Slide 6- Aine

They co-injected eGFP mRNA and Gb cad dsRNA into eggs.

As a negative control, GFP mRNA and double strained red2 RNA were also co-injected into egg as
shown in A-C.

Arrows and arrowheads indicate anterior and posterior limits of a germband, respectively.
In the Control, embryo developed normally at a morphological level.
In eggs D–F, the embryo showed class I phenocopy at a morphological level.
Development of injected embryos was retarded by a few hours, compared with normal embryos at
48 hAEL.
No significant difference in length of the embros was observed until 40 hAEL, indicating germ anlage
in Caudal RNAi embryos. But, by 60 hAEL class I embryos were shorter than the control.

G-N, stained with Fuschin to observe detailed morphology.

No sig difference in control and class 1 at 40 hAEL.
By 48 hAEL, caudal RNAi embryos were shorter than the control, while prospective gnathal and
thoracic regions remain undeveloped.
This indicated that caudal is not necessary for formation of germ anlage, but is for development of
gnathal and thoracic regions and for posterior elongation.

Slide 7- Aine
These images are the observed expression patterns in early Caudal-RNAi embryos of hunchback and
kruppel (gap genes), a pair rule gene (even skipped) and a segment polarity gene (wingless)
The silencing of caudal confirmed by WISH
In all cases the expression pattern of genes is altered by caudal silencing.
o Expression is lost in Caudal (expected), kruppel and eve
o Kruppel change is shifted to a more posterior region with either dramatic reduction of expression (F
about 29%) or less dramatic reduction (L- 25%)
o hb looks uniformly expressed in RNAi knockouts
o wg has no expression along the centre of the embryo. Reductions is caudal didn’t affect wingless in
the posterior growth zone- suggests that caudal may be a downstream gene in the
Wingless/Armadillo signally pathway.
o Embryo M. Caudal expression in Armadillo-RNAi embryos show.
o Suggests that Wingless/armadillo signalling involved in regulation of caudal in posterior growth
zone.
 Caudal control (M), shows expression of cad in Posterior,
 In armadillo RNAi, there is a loss of this expression, suggesting armadillo is upstream of caudal

Slide 8- Áine
This is a proposed regulatory networks in segmentation genes in Drosophila and Gryllus
In Drosophila
 Bcd organises anterior patterning in concentration dependent way
 Activates gap genes hunchback and kruppel
 Caudal required for patterning the posterior embryo. Regulates the gap gene Knirps and giant
In Gryllus
 Caudal may organize patterning of gnathum and thorax by activating gap genes hunchback and
kruppel.
 In posterior growth zone (box on the right), wingless and armadillo signalling may regulate
expression of caudal, leading to posterior growth of Gryllus embryo.
 It remains unclear whether maternal caudal is involved in the processes.
 Slide 9 (Alix)

Slide 10
In short- or intermediate-germband insects, the posterior segments form sequentially from the
cellularized posterior growth zone, suggesting that the process might require a cell–cell
communication pathway to generate the segments.
This doesn’t occur in drosophila, where all segments specified simultaneously by a genetic cascade
of transcription factors at the syncytial blastoderm stage
 Caudal expression is present in Posterior growth zone
 Abdominal segments were deleted or severely abnormal in Caudal RNAi embryos
 This suggests caudal has a function in abdominal formation in later stages
 Abdominal defects due to inhibition of proper posterior germ band growth (as shown by
morphological analysis at early stages) suggests caudal involved in proliferation of posterior germ
band

Slide 11
 Caudal expressed in overlapping manner with wingless in posterior growth zone until segmentation
complete- suggests an interaction]
 Defects in caudal RNAi embryos like Armadillo RNAi embryos
 Wingless expression was unaffected in Caudal RNAi embryos but almost completely eliminated in
armadillo RNAi embryos, suggest posterior growth is regulated by caudal expression which in turn is
regulated by wingless/armadillo signalling in the posterior growth zone.
 Expression patterns of wingless and caudal in posterior growth zone similar to orthologues in short-
germband insects, model may be conserved.
 Expression of cad also similar to vertebrate Cdx genes in posterior parts of the primitive streak. E.g.
Wnt3 in the mouse important regulator of Cdx1 expression: the Wnt/Cdx may be conserved from
Wingless/Caudal interaction.