Expression pattern of efhc2 in zebrafish embryos
Whole-mount mRNA in situ hybridization (WISH) and semi-quantitative RT-PCR were performed to study the spatio-temporal expression pattern of efhc2 during zebrafish development. Expression of efhc2 was first observed at 6 hpf by RT-PCR and express during the early development period (Fig. 1a). WISH data showed that efhc2 mRNA was expressed ubiquitously at 6 hpf (Fig. 1b). By 9 hpf, its expression was localized to kupffer’s vesicle (KV), a transient organ containing ciliated cells (Fig. 1c). At 12 hpf, efhc2 is expressed in the intermediate mesoderm, notochord and otic vesicle (Fig. 1d). In 24 hpf embryos, efhc2 is expressed in pronephros, olfactory placode, notochord, otic vesicle, epiphysis, and tail bud. efhc2 is not expressed in the glomerulus and neck segments of the pronephros, but its expression starts at the PCT. More intense expression of efhc2 was found in the proximal part of PCT compared to the distal part of the same segment (Fig. 1e, f). Strongest expression of efhc2 can be seen in the pronephric tubule segments; PST and DE. Its expression is low in DL and PD (Fig. 1e–g). Histological analysis of two colour WISH of efhc2 and pdzk1 [7] confirmed the expression of efhc2 in pronephros (Fig. 1h–j). Both 36 hpf and 48 hpf embryos showed expression of efhc2 in the pronephros, olfactory placode, notochord, otic vesicle, epiphysis, and tailbud (Fig. 1k, l). At 72 hpf, efhc2 expression was observed in neuromast cells and olfactory placode (Fig. 1m). Expression pattern indicates the association of efhc2 with pronephros morphogenesis. Thus, we sought to address the function of efhc2 in pronephros development in zebrafish.
Knock-down of Efhc2 reveals its role in pronephros development
Zebrafish efhc2 gene contains 16 exons. A morpholino antisense oligo targeting exon-3/intron-3 splice donor junction was designed to block the pre-mRNA splicing of efhc2 (efhc2-Mo). Corresponding mis-match morpholino antisense oligo (efhc2-MM) containing 5 mis-matches compared to efhc2-Mo that was predicted not to affect normal splicing of efhc2 pre-mRNA was used as a control. efhc2-Mo was injected in different quantities (1–4 ng) into one-cell stage embryos and its effect on RNA splicing was checked at 24 hpf by RT-PCR using forward and reverse primers corresponding to exon-1 and exon-4 respectively. As expected, RT-PCR amplification of efhc2 cDNA prepared from isolated RNA from 24 hpf wild-type and efhc2-MM injected control embryos resulted in a 651 bp product, whereas the efhc2-Mo injected embryos showed a 311 bp product (Fig. 2a). Both 651 bp and 311 bp fragments were cloned into pCR-BluntII-Topo vector and sequenced. The sequencing data confirmed that injection of efhc2-Mo results in deletion of a 340 bp fragment containing both Exon-2 and 3 (Additional file 1: Figure S1). This indicates that injection of efhc2-Mo leads to mis-splicing of efhc2 mRNA whereas injection of efhc2-MM has no effect on normal splicing. Based on above observations, 2 ng of efhc2-Mo was injected into embryos for further characterization of its loss-of-function. Same quantity of efhc2-MM was used as control. The un-injected or efhc2-MM injected embryos did not show any morphological defects (Fig. 2b). The efhc2-Mo injected embryos, however, exhibited phenotypic defects such as slightly curved body, mild pericardial oedema and hydrocephalus typically observed in embryos where pronephros development and function is impaired (Fig. 2b). Similar morphological defects were observed when an independent morpholino antisense oligo designed to block efhc2 translation (efhc2-ATG-Mo) was injected (Additional file 2: Figure S2A).
Knock-down of Efhc2 results in impaired pronephros function
Next, we asked if knock-down of Efhc2 affects the function of the nephrons. Functional zebrafish nephrons of pronephros can easily clear 40 kDa dextran injected into the cardinal vein [16]. The efhc2-Mo and efhc2-MM morphants were injected with fluorescein isothiocyanate 40 kDa dextran at 48 hpf and the embryos were visualized at 72 and 96 hpf. efhc2-MM injected embryos were able to clear it and the dextran was not visible at 72 or 96 hpf. However, abundant fluorescent dextran was seen in embryos where Efhc2 was knocked-down (Fig. 2c). Accumulation of 40 kDa dextran also led to severe pericardial oedema in these embryos (Fig. 2c). These data indicate that Efhc2 is crucial for pronephros function.
Efhc2 knock-down affects segmentation of distal part of the pronephros
The nephrons are divided into different segments that reflect their function and are highly conserved among vertebrates [6, 10]. Eight distinct segments can be seen in a nephron of a zebrafish embryo [7]. We asked if loss-of-Efhc2 leads to aberrant segmentation of the nephrons. Sodium-dependent phosphate transporter slc20a1a is expressed in the pronephros aligning with 5th to 8th somite at 24 hpf and from 3rd to 7th somite at 48 hpf demarcating the PCT. Transient receptor potential cation channel gene trpm7 is expressed in part of the nephron adjacent to 9th to 11th somite at 24 hpf and from 8th to 11th somite demarcating the PST segment of the pronephros. The expression domains of slc20a1a and trpm7 were identical in both efhc2-Mo and efhc2-MM injected embryos (Fig. 3a and Additional file 3: Figure S3A). This indicates that knock-down of Efhc2 does not influence segmentation of the proximal part of the pronephric tubule consisting of PCT and PST. The expression of sodium/potassium/chloride transporter slc12a1 in the pronephros next to 12th and 13th somite at 24 hpf and 12th to 14th somite in 48 hpf demarcates the DE segment of the nephron. This expression domain of slc12a1 was not changed in efhc2-MM injected embryos. However, Efhc2 knock-down led to the expression of slc12a1 in 12th to 15th somite in both 24 and 48 hpf embryos (Fig. 3b and Additional file 3: Figure S3A). This expression domain of slc12a1 in Efhc2 knock-down embryos indicates that loss-of-Efhc2 function results in the expansion of DE distally. The DE expansion was observed in 53% (51/97) at 24 hpf and 69% (49/71) at 48 hpf in Efhc2 knock-down embryos (Fig. 3b and Additional file 3: Figure S3A). A second morpholino designed to block efhc2 translation (efhc2-ATG-Mo) also lead to expansion in DE expression domain by 3 somites at 48 hpf in 59% (16/27) embryos as marked by slc12a1 expression (Additional file 2: Figure S2B).
The efhc2-Mo morphants showed significantly reduced DL (Fig. 3b and Additional file 3: Figure S3A). The expression of DL marker sodium/chloride transporter slc12a3 was confined to pronephros adjacent to 16th and 17th somite in 24 hpf and 48 hpf embryos injected with efhc2-Mo. Whereas, the position of DL segment was normal in efhc2-MM injected embryos, where it was expressed next to 14th to 17th somite in 24 hpf and 15th to 17th somite in 48 hpf embryos (Fig. 3b and Additional file 3: Figure S3A). The DL reduction in efhc2-Mo morphants was 53% (54/102) at 24 hpf and 55% (42/77) at 48 hpf (Fig. 3b and Additional file 3: Figure S3A). This result was confirmed by injection of efhc2-ATG-Mo, which resulted in reduction of DE domain in morpholino injected embryos. The slc12a3 expression domain was reduced to 16th and 17th somite at 48 hpf in 59% (16/27) embryos as compared to normal expression from 15th to 17th somite (Additional file 2: Figure S2B). Stanniocalcin (stc1) is expressed in CS (next to somite number 15) and is involved in calcium and phosphate homeostasis. The expression level and position of stc1 was affected by knock-down of Efhc2. stc1 expression was reduced in efhc2-Mo injected embryos as compared to embryos injected with control efhc2-MM morpholino. Its localization was shifted distally next to 16th somite, whereas efhc2-MM injected embryos showed normal expression of stc1 adjacent to 15th somite. The CS reduction in efhc2-Mo morphants was 50% (43/85) (Fig. 3b and Additional file 3: Figure S3A). efhc2-ATG-Mo injected embryos also showed the same results as seen with efhc2-Mo injected embryos. The expression level of stc1 was reduced and the expression domain was sifted from 15th somite to 16th somite in efhc2-ATG-Mo injected embryos (Additional file 2: Figure S2B). To confirm the specificity of phenotype caused by morpholino-mediated knock-down of Efhc2, we co-injected zebrafish efhc2 mRNA with efhc2-Mo. To check overexpression phenotype, we injected 100–300 pg of efhc2 mRNA/embryo. Embryos injected with 100–200 pg mRNA develop normally. However, more than 200 pg mRNA injection leads to severe developmental defects (Additional file 4: Figure S4). Hence, we have injected 200 pg efhc2 mRNA for rescue experiments. Co-injection of zebrafish efhc2 mRNA partially rescued the effect of efhc2-Mo mediated knock-down of endogenous Efhc2. We found that the DE segment defect was rescued by 62.5% (20/32) at 24 hpf and 56% (24/43) at 48 hpf. DL segment defect was rescued by 62.07 (18/29) at 24 hpf and 57% (24/42) embryos at 48 hpf. More than 92.5% of efhc2-Mo morphants were rescued for CS segment development (Fig. 3b, c). Thus, our data suggests that the pronephros segmentation phenotype is Efhc2 knock-down specific.
Taken together, our experiments suggest that loss-of-Efhc2 function leads to expansion of DE distally and reduction of CS and DL segments of the distal pronephric tubule in zebrafish embryos (Fig. 3, Additional file 2: Figure S2 and Additional file 3: Figure S3). Hence, Efhc2 is essential for normal segmentation of the distal part of the pronephros.
Efhc2 has no influence on Retinoic acid mediated segmentation of zebrafish pronephros
Our findings reveal an important role for Efhc2 in the segmentation of distal part of zebrafish pronephros. Several reports suggest that RA signaling is important for nephron segmentation in vertebrates [4, 7]. RA signaling is required for the development of PCT and PST and is thought to inhibit the formation of distal segments such as DE and DL in zebrafish [15]. Exogenous treatment of RA results in a pronephros with expanded PCT and PST. The DE and DL segments are either reduced or shifted distally dependent on the time and concentration of RA treatment [7]. Conversely, inhibition of RA synthesis by DEAB (4-diethylaminobenzaldehyde) results in reduced proximal segments and expanded distal segments [7]. Hence, we asked if Efhc2 plays any role in RA mediated inhibition of distal pronephric segment formation. The uninjected control or efhc2-Mo injected embryos were treated with exogenous RA (1 × 10−7 M) from 9 to 16 hpf and the formation of distal pronephric tubule segments were checked at 24 and 48 hpf. As reported by other groups, RA treated wild-type embryos had the DE segment shifted distally compared to vehicle (DMSO) treated embryos. The RA treated embryos expressed DE marker slc12a1 in a domain next to 14th to 16th somite compared the DMSO treated controls that showed normal expression domain next to 12th to 14th somite. Injection of efhc2-Mo did not shift the DE segment and the anterior domain of slc12a1 was still localized to 12th somite. These embryos, however, had a slightly expanded DE segment. The DE segment shifted distally and expanded when efhc2-Mo injected embryos were treated with RA (Fig. 4a and Additional file 5: Figure S5). Essentially, RA treatment of Efhc2 knock-down embryos exhibited a phenotype that was a combination of both RA treatment and Efhc2 knock-down (Fig. 4a, b).
Next, we examined the effect of RA treatment on DL and CS segments in embryos lacking Efhc2. DMSO treated wild-type embryos showed expression of DL marker slc12a3 next to 15th to 17th somite, which was shifted distally and reduced to an expression domain next to 17th and 18th somite when treated with RA. The efhc2-Mo injected embryos had the slc12a3 expression domain reduced in nephron next to 16th and 17th somite. However, when these Efhc2 knock-down embryos were subjected to RA treatment, the DL segment was dramatically reduced to only one somite length and occupied the distal most part of the pronephric tubule next to 18th somite (Fig. 4a and Additional file 5: Figure S5). RA treatment of embryos lacking Efhc2 had a similar effect on the CS. The CS segment marker stc1, which is normally expressed next to 15th somite, was reduced and shifted distally to 18th somite in the RA treated and 16th somite in efhc2-Mo injected embryos. This expression of stc1 was completely abolished in more than 90% embryos that lacked Efhc2 and were treated with RA (Fig. 4a).
In summary, efhc2-Mo injected embryos show two distinct and opposing effects in response to exogenous RA treatment within the distal pronephric tubule segments. RA treatment of embryos lacking Efhc2 function expands the DE segment whereas, under same conditions, the CS and DL segments are much reduced (Fig. 4b and Additional file 5: Figure S5). However, the effect of RA treatment of Efhc2 knock-down embryos reflects a combination of the effects of Efhc2 knock-down and exogenous RA treatment. Hence, the effect of RA and Efhc2 on pronephros segmentation may be independent of each other.
Next, we checked the effect of lack of RA signaling on Efhc2 mediated segmentation of distal part of the nephron. RA synthesis was inhibited by treatment of embryos with 4-diethylaminobenzaldehyde (DEAB) (1 × 10−5 M) or 3,7-dimethyl-2,6-octadienal (citral) [17] (2.5 × 10−5 M) from 9 to 16 hpf, and the effect of this treatment on pronephros segmentation was monitored at 24 and 48 hpf using segment-specific marker gene expression. DEAB treatment resulted in nephrons that had the DE domain shifted proximally and the DE marker slc12a1 was expressed in a region next to 9th to 12th somite as compared to its expression next to 12th to 14th somite in vehicle (DMSO) treated embryos (Fig. 5a). However, DEAB treatment of Efhc2 knock-down embryos resulted in slc12a1 expression in nephron next to 8th to 13th somite (Fig. 5a and Additional file 5: Figure S5). Blocking RA synthesis by citral also resulted in similar changes in DE marker expression. Citral treatment of Efhc2 knock-down embryos resulted in slc12a1 expression in nephron next to 8th to 14th somite (Fig. 6a). This indicates that inhibition of RA synthesis by DEAB or citral in combination with Efhc2 knock-down leads to embryos with expanded DE segment as seen in efhc2-Mo morphants and a slight shift of this domain to proximal part of the embryos as seen in DEAB or citral treated embryos (Figs. 5b and 6b).
Inhibition of RA signaling by DEAB or citral results in expansion and slight proximal shifting, and Efhc2 knock-down results in reduced DL segment. The DL marker slc12a3 was expressed next to 13th to 17th somite in DEAB or citral treated embryos as compared to its normal expression domain of 15th to 17th somite in vehicle treated embryos. slc12a3 was expressed in 16th to 17th somite in Efhc2 knock-down embryos (Figs. 5a, 6a and Additional file 5: Figure S5). DEAB treatment of Efhc2 knock-down embryos resulted in slc12a3 expression in 14th to 17th somite. Citral treatment of Efhc2 knock-down embryos resulted in slc12a3 expression in nephron next to 15th to 17th somite. This indicates that DEAB or citral treatment of Efhc2 knock-down embryos had resulted in DL segment that had shifted proximally compared to both wild type or Efhc2 knock-down embryos. The DL segment in these embryos was expanded when compared to vehicle treated or Efhc2 knock-down, but there was a reduction in DL domain when compared to DEAB or citral treatment alone. These effects of RA signaling inhibition on DE and DL in Efhc2 knock-down embryos is opposite of the effects seen upon exogenous RA treatment. These data support the observation that exogenous RA treatment or inhibition of RA synthesis is able to exhibit their effects in absence of Efhc2.
The CS segment present next to 15th somite was shifted proximally and expanded upon DEAB treatment. Expression of CS marker stc1 was much reduced and was shifted distally to 16th somite after Efhc2 knock-down. DEAB treatment of embryos lacking Efhc2 resulted in enhanced stc1 expression in somite 10th to 11th (Fig. 5a and Additional file 5: Figure S5). Citral treatment of Efhc2 knock-down embryos also resulted in enhanced expression of stc1 in nephron next to 10th to 11th somite (Fig. 6). This effect of DEAB or citral on efhc2-Mo morphants is exact opposite of RA treatment of same embryos where stc1 expression was completely abolished. Taken together, these results indicate that RA signaling is able to control the formation of CS in absence of Efhc2. Hence, although both RA and Efhc2 affect distal segmentation of pronephros, their functions are not interdependent.
Efhc2 is required for formation of multi-ciliated cells (MCC)
Multi-ciliated cells (MCC) are present along the PCT, PST, DE and anterior part of DL segments of pronephros in zebrafish embryos [16]. These cells can be identified by WISH using ciliogenesis genes such as odf3 and rfx2 [18]. We asked if knock-down of Efhc2 had any effect on MCC formation. The control efhc2-MM injected embryos expressed odf3 corresponding to 2nd to 15th somite, whereas the efhc2-Mo morphants had odf3 expression next to 8th to 15th somite (Fig. 7a). This suggests that embryos injected with efhc2-Mo had a much-reduced domain of odf3 expression compared to embryos injected with mis-match control. The MCC reduction in efhc2-Mo morphants was 59% (63/106) at 24 hpf and 61% (49/80) at 48 hpf (Fig. 7a and Additional file 3: Figure S3). This effect of Efhc2 knock-down was rescued by co-injection of efhc2 mRNA in 68.3% (26/33) embryos at 48 hpf (Fig. 7a). Injection of efhc2-ATG-Mo also lead to reduction in MCC formation. Fifty five percent embryos (21/38) had the odf3 expression level and domain reduced (Additional file 2: Figure S2). These observations indicate that Efhc2 positively regulates pronephric multi-ciliated cell development (Fig. 7a and Additional file 2: Figure S2).
It is known that RA signaling promotes MCC formation [16]. Exogenous treatment of RA enhances MCC number and these cells are expressed slightly distally in response to RA treatment. Conversely, treatment of embryos with RA inhibitor DEAB reduced MCC number. Knock-down of Efhc2 resulted in reduced MCC number and the expression domain of MCC marker odf3 was also reduced. Hence, we asked if Efhc2 plays any role in RA mediated MCC formation. The wild-type and Efhc2 knock-down embryos were treated with RA (1 × 10−7 M) or RA inhibitors DEAB (1 × 10−5 M) or citral (2.5 × 10−5 M) from 9 to 16 hpf and development of MCC in these embryos were examined using the expression of MCC marker odf3 at 48 hpf. The RA treated wild-type embryos showed an increase in MCC number and expansion odf3 expression domain as compared to DMSO treated embryos (Fig. 7b). The expression of odf3 is seen in pronephros adjacent to 2nd to 16th somite in 48 hpf RA treated embryos. However, efhc2-Mo injected embryos treated with same concentration of RA showed expression of odf3 next to 5th to 16th somite in 48 hpf embryos as compared to expression of odf3 from 8th to 15th somite in Efhc2 knock-down alone (Fig. 7b and Additional file 5: Figure S5). This indicates that RA was able to partially compensate for loss-of-Efhc2 function in the development of MCC. As compared with RA treated embryos, wild-type embryos treated with DEAB showed reduced MCC formation (Fig. 7c and Additional file 5: Figure S5). The expression of odf3 is seen adjacent to 2nd to 13th somite in 48 hpf embryos as compared to DMSO treated embryo, where its expression can be seen from 2nd to 15th somite (Fig. 7c). efhc2-Mo morphants treated with DEAB show almost or complete loss of odf3 expressing multi-ciliated cells (Fig. 7c and Additional file 5: Figure S5). Treatment of Efhc2-knock-down embryos with another RA inhibitor citral, also lead to similar reduction of number and domain of odf3 expressing MCC (Fig. 7d). This indicates that the inhibition of RA synthesis by DEAB or citral leads to a dramatic loss of MCC formation in efhc2-Mo injected embryos. This could be interpreted as loss of Efhc2 and RA signaling has a synergistic effect on reduced MCC formation. Taken together, our results suggest that RA and Efhc2 synergistically regulate MCC development (Fig. 8).