse of the longitudinal tracts into a single fused tract at the midline. The midline precursor cells are displaced ventrally and do not differentiate properly. The posterior commissure sometimes forms and the anterior commissure is always initially formed, but the commissures are narrower and are not as straight or regular in shape. The commissures disappear as the midline precursors displace.
The presence of Ecol\lacZsim.7.8
construct shows that the midline cells are disorganised compared to wild type at stage 11 and at stage 13 are clustered near the ventral surface of the embryo leading to the collapse of the two lateral CNS hemiganglia and fusion of the longitudinal connectives.