In control stage 15 embryos, the anterior and posterior lateral trunks (Lta and Ltp, respectively) from adjacent segments are joined. In contrast, gaps occur between the Lta and Ltp in stage 15 homozygous jing3 embryos.
By stage 15, in control embryos, tracheal ganglionic cells and branches enter the ventral nerve cord (VNC) toward the central nervous system (CNS) midline. In contrast, three ganglionic branch (Gb) phenotypes are observed in in jing3 homozygotes: (1) Gbs do not reach the CNS midline appearing truncated; (2) Gbs show breaks; and (3) Gbs are absent from ventral positions
In contrast to control stage 13 embryos, the dorsal branch (Db) in jing3 embryos appear truncated and there are breaks in the dorsal trunk (Dt) as well. In stage 15 homozygous jing3 embryos, there are Dt gaps and Db breaks, Dt constrictions and truncated Db. The aberrant tracheal patterning in association with jing3 does not appear to be caused by cell loss. An additional phenotype observed in jing3 mutant embryos is the aberrant presence of Db cells in adjacent tracheal metameres instead of near the dorsal midline as in control embryos.
Fas2-positive longitudinal axons aberrantly cross the central nervous system midline in stage 16 homozygous embryos.
robo-positive and Fas2-positive longitudinal axons cross the central nervous system midline in jing3/Df(2R)ST1 stage 16 embryos.
Longitudinal glia inappropriately occupy positions at the central nervous system midline in stage 12 jing3/Df(2R)ST1 embryos.
jing3/Df(2R)ST1 embryos have a normal number of glia in the ventral nerve cord at stage 12, but the number of glia is reduced compared to controls at stage 15.
37.8% of stage 14 homozygous embryos have a reduced number of midline glial cells. jing3/Df(2R)ST1 embryos have a reduced number of midline glial cells. The embryos have breaks in the dorsal trunk of the tracheal system at stage 15.
The initial formation of the circumesophageal connectives appears normal during stage 12 in homozygous embryos, as both descending and ascending axons meet and fasciculate to form a connective. However, the preoral commissure is not pioneered in stage 12/0 homozygous embryos and formation of the preoral commissure has still not occurred in stage 15 homozygous embryos. The postoral tritocerebral commissure and circumesophageal connectives are reduced compared to wild type in stage 15 homozygous embryos and the connection between the brain and the ventral nerve cord is absent. The number of apoptotic glia in stage 11 homozygous embryonic brains is not significantly altered compared to wild type. At stage 15, mutant brains contain an increased number of apoptotic glia and a reduction in the total number of glia in the brain compared to wild type. Stage 15 mutant embryos show an approximately 26% reduction in the number of cas-expressing neurons in the brain compared to wild type, while cell death of these neurons is increased in the mutant brains. The number of sim-positive cells in the brain is reduced by about 50% in stage 15 mutants compared to wild type.
In jing3 homozygous embryos, commissural growth cones are often in the midline by stage 12. By stage 14, mutants show losses of longitudinal connections and reduced commissures compared to wild-type. The longitudinal fascicles stall within segment boundaries causing breaks in the longitudinal tracts in 95% of mutants. A subset of normally ipsilateral axons of the most medial fascicle project instead contralaterally, suggesting that midline repulsive mechanisms are perturbed. In wild-type embryos, one or two apoptotic glia are seen within the entire nerve cord of a stage 12 embryo. By contrast, every nerve cord segment in jing3 mutant embryos contains apoptotic glia. Loss of VUMs, MNB MP1, vMP2 and dMP2 neurons are seen. Ectodermal segmentation appears relatively normal in mutants. Compared to about three in wild-type embryos, about 20 apoptotic cells are seen in stage 11 mutant embryos. There is also an increase in the number of apoptotic profiles surrounding the tracheal pits in mutants. Cell death is observed throughout embryogenesis in all tracheal branches in mutants. Homozygotes exhibit tracheal defects; tracheal cells invaginate but the tracheal branches do not migrate properly anteriorly across en-positive stripes. By stage 15, parts of the dorsal trunk, the dorsal branch and transverse connectives are missing and correlate with a loss of cells by apoptosis. In addition the visceral branch does not form in mutants. The overall embryonic pattern of cell death is similar to wild-type, suggesting the CNS and tracheal phenotypes are specific to those systems.