function embryos
7.2. The CNS phenotype in /n/rror mutant embryos
7.2.4. Loss of Mirror function leads to defects in the CNS
Most neurons in the ventral nerve cord are interneurons, and many of these
intemeurons have long axons that are organised in a characteristic ladder-like pattern,
showing two longitudinal tracts, which are connected via two commissural tracts per
segment. It has been previously noted that the overall pattern o f commissures and
connectives of the CNS is abnormal in mirror mutant embryos (McNeill, unpublished; see also chapter 6, flg.6.6.F+H). In order to further characterise this observation, stage
14 and 16 Iroquois and wild type embryos were stained with the BP 102 antibody. Typically the axonal projections cross the midline in one of the commissures before
they extend rostrally or caudally in one of the longitudinal pathways (fig.7.13.A). The
anterior commissure develops after, but contains almost twice as many axon bundles as
does the posterior commissure. Initially the commissures are in close proximity to each
other at the midline and only get separated as a result of cell migration and cell adhesion
events between midline glial cells, the RPl and the RP3 neurons. This separation takes
place normally during embryonic stage 12 (reviewed in Goodman and Doe, 1993;
Chapter 7: Mirror confers neurohiast identity'
at least stage 14. In addition, the anterior and posterior commissures are usually thinner
in these embryos than in wild type embryos, suggesting that axon projections are
missing (fig.7.13.B). The longitudinal axon pathway is pioneered for much of its length
by the ascending pCC growth cone starting at stage 12. The pCC growth cone appears
to contact and extend directly towards one o f the medial rows of longitudinal glial cells,
named LG5 (Jacobs and Goodman, 1989). Finally the pathway becomes complete as
several other growth cones meet and their axons fasciculate (fig.7.13.C). In Iroquois
mutant embryos the longitudinal pathway is often interrupted or does not form to
connect the segmental ganglia in the CNS (fig.7.13.D). A similar phenotype has been
described in embryos with a mutation in the gene longitudinals lacking (lola). In these embryos the LG5 cell is bom, migrates and divides as normal but the interaction with
growth cones that extend along the longitudinal pathway is perturbed (Giniger et al.,
1994; Seeger et al., 1993). Moreover, Iroquois mutant embryos frequently show a hole in the CNS (fig.7.13.D).
To conclude. Mirror loss-of-function embryos exhibit several defects during the
development of the CNS. Clusters of Engrailed expressing cells are altered. In particular
cells within the ML cluster are either absent, mislocated or fail to express Engrailed.
Two or three cells of this cluster normally give rise to serotonin expressing neurons.
These cells are usually characterised by their expression of the Eagle protein. A
preliminary attempt to locate this serotonin precursor cells by using an anti-Eagle
antibody suggests that these cells have most likely adopted a different cell fate and
don’t express the antigen anymore or are absent rather than mislocated. The axonal
defects of Mirror loss-of-function embryos resemble those o f the lola mutant phenotype. It would be interesting to investigate if the LG5 has also lost cell adhesion
properties or if it is missing in Mirror mutant embryos. Finally, it should be mentioned
*
Figure 7.13. CNS phenotype o f Iroquois mutants. Axon tracts within the ventral nerve cord o f wild type and Iroquois mutant embryos labelled with mAh BP 102 at stage 14 and 16. A,C) The wild type embryo demonstrates that axons are normally organised into two longitudinal tracts, which are connected via two commissural tracts per segment. B,D) /ro-mutant embryos exhibit a loss o f the longitudinal connections (arrow) o f the CNS and commissures appear thinner (B) than in wild type. The severe defects in the commissural tracts o f Iro' embryos are usually more outstanding at earlier stages o f development (compare B and D). In addition, Iro' mutants often have holes in the CNS between the commissures o f two segments (D). All panels are ventral views o f the embryos and are shown anterior to the right.
Chapter 7; Mirror confers neurohiast identity^
that a transient hole occurs normally in the epidermis of wild type embryos during the
ventral closure event at stage 12. Closure of this hole implicates cell migration and
adhesion of ventral epidermal cells which seam the midline in a non-random process by
interdigitation from anterior to posterior (reviewed in Martinez Arias, 1993). It might be
that these are the observed holes in the ventral epidermis which failed to close in
Iroquois mutant embryos. Alternatively it might be the same holes, which have been observed in the disorganised ventral midline of AS-C mutant embryos. In this case, it
has been proposed that the median neuroblast does not develop and leaves a gap at its
location (Cabrera et ah, 1987).
Discussion
The microarray analysis showed that over-expression of Mirror reduces the level
of Kriippel transcript in a manner characteristic for “slow Mirror response” genes. Downregulation of Kriippel transcript is already detected after 30 minutes of ubiquitous Mirror activity. The in vivo analysis confirmed expression o f Krüppel and Mirror in mutually exclusive domains in the ventral nerve cord during the embryonic stages 11-
14. Krüppel expression in the CNS is highly dynamic and complex. UAS controlled
over-expression o f Mirror reduces Krüppel expression in the neuroectoderm and
individual muscle precursor cells (not shown). Furthermore, loss of Mirror activity leads
to ectopic Krüppel expression in the embryo. The phenotype is variable and is
occasionally manifested in the entire Mirror domain. Krüppel expression is consistently
observed in a single medial row neuroblast per hemisegment, the NB2-2. Several other
defects in the developing CNS were also observed in Mirror loss-of-function embryos,
which might be independent o f ectopic K riippel expression. The expression of Engrailed and Eagle is altered in these embryos in medial and lateral rows of NBs. It is
noteworthy to mention that medial and lateral rows are also preferentially affected by
the loss of lethal o f scute expression (Martin-Bermudo et al., 1991) or by removal of the whole AS-C (Jimenez and Campos-Ortega, 1990), which suggests that Mirror and the
AS-C might act in the same pathway. Taken together, this data suggests that Mirror is
required to confer neuroblast identity. Neuroblast identity depends on the combination
o f genes it expresses and is tightly correlated with the time and position at which the
neuroblast forms. The identity of a NB can be revealed by the characteristic lineage of
GMC and neurons it will form. Furthermore, local cell-cell interactions have been
shown to contribute to the cell identity of neuroblasts (Goodman and Doe, 1993). This
Chapter 7: Mirror confers neurohiast identity^
possible to determine if NB2-2 or other NBs adopt a new fate in the absence of Mirror
function. It seems that neurons and glia cells are lost in mirror mutant embryos (see also chapter 4). To assess if loss of Mirror function leads to increased cell death in the
embryo, acridine orange staining were carried out. Acridine orange is a dual-
fluorescence dye that interacts with DNA and RNA and is commonly used as a marker
for apoptotic cell death in Drosophila. Embryos which lack Mirror function showed increased staining in a segmented pattern at stage 12 and staining did increase further in
later stages (McNeill, unpublished). Thus, cell death could also account for the observed
loss of Eagle and Engrailed positive cells.
The commissural and longitudinal axon pathways of the CNS are disrupted in
Mirror loss-of-function embryos. This indicates that Mirror plays an important role for
the proper formation of the axonal network. Indeed, Mirror is expressed in the median
neuroblast and other midline cells in the anterior part of each segment. It is this area
from which the midline glial cells develop from a set of 2-3 progenitors (Bossing and
Technau, 1994). The midline glial cells migrate along cell processes o f the ventral
unpaired median neurons (VUM) to separate anterior and posterior axon commissures
(Klambt and Goodman, 1991). If this migration is blocked, a typical fused commissure
phenotype develops (Stollewerk and Klambt, 1997). This might be the case in Iroquois
mutant embryos at stage 14. However, the separation of the commissures still seems to
take place but in a retarded manner and is often incomplete. In this context, it might be
noteworthy that Krüppel is normally expressed in the midline precursor cells between
the commissures and connectives of the nervous system.
The observed defects in the longitudinal pathway persist in later stages. Again,
the specialised midline cells have an essential role in regulating the neurons that project
their axons on one side (reviewed in Tear, 1999). Specific staining of axon bundles
(fascicles) using Fasciclin I (FasI) and Fasciclin II (FasII) antibodies should be useful to
further characterise the CNS phenotype. FasI is expressed on the surface of a subset of
commissural pathways in the CNS and all sensory axon pathways in the PNS (Zinn et a i, 1988), whereas FasII specifically labels the medial MPI fascicle and other axons in the longitudinal pathway (Grenningloh et al., 1991).
During the course of this project, the preferred site for Mirror binding has been
identified using a site selection assay. Mirror binds to a palindromic DNA sequence
(acanntgt), which is different from the previously identified binding site for Araucan
(Aphrodite Bilioni and Helen McNeill, unpublished). In vivo assays have shown that the palindrome is indeed functional. Repeats of the palindrome fused to a lacZ reporter
construct, which is under the control of a basal promoter, have been injected in flies.
Staining with p-galactosidase of eye imaginai discs revealed repression of the reporter
transgene in the dorsal half of the eye, which correspond to the Mirror expression
domain (Aphrodite Bilioni and Helen McNeill, unpublished). Therefore, the presence of
this palindrome in regulatory elements of genes identified in the microarray screen
would argue in favor of a direct regulation by Mirror. The bioinformatic search to match
potential target genes from the over-expression analysis which contain this site within a
regulatory element is currently under way. Strikingly, three copies of the palindrome are
located in the NS2 element in the central region (within 170bp) of the Krüppel intron. The N Sl element also contains the identified Mirror binding site. N Sl and NS2 are
required to drive lacZ expression of a reporter construct in the Krüppel pattern in the nervous system. Currently in vivo reporter assays using the Krüppel intron with wild type and mutated palindromes are under way to further strengthen the argument that
Mirror acts directly to shape the Krüppel expression pattern in the developing CNS. Thus, Krüppel represents the most promising candidate so far to be directly
Chapter 7; Mirror confers neurohiast identity
regulated by Mirror,