To determine if cdh11 could act as a potential mediator of Qkia’s function in Left-Right patterning, we next analysed the effect of Cdh11 depletion on organ laterality.
We used a previously described splice blocking morpholino (cdh11MO) (Clendenon et al., 2009) and a homozygous cdh11 mutant line carrying the cdh11sa14413 allele.
This allele contains a premature stop codon upstream of the cdh11 transmembrane and intracellular domain coding region, and therefore is not expected to produce a functional protein (Fig. II.M1c,d).
In regard to cardiac laterality, we observed that 37% of cdh11 morphants had unlooped heart tubes and 11% had reversely looped heart tubes (L loop) at 48-50 hpf (Fig. II.6a,b). Additionally, at 30 hpf, 19% of cdh11 morphants displayed unjogged heart tubes and 13% displayed reversely jogged heart tubes (Right jog) (Fig. II.6d,e). However, no significant laterality defects were detected in the heart tubes of cdh11 mutants, both at 48-50 hpf and at 30 hpf (Fig. II.6a,c,d,f). In cdh11 morphant embryos we also observed that the lateral positions of the liver and pancreas were reversed in a small percentage of embryos (17%) (Fig. II.6g,h).
However, as with the heart tube, no significant laterality defects were observed in the liver and pancreas of cdh11 mutant embryos (Fig. II.6g,i).
Taken together these results indicate that the establishment of heart, liver and pancreas laterality is compromised under cdh11 knockdown conditions, but not under cdh11 knockout conditions.
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Fig. II.6 – cdh11 morphants, but not cdh11 mutants, display organ laterality defects.
(Figure legend on the next page)
(g) Normal Bilateral Left jog mild Left jog No jog
Left
69 Fig. II.6 – cdh11 morphants, but not cdh11 mutants, display organ laterality defects.
(a) Ventral views of representative embryos illustrating the different heart laterality phenotypes observed at 48-50 hpf after WISH for myl7. (b,c) Quantification of the heart tube laterality phenotypes observed (b) under cdh11 knockdown conditions (cdh11MO) and (c) in cdh11 mutant embryos (cdh11sa14413). The respective control conditions are indicated.
(d) Dorsal views of representative embryos illustrating the different heart laterality phenotypes observed at 30 hpf after WISH for myl7. (e,f) Quantification of the heart laterality phenotypes observed under (e) cdh11 knockdown conditions (cdh11MO) and (f) in cdh11 mutant embryos (cdh11sa14413). The respective control conditions are indicated. (g) Schematic representation of the different phenotypes observed regarding the laterality of the Liver (L) and Pancreas (P). Visceral organ laterality was assessed (h) in sox17:GFP embryos at 48 hpf, (i) after WISH for foxa3 at 50 hpf. (h,i) Quantification of the liver and pancreas laterality phenotypes observed (h) under cdh11 knockdown conditions (cdh11MO) and (i) in cdh11 mutant embryos (cdh11sa14413). The respective control conditions are indicated.
To determine if the organ laterality defects observed in cdh11 morphants derived from a disruption of asymmetric Nodal signalling, we analysed the expression of the laterality associated genes spaw and pitx2 in the LPM of cdh11 morphants. The left side specific LPM expression of pitx2a appeared to be randomized in cdh11 morphants, with 33% of embryos displaying right side expression, 24% displaying either bilateral expression or no expression in the LPM and 43% displaying left side expression (Fig. II.7a). Similarly, expression of the left side specific LPM gene spaw was randomized in cdh11 morphant embryos, with 40% of morphants displaying right side expression, 20% displaying either bilateral expression or no expression in the LPM and the remaining 40% displaying left side expression (Fig. II.7b). These results indicate that the cdh11 morphant phenotype stems from a disruption upstream of LPM Nodal signalling, probably at the level of asymmetric signal establishment in the KV.
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Fig. II.7 – Expression of Left-Right patterning genes is affected in the LPM of cdh11 morphants.
(a) WISH analysis of pitx2 expression in the lateral plate mesoderm of 24 ss control and cdh11 morphant embryos. (b) WISH analysis of spaw expression in the lateral plate mesoderm of 20 ss control and cdh11 morphant embryos.
To ascertain if cdh11 could influence Left-Right patterning by contributing to asymmetric signal establishment in the KV, we next set out to determine if cdh11 is expressed in this organ. Through WISH we were able to verify the previously reported expression of cdh11 in the intermediate mesoderm and neural tube at the 8 ss, as well as in the otic vesicle and inner ear at the 20 ss and 24 hpf, respectively (Fig. II.8a-g) (Franklin and Sargent, 1996, Clendenon et al., 2009). However, this technique did not enable a clear detection of cdh11 expression in tissues with a known involvement in Left-Right patterning, specifically, the KV at the 8ss and the LPM at the 20ss and at 24hpf (Fig. II.8a-g).
To account for a potentially reduced sensitivity of the WISH method to low levels of gene expression, we resorted to a complementary approach to determine if cdh11 is expressed in the KV. Specifically, we performed fluorescence activated cell sorting (FACS) of 8 ss sox17:GFP transgenic embryos, which label the KV and the endoderm at this developmental stage. Total RNA was subsequently extracted from the sorted cells and, following reverse transcription, the expression of cdh11 was detected by PCR amplification. The expression of dand5 was also analysed and used as a marker for KV cells. This approach allowed us to detect cdh11 expression
0
71 in cells sorted for high levels of GFP expression (GFP++), with these cells also expressing dand5 (Fig. II.8h,i,j). Therefore, even though further experiments are required to validate this expression, the results obtained suggest that cdh11 is expressed in KV cells at the 8 ss.
Taken together, our results reveal that under cdh11 knockdown conditions internal organ laterality is compromised, most likely due to a disruption of the establishment of early asymmetric cues in the KV.
Fig. II.8 – cdh11 expression in wildtype embryos and FACS sorted sox17:GFP cells.
(a-g) WISH for cdh11 in wildtype embryos (a,b) at the 8 ss, (c,d) at the 20 ss and (e,f,g) at 24 hpf. (a,c,e,f) Whole-mount embryos, dorsal view, anterior to top. (b) Transversal section at the KV level (B). (d) Transversal section at the otic vesicle level (D). (g) Transversal section at the inner ear level (G). (b,d,g) Dorsal to top. (a-g) IM, Intermediate mesoderm;
NT, Neural Tube; KV, Kupffer’s vesicle; Ov, Otic vesicle; MB, Midbrain; IE, Inner Ear. (h) FACS profile for sox17:GFP 8 ss embryos. Cells were sorted based on GFP levels. (i) PCR detection of dand5 in samples obtained from sox17:GFP cell sorting, 8 ss whole embryos and 2 hpf whole embryos. (j) PCR detection of cdh11 in samples obtained from sox17:GFP cell sorting, 8 ss whole embryos and 2 hpf whole embryos. (i,j) The 8 ss whole embryo condition was used as a positive control and the 2 hpf whole embryo condition was used as a negative control for both cdh11 and dand5 expression. ntc, no template control; m1,
GFP- GFP+ GFP++ 8ss 2hpf ntc GFP- GFP+ GFP++ 8ss 2hpf ntc
dand5 cdh11
(h) (i) (j)
m2 m1
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