Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development

Abstract

Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.

Keywords: Epithelial cell fate; Kidney; Multiciliated cells; Nephrogenesis; Notch signaling; Pronephros; Retinoic acid; etv4; etv5a.

Figure 1. etv5a transcripts show early expression in the renal progenitor field where the MCC domain later emerges

(A) Schematic of the fully segmented zebrafish pronephros at 24 hpf, shown in both a lateral and dorsal view. Enlargement depicts the somite map of transporter cell segments and the MCC domain. (B) WISH at the 15 ss reveals that the etv5a (purple) expression domain correlates to the rostral and central regions of the renal progenitor field, demarcated by the marker slc4a4a (purple). slc12a3 (purple) denotes the caudal region, and somites are stained by smyhc1 in red. Black brackets highlight etv5a, slc4a4a, and slc12a3 expression domains in the pronephros. (C) The expression domain of etv5a is restricted in the proximal tubule during nephrogenesis, where the MCC domain, marked by odf3b (purple), expands. Insets show magnified view of etv5a and odf3b domains in the pronephros, denoted by the black bars. At the 28 ss, the etv5a domain is largely restricted to the PST (black bar), with weak expression in neighboring pronephros segments (black dots). The expression domain of odf3b at the 28 ss stage spans a caudal portion of the PCT (black dots), the PST (black bar), and the DE segment (black dots). (D) Schematic of etv5a cDNA depicting the two functional domains, acidic (light green) and ETS (dark grey), of the Etv5a protein. Abbreviations: P (podocyte), N (neck), PCT (proximal convoluted tubule), PST (proximal straight tubule), DE (distal early), CS (corpuscle of Stannius), DL (distal late), PD (pronephric duct), C (cloaca), ss (somite stage), etv5a (ets variant 5a), slc4a4a (solute carrier family 4 (sodium bicarbonate cotransporter), member 4a), slc12a3 (solute carrier family 12 (sodium/chloride transporter), member 3), smyhc1 (slow myosin heavy chain 1), odf3b (outer dense fiber of sperm tails 3b), TAD (transactivation domain).

Figure 2. etv5a morphants have possible kidney dysfunction

(A) Live time course demonstrates the formation of an edema (black arrowhead), indicative of fluid imbalance and possible renal dysfunction, by 120 hpf in etv5a morphants (etv5a MO) compared to control siblings. etv5a morphants also have a smaller, darker head (black arrow) than the control embryos. (B) Control embryos demonstrate proper renal function by clearing FITC-dextran over time. etv5a morphants continue to accumulate fluorescence (white arrow), suggesting improper renal clearance. White asterisks denote uptake of dextran by the PCT of both the control and etv5a morphant. (C) Whole mount IF in combination with FISH demonstrates that etv5a morphants have fewer odf3b⁺ cells (red) than control siblings at 24 hpf. Insets show digital zoom of single odf3b⁺ cells (white arrowhead) associated with multiple cilia (green) marked by α-tubulin in both the control and etv5a morphants. Nuclei are stained by DAPI in blue. Abbreviations: MO (morpholino), hpf (hours post fertilization), hpi (hours post injection).

Figure 3. etv5a is required for MCC development

(A) At 24 hpf, WISH analysis demonstrates etv5a loss of function via morpholino (MO) knockdown (etv5a MO, etv5a MO2) and deletion of the acidic domain (etv5a^Δacidic) resulted in reduced expression of the MCC markers odf3b and centrin 4 (cetn4) (purple) in the pronephros. Insets are a magnification of the MCC domain in both a lateral (top) and dorsal (bottom) view. (B) odf3b expression via WISH in 24 hpf embryos co-injected with etv5a capped RNA (cRNA) and each morpholino (MO) was not as reduced as MO injection alone. Injection of etv5a cRNA also did not produce a great change in odf3b expression in the pronephros. (C) Quantification demonstrates a significant decrease in average MCC number for cetn4 in all three etv5a knockdown versions. (D) etv5a loss of function (etv5a MO, etv5a MO2, and etv5a^acidic) produced a significant reduction in the average MCC number compared to the control, as marked by odf3b. Co-injection of etv5a cRNA and MO partially rescued the MCC phenotype seen in embryos injected with MO only, where injection of etv5a cRNA alone did not produce a significant change in average MCC number compared to the control. Images and quantification are representative of at least 50 embryos, and error bars denote standard error. P-values: *****p<0.001, ****p<0.002, **p<0.02

Figure 4. Redundancy between etv5a and etv4 in MCC formation

(A) In embryos injected with either etv5a MO, etv4 MO, or a combination of etv5a MO and etv4 MO, there is reduced expression of odf3b (purple), where the greatest loss appears in the absence of both etv5a and etv4 transcripts. (B) A significant decrease in average MCC number results in the loss of etv5a, as well as the loss of etv4. Injection of etv5a and etv4 together caused a greater decrease in the average number of MCCs. Representative images are shown for the quantified groups (n>50 for each), and standard error is depicted by the error bars. P-values: *****p<0.001, ****p<0.002

Figure 5. RA signaling acts upstream of etv5a to promote MCC fate

(A) WISH analysis on 24 hpf embryos demonstrates an increased etv5a expression domain after treatment with exogenous retinoic acid (+RA). Conversely, treatment with the pan-RA inhibitor DEAB completely ablates etv5a expression in the pronephros. Insets show a magnified view of the pronephros, where black bars denote the etv5a domain. (B) Exogenous RA significantly increased average etv5a length (μm) in the pronephros, but etv5a expression is lost after DEAB treatment. (C) Exogenous RA increases odf3b expression in both control and etv5a morphant embryos (etv5a MO), however etv5a morphants still appear to have a reduction of odf3b transcripts when compared to control siblings analyzed by WISH. odf3b expression is greatly reduced in both control and etv5a morphant embryos treated with DEAB. A magnified lateral (top) and dorsal (bottom) view of the odf3b domain is presented in the insets. (D) Quantification shows a significant increase of average MCC density/somite in etv5a morphants + RA compared to etv5a morphants, and that treatment with RA rescues average MCC density in etv5a morphants to the control value. Morphants treated with DEAB have a significantly lower MCC density/nephron* than control embryos treated with DEAB. At least 50 embryos were analyzed for each treatment, and the error bars represent standard error. P-values: *****p<0.001

Figure 6. Notch signaling acts upstream to inhibit etv5a and restrict MCC fate

(A) WISH staining demonstrates expanded etv5a and odf3b domains after treatment with the Notch-inhibitor DAPT. A magnified lateral view of the etv5a domain with expression denoted by the black bars can be seen in the insets. odf3b domain is shown both laterally (top) and dorsally (bottom) in the insets. (B) Quantification of etv5a expression length (μm) in the pronephros shows a significant increase in DAPT-treated embryos. (C) MCC density is significantly increased in control and etv5a morphants (etv5a MO) both treated with DAPT, however there is still a significant difference in MCC density between DAPT and etv5a MO + DAPT embryos. Error bars represent standard error. P-values: *****p<0.001; **p<0.10

Figure 7. Working model of etv5a AND etv4 function in MCC patterning during nephrogenesis

Interplay between retinoic acid (RA), etv5a, and Notch signaling in the renal progenitor field mediates multiciliated cell (MCC) formation during nephrogenesis. etv5a responds downstream of RA signaling to promote MCC fate, although it is likely that etv5a is not the only target of RA in this pathway. Conversely, Notch signaling inhibits etv5a activity to restrict MCC formation and favor transportive cell identity. In addition, etv4 promotes MCC fate, although it is not resolved if etv4 acts within renal progenitors or neighboring tissues, and whether other known MCC specification factors impact etv4 in other embryonic locales to affect pronephros development. Abbreviations: P (podocyte), N (neck), PCT (proximal convoluted tubule), PST (proximal straight tubule), DE (distal early), CS (corpuscle of Stannius), DL (distal late), PD (pronephric duct), C (cloaca).