4.3. Candidate Notch Targets: ETS Transcription Factors Etv5a/4Several transcriptional regulators are requisite for MCC genesis in the zebrafish pronephros. Of these, Etv5a/4 are necessary to support MCC fate choice [58]. We initially hypothesized that Etv5a controls MCC fate because etv5a was co-expressed in renal precursors that express MCC marker odf3b [58]. Moreover, there was precedence from prior work implicating Etv5a in tissue patterning and ciliogenesis [62]. Knockdown studies, as well as over-expression of a dominant negative construct, revealed that etv5a is required for MCC formation in zebrafish pronephros [58]; etv5asa16031+/− and etv5asa1603−/− embryos also had reduced MCC numbers, confirming the knockdown findings and identifying a genetic model for further studies [51]. In each case, loss of etv5a led to a significantly reduced MCC contingent [51,58]. This number was further reduced in etv5a-deficient embryos that were deficient in the ubiquitously expressed, related family member etv4 [58]. These results establish that Etv5a and Etv4 have redundant roles in MCC formation.Interestingly, etv5a expression is negatively regulated by Notch signaling to partly constrain MCC number [58]. Given the central role of Notch in MCC fate, we examined if Notch interacts with etv5a. Notch signaling inhibits etv5a to restrict MCCs as DAPT treatment expanded the etv5a expression domain in the pronephros as well as increased MCC numbers [58]. In addition, etv5a-deficient embryos treated with DAPT had significantly fewer MCCs than DAPT treatment alone [58]. This indicates that Notch, either directly or indirectly, serves as a negative regulator of etv5a. Further, the relationship between Notch signaling, etv5a, and MCC formation was evaluated using the aforementioned transgenic line Tg(hsp70:gal4; uas:notch1a-intra) that expresses the NotchICD1a under temporal control mediated by heat-shock. In this context, there was a dramatic reduction in the length of the etv5a expression domain in NICD+ embryos and MCC number, further supporting the conclusion that Notch inhibits MCCs partly through affecting expression of etv5a [58]. 4.4. The Iroquois (irx) Transcription Factor irx2aThe Iroquois family of transcription factors have been established as essential regulators of embryogenesis, specifically in the processes of patterning [63,64]. Of this family, irx3b and irx1a are expressed in PST-DE and DE regions of the zebrafish pronephros, respectively, and these factors are essential components of the gene regulatory network that regulates genesis of DE lineage [49,65,66,67,68,69]. Yet another member of this family, irx2a, is expressed in the PST-DE segments of the nephron and is essential for proper development of pronephric cell types, including MCCs [70]. Interestingly, irx2a colocalized with a subset of odf3b+ cells at 24 hpf, but co-expression presented as a range in which some cells were independently irx2a+ or odf3b+ [70]. This modulation of irx2a expression suggests that perhaps irx2a marks MCC precursors, and the observed diminished expression is a result of MCC maturation [70]. This hypothesis was further supported as deficiency models of irx2a in the zebrafish resulted in a decreased number of MCCs as well as decreased expression domain of etv5a [70]. Changes in retinoic acid signaling also affected irx2a expression as treatment with exogenous RA expanded the irx2a domain and shifted it caudally, while inhibition with DEAB shifted the irx2a domain rostrally and caused it to be significantly decreased in length [70]. Currently, these data place irx2a downstream of RA and upstream of etv5a in the MCC regulatory pathway, yet additional studies are essential to determine the exact nature of the interactions amongst these regulators. 4.5. Prostaglandin Signaling Regulates MCC Specification and DifferentiationSeveral studies have illuminated important roles for prostaglandin signaling in MCC progenitor fate choice and subsequently in proper MCC differentiation. Prostaglandins (PGs, or prostanoids) are small lipid-derived molecules that regulate cellular activities in an autocrine or paracrine fashion. PGs are produced through several steps, beginning with phospholipases releasing arachidonic acid (AA) from membrane lipids. From here, AA is converted into prostaglandin intermediate PGH2 by cyclooxygenases [71]. There are two primary cyclooxygenases in vertebrates: COX-1, which is more common and functions to mediate the homeostatic functions of PGs, and COX-2, which is less common as it appears to be active only after being induced. Both COX enzymes are endoplasmic-reticulum- or nuclear-membrane-bound and function as homodimers with one catalytic and one regulatory subunit. The intermediate PGH2 is then further modified by specific synthases into one of the following PGs: PGE2, PGF2α, PGD2, or PGI2 [72]. Generally, this derivation of specific PGs occurs within the cell. However, it is possible for transcellular synthesis with COX and synthase activity to occur in other cells [72,73]. Additionally, prostanoids can diffuse or be transported out of the cell and into neighboring cells via diffusion or specialized transport proteins (ABCC4, MRP4, SLCO2A1), where they bind to their specific G protein-couple receptors (EP1-4, FP, DP and CRTH2, and IP, respectively) [74,75,76]. It is important to note that, in high enough quantities, it is also possible for prostanoids to bind non-specifically to other PG receptors [77]. However, bioactive PGs are usually found in low concentrations in vivo, in part due to their short half-lives, and bind to their specific receptors [72].After PGs bind their respective receptors, they are involved in several biological processes, including Gα-dependent signaling cascades (such as cAMP), MAPK, and PPAR signaling [72]. Proper balance of PG concentration is regulated not just by COX-initiated synthesis but also by degradation via 15-hydroxyprostaglandin dehydrogenase (12-PGDH) [71]. Importantly, PG receptors are found on many cell types. This explains the variety of cell types and corresponding functional effects associated with PG signaling [71]. Interestingly, PGE2 can be produced by many cell types and has been recognized to activate neutrophils, macrophages, and mast cells in inflammation while also being involved in fibroblasts and epithelial cells in other contexts [78,79,80,81,82].Major inroads in understanding the developmental roles of prostanoids have been afforded through zebrafish-based research. Because zebrafish develop ex utero, this prevents maternal PGs from affecting embryonic development, unlike mammalian models where maternal contributions have prevented researchers from delineating PG requirements in embryogenesis [83]. Moreover, zebrafish Cox genes are very similar to their mammalian counterparts and are maternally deposited, further pointing to their importance in early development [83,84,85]. Overall, Cox activity has been noted as early as 3 hpf, prior to MCC specification in the pronephros and other tissues [86]. The essential components of prostaglandin signaling are also expressed in the developing pronephros, including receptors ptger2a and ptger4a [87] and Cox1 encoded by ptgs1 [88,89,90]. Additional Cox enzymes, ptgs2a and ptgs2b, are expressed in the tissues immediately surrounding the pronephros (like the cloaca and somites), which could also serve as a source of prostanoids if excreted [88,89].Prostaglandins have been linked to ciliary function for decades, including modulation of beat frequency in human airway cilia and other ciliated cells [91,92,93,94,95,96]. More recently, PGE2 was linked specifically to ciliogenesis as a mutation in the ABCC4 transporter in zebrafish resulted in hallmark ciliopathic phenotypes, such as body curvature, alterations in fluid homeostasis, and laterality defects [97]. ABCC4 localizes to the ciliary membrane of various cells, including the zebrafish Kupffer’s vesicle (KV), olfactory placode, and otic vesicle, as well as human retinal pigmentation epithelial 1 (hRPE1) cells and murine inner medullary collecting duct 3 (IMCD3) cells, and is essential for PGE2 signaling to drive intraflagellar transport (IFT) [97]. IFT is a highly regulated process driven by microtubule-based axoneme track and motor proteins, and its dysregulation often results in blunted or bulging cilia [98]. PGE2 specifically drives cAMP signaling, which, in turn, regulates anterograde IFT [97,99]. These findings have been applied to rescue cilia length in EP4-deficient cells and other ciliopathic models [90,97]. Additionally, prostaglandins have been recently proposed as a therapeutic for nephronophthisis as agonism of PG receptors rescues defective ciliogenesis [100].In addition to cilia formation, PGE2 plays an important role in MCC fate choice. Cox1- and Cox2-deficient zebrafish embryos exhibit decreased numbers of pronephric MCC progenitors, marked by expression of Notch ligand jag2b and transcription factor pax2a at the 24 ss [51]. This decrease persists through at least 28 ss and is also associated with a decrease in the number of cells that express MCC differentiation marker odf3b [51]. Even though MCCs (odf3b+) are distributed along several segments (end of PCT, throughout PST, and DE), deficiency of Cox1/2 appeared to only affect MCCs in the proximal segments as the number of transporter cells increased at the expense of MCCs [51]. Even in the case that prostaglandin-deficient animals activated expression of mature MCC markers, they were not necessarily mature, as evidenced by the increase in the number of unciliated basal bodies [51]. Supplementation with dmPGE2 (a stable form of PGE2) could rescue MCC number in Cox1, Cox2, and double Cox1/2 deficiency, suggesting that PGE2 was indeed the major prostanoid of importance in the context of MCC genesis in the nephron [51]. These studies reveal that prostaglandin signaling, especially via PGE2, is essential for both MCC specification and cilia formation and maturation.Furthermore, there is compelling evidence that prostaglandin signaling acts downstream of transcription factor etv5a during renal MCC development. This notion is supported by the finding that dmPGE2 supplementation partially rescues MCC number in the nephrons of etv5a-deficient zebrafish [51]. Interestingly, the proximal promoters of zebrafish cox1 and cox2 contain putative Etv5 binding sites [51]. In murine in vitro studies, Etv5 increases the transcriptional activity of the Cox2 promoter [101]. Taken together, this reasonably suggests a mechanism by which Etv5, or possibly a related family member such as Etv4, may directly regulate prostanoid biosynthesis to induce MCC fate choice. However, future studies are still needed to examine these possible molecular interactions in renal progenitors. 4.6. Modulation of Prostanoid Biosynthesis by ppargc1aWhile prostaglandin signaling components have been well-characterized, the transcriptional network controlling this essential pathway is relatively understudied in multiciliogenesis and limited to the links with Etv5a discussed in the previous paragraph. This void has begun to fill with recent studies that identified ppargc1a, an essential coactivator of the PPAR pathway, as a key regulator of nephron formation [90,102,103]. Zebrafish deficient in ppargc1a exhibit many of the pleiotropic defects associated with defective cilia—body curvature, aberrant left–right symmetry, and pronephric cysts [90]. Consistent with these phenotypes, ppargc1a mutants had a decreased number of renal MCCs, and pronephric cilia of both multi- and mono-ciliated cells were shorter [90]. However, the number of basal bodies in each region of the nephron remained unaffected by ppargc1a deficiency, although there were fewer ciliated basal bodies [90]. These phenotypes (e.g., decreased MCC number and ciliated basal bodies) were strongly reminiscent of those observed in Cox deficiency models. Interestingly, ppargc1a deficiency also leads to decreased expression of ptgs1 and endogenous levels of PGE2, and supplementation of either ptgs1 transcripts or dmPGE2 was sufficient to rescue the ciliopathic phenotypes [90]. This suggests that prostaglandin signaling is under the regulatory control of ppargc1a. While the presence of putative PPAR binding sites upstream of the ptgs1 open reading frame suggests that ppargc1a is likely acting in tandem with PPAR transcription factors, future experiments may look to interrogate the exact relationship between PPAR and prostaglandin signaling in the context of MCC genesis.The ppargc1a deficiency phenotypes affect cilia formation of both MCCs and mono-ciliated transporter cells but push cells towards mono-ciliated cell fate. These two characteristics—cilia formation and MCC number—are not inextricably linked, as suggested by the unique phenotypes of IFT-specific-deficient animals. For example, knockdown of ift88 results in decreased cilia length in the pronephros, while the number of MCCs remains constant [90]. While supplementation of either ptgs1 transcripts or dmPGE2 can rescue the ppargc1a deficiency phenotypes, future studies are needed to parse out other gene regulatory network components that contribute to cilia outgrowth or MCC fate. Certainly, other factors of interest include but are not limited to the aforementioned etv5a, irx2a, mecom, and Notch signaling components. However, these factors are likely to be subsets of the regulatory network. Approaches to discover the missing players are one of the many future opportunities to build our understanding of these developmental events.