Insulin-Like Growth Factor-Binding Proteins of Teleost Fishes

The insulin-like growth factor (Igf) binding protein (Igfbp) family has a broad range of physiological functions and a fascinating evolutionary history. This review focuses on the Igfbps of teleost fishes, where genome duplication events have diversified gene repertoire, function, and physiological regulation—with six core Igfbps expanded into a family of over twenty genes in some lineages. In addition to briefly summarizing the current state of knowledge on teleost Igfbp evolution, function, and expression-level regulation, we highlight gaps in our understanding and promising areas for future work.


ORiGiNS OF THe CORe igfbp SUBTYPeS
Gene duplication and subsequent divergence is central to the evolutionary "narrative" of the Igfbp family. While it is long-established that many vertebrates possess six ancestral subtypes (Igfbp-1, -2, -3, -4, -5, and -6), with the primary cDNAs first reported over 25 years ago [e.g., Ref. (7,8)], their evolutionary origins were elucidated more recently. An important study reported in 2011 (9), built on past work revealing linkage between Igfbp genes and Hox clusters [e.g., Ref. (10); Hox clusters being well-established markers of genome duplication events], to present a realistic scenario for the origin and expansion of core Igfbp subtypes. The hypothesis is that an ancestral Igfbp gene was duplicated in tandem during an early stage of vertebrate evolution to produce a pair of Igfbp genes (9,10). Subsequently, two genome duplication events in the ancestor to extant vertebrates (11) led one gene to give rise to Igfbp-1, -2, and -4, and the other to Igfbp-3, -5, and -6. A single Igfbp is present in amphioxus, a chordate that did not undergo the same duplications, and this FiGURe 1 | Expansion of the core insulin-like growth factor (Igf) system-including Igfbps-during teleost evolution. (A) Simplified depiction of Igf system. (B) Core Igfbp system components (i.e., proteins encoded by distinct genes) in different vertebrate groups, contrasting a typical mammalian system with that of two teleost lineages. For teleosts, % identity is shown for paralogous amino acid sequences. For Atlantic salmon, the underlined % identities highlight paralog pairs residing in regions of the genome that experienced a delay in cytological rediploidization after genome duplication (24), a process required for paralogs to diverge in sequence on distinct chromosomes-hence, these genes have had less evolutionary time to diverge, leading to extremely high identity. Phylogenetic relationships of the Igfbp families from these different lineages, along with another group of teleosts that experienced a separate lineage-specific genome duplication event, are depicted in molecule failed to bind Igf-I or Igf-II, indicating that Igf-binding is either a vertebrate-specific function (12), or was secondarily lost. The same study confirmed that Igf-independent functions had evolved before vertebrates (12). It is also important to remember that the diversification of the core vertebrate Igfbp system occurred alongside expansions in other key gene families within the Igf system, including both hormones (13) and receptors (14). It now seems certain that the early vertebrate genome duplication events were crucial for the evolution of distinct insulin and Igf systems [e.g., Ref. (15)]. For the remainder of this review, we focus on the Igfbp system of teleosts, where additional genetic expansions-some dramatichave been recently characterized.
Several teleost lineages experienced additional rounds of genome duplication. This includes a well-studied event ~95 million years ago in the salmonid ancestor (21,22) that caused dramatic genetic expansions within the Igf system (summarized in Figure 1). For example, we reported in 2013 that salmonids retain at least 19 unique Igfbp genes, with salmonid-specific paralogs of igfbp-1a, -1b, -2b, -3a, -3b, -5b, -6a, and -6b (16). We proposed a nomenclature with either "1" or "2" after the "a" and "b" teleost symbols (e.g., "igfbp-1a1" and "-1a2"). Several of these Igfbp pairs are highly divergent compared to the genome-wide average for paralogs retained from the salmonid genome duplication event (Figure 1; e.g., sharing <80% amino acid identity, compared to an average of ~93% across thousands of paralog pairs) (23). This points to functional divergence at the protein level that remains entirely unexplored.
More recently, an improved understanding of the complexities of genome evolution following the salmonid genome duplication, which was a spontaneous genome doubling event (or "autotetraploidization") (21,24), led to the discovery of salmonid Igfbp paralog pairs for igfbp-4, igfbp-2a, and igfbp-5a (24), which share extremely similar sequences and were previously unrecognized or ignored as alleles (see Figure 1; legend contains additional information). Thus, some salmonid species, including the commercially important Atlantic salmon (Salmo salar), possess 22 unique igfbp genes, with 11 paralog pairs (Figure 1), some of which may have evolved adaptively (25). Remarkably, every possible Igfbp paralog generated from the salmonid-specific genome duplication was evidently maintained, despite the genome-wide paralog retention rate being around 50% in the same species (21). We and others have also identified expansions to other core gene components of the Igf system due to the salmonid genome duplication, including Igf-I [e.g., Ref. (24,26)], Igf-II (25), and Igf-1R (27). These paralogs remain of substantial interest, but we are at an early stage of understanding their roles in salmonid biology.
Additional lineage-specific genome duplication events have occurred in several teleost groups, including the ancestor to goldfish (Carassius auratus) and common carp (Cyprinus carpio). This event is younger than the salmonid-specific genome duplication event, occurring around 8-12 million years ago (28,29). It also involved a distinct mechanism ("allotetraploidization"), where two species hybridized before genome duplication (28). This event created a large set of paralogs (28), some of which are known to have experienced functional divergence [e.g., Ref. (29,30)]. However, no accompanying expansions to Igfbp repertoire are yet reported. To explore this knowledge gap, we performed a bioinformatic and phylogenetic analysis, revealing that common carp retains at least 17 unique igfbp genes, including paralog pairs for igfbp-1a, -1b, -2b, -3a, -5a, -5b, -6a, and -6b (see Figure 2; methods provided therein). To avoid confusion with the salmonid-specific paralogs, we suggest "α"/"β" is added to the existing teleost nomenclature when these duplicates are studied in the future (e.g., igfbp-1aα/-1aβ) (Figure 2). The results confirm that salmonids are not unique among teleosts in retaining a highly expanded Igfbp repertoire. In fact, as many vertebrate groups have experienced lineage-specific genome duplication events, both fishes and non-mammalian groups, including anuran frogs [e.g., Ref. (31)], it seems likely that many other species possess expanded Igfbp repertoires, contributing additional complexity to their growth regulation.

PHYSiOLOGiCAL ROLeS OF TeLeOST igfbps
Many studies have investigated the physiological roles of Igfbp genes in the teleost lineage. Barring a few model species (e.g., zebrafish), it has been historically challenging to perform functional analyses in most teleosts, although this is changing in light of emerging genome-editing methods (see Perspectives and Future Work). Hence, while in mammals, Igf-dependent and Igf-independent functions have been widely demonstrated, the majority of studies in teleosts have failed to reach similar levels of functional insight. In fact, most work has focused on expression-level regulation of igfbp genes or proteins under a diverse set of experimental stimuli. It is also important to note that most teleost Igfbp research has focused on aquaculture species of high commercial value, including the salmonid, perciform (perch-like fish), pleuronectiform (flatfish), cypriniform (carp spp. and relatives), and siluriform (catfish) groups. This has led to a bias toward physiological processes relevant to commercial production, especially growth, muscle development, stress, and disease resistance. In this section, we briefly summarize the literature on teleost Igfbp function and regulation, considering the core vertebrate subtypes separately. We make attempts to distinguish Igfbp paralogs according to the evolutionary histories and nomenclature described above, although this is often not possible as many studies failed to distinguish paralogs, especially for the most recently discovered genes.

igfbp-1: A Negative Regulator of Teleost Growth
In mammals, Igfbp-1 is mainly produced in the liver and secreted to circulation, where it acts to limit Igf signaling in catabolic contexts, such as fasting, stress, and hypoxia (39). It is widely considered a negative regulator of somatic growth, reproduction, and development (4,40); and interacts with cell surface integrins to stimulate cellular motility (41). It has Igf-dependent and Igf-independent functions, along with important roles in the regulation of metabolism [reviewed in Ref. (42)].
Several teleost studies have reported protein or transcript level upregulation of Igfbp-1 genes during catabolic a process, which probably serves to downregulate growth by sequestering Igfs from Igf-1Rs, allowing allocation of resources to metabolic processes essential for survival. Consistent with these findings, overexpression of igfbp-1a (and igfbp-1a in zebrafish) in cypriniform embryos (17,54,59) caused growth and developmental retardation. Nutrient deprivation has been shown to increase circulatory Igfbp-1 proteins and igfbp-1 gene expression in liver (for both teleost paralogs, when distinguished) and skeletal muscle igfbp-1a expression, which is reversed by a return to anabolic conditions (50,51,55,(60)(61)(62)(63)(64)(65). It has also been shown that dietary amino acid deficiency can upregulate igfbp-1 gene expression through a not well-described mechanism (64). Teleost igfbp-1 genes are also negatively regulated by growth hormone (Gh) FiGURe 2 | Independent evolutionary expansions to the Igfbp family of teleosts. A phylogenetic analysis was performed, including 71 unique Igfbp amino acid sequences from a standardized set of taxa: Human Homo sapiens ("Hs"), coelacanth Latimeria chalumnae ("Lc"), zebrafish Danio rerio ("Dr'"), common carp Cyprinus carpio ("Cc"), Atlantic salmon Salmo salar ("Ss"), and northern pike Esox lucius ("El," from a sister lineage to salmonids that did not undergo the salmonid-specific genome duplication). Accession numbers are given for all sequences, which were gathered from the NCBI RefSeq database, facilitated by BLAST analyses (32). The sequences were aligned using Mafft V.7 (33) with default settings. Alignment quality filtering was done using the Guidance2 algorithm (34) to remove the least confidently aligned regions. This led to a high-confidence alignment of 212 amino acids positions (SI file 1). The alignment was used in Bayesian phylogenetic analysis, using methods published elsewhere (35). Briefly, this was done in BEAST v. 1.8 (36) using the best-fitting amino acid substitution model (JTT+G+I), determined by maximum likelihood via the IQ-tree server (37), along with a relaxed molecular clock model (38), allowing probabilistic estimation of the trees root [consistent with previous studies (9,16)]. The tree is annotated to show genome duplication events in teleost evolutionary history, including in the teleost ancestor ("3R") and additional events in the salmonid and carp lineages. The nomenclature for salmonid and carp paralogs is given as described in the text. Branch support values (posterior probability) are highlighted by circles placed on nodes, with colors matching a legend. Minor inconsistencies in branching patterns in some Igfbp clades (e.g., relating to the 3R or salmonid 4R) compared to other studies (16), can be explained by the short alignment length. Nonetheless, the tree demonstrates independent expansions within the salmonid and carp Igfbp repertoire, additional to paralogs retained in many other teleosts. and Igf-I, consistent with a negative role in growth regulation (44,46,48,53,57,62).
Igfbp-1 expression in teleosts is also modulated by hormones others than the Igfs. For instance, igfbp-1a evidently plays a role in zebrafish sexual maturation, in a way that seems to differ from its classical anti-proliferative role. Specifically, igfbp-1a expression increases in response to T3 and the follicle stimulating (Fsh) hormones (well-known for stimulating spermatogonia proliferation) (58). The female sex hormone 17β-estradiol also increased Igfbp-1 secretion in striped bass liver explants (46).
Stress is also known to induce igfbp-1 gene expression in teleosts. Cortisol, the main stress hormone in vertebrates (66,67), and other synthetic glucocorticoids such as dexamethasone increases both Igfbp-1a and Igfbp-1b circulatory levels, as well as gene expression in liver and cultured myotubes (43,57,62,68,69). Environmental stressors, such as hypoxia, confinement, temperature, heavy metals, and salinity, were also found to increase igfbp-1 expression (17, 49-53, 56, 61, 70, 71). It is also possible that igfbp-1 upregulation in response to food deprivation (see above) is related to a rise in cortisol, as some studies have found increasing levels of circulatory glucocorticoids in response to fasting (70,72,73). There is also emerging evidence from salmonids that igfbp-1a1 upregulation during infection serves a role in linking growth to innate immunity, potentially promoting downregulation of growth in favor of a more effective immune response (74). This expression response represents an example of salmonid-specific divergence in the regulation of Igfbp paralogs, as igfbp-1a2 was unaltered by infection in the same study (74).

igfbp-2: A Major Circulatory igfbp in Teleosts
In mammals, Igfbp-2 is highly expressed during embryonic stages, and more lowly expressed in adult tissues, with highest levels in liver, adipocytes, the central nervous and reproductive systems, heart, and kidney (75). Mice embryos overexpressing Igfbp-2 show a reduced growth rate, likely through reduced Igf availability (76). However, Igfbp-2 deletion in mice embryos does not have any significant effect on growth or development (77), which may indicate compensatory effects with other Igfbps. While the functional roles of Igfbp-2 remain relatively poorly established, recent studies have linked it to several pathological states. For example, Igfbp-2 may act as a tumor promoter (78) by suppressing epidermis growth factor receptor nuclear signaling (79). There is also increasing evidence that Igfbp-2 plays a role in mammalian bone formation (80).
Igfbp-2b is the third main circulatory Igfbp in salmonids and probably other teleost species (41 kDa form) and the main Igf carrier (6,81,82). For a long time, Igfbp-2b was wrongly considered to be Igfbp-3 (83) due to its similar physiological regulation to Igfbp-3 in mammals [e.g., Ref. (83)]. Teleost igfbp-2 genes are expressed in a range of tissues [e.g., Ref. (16,(84)(85)(86)] with zebrafish igfbp-2a and igfbp-2b having different spatiotemporal patterns during early development, and each being expressed in liver in adults (18). In adult salmon, igfbp-2a was expressed across multiple tissues, with especially high abundance in liver, whereas igfbp-2b1 and igfbp-2b1 were liver-restricted (16). Overexpression of igfbp-2a and igfbp-2b causes a reduction in growth and developmental rate in early-stage zebrafish (18,87), suggesting an equivalent role to that observed in mammals. Similar to Igfbp-1, past work has suggested a role for Igfbp-2 in teleost sexual maturation, with igfbp-2 mRNA being expressed in pre-ovulatory ovaries of rainbow trout, and upregulated in response to female sex hormones (83).
Mixed results exist on the regulation of teleost igfbp-2 genes by nutritional status. For example, some past studies showed that igfbp-2 genes are downregulated or unchanged in liver and skeletal muscle of fasted fish (55,(88)(89)(90), which does not support an obvious role in growth inhibition. By contrast, a significant increase of igfbp-2 expression was observed in fasted zebrafish (91), although this same effect was not clearly observed in a later study of zebrafish that distinguished igfbp-2a and -2b (65). In Atlantic salmon, a significant decrease in igfbp-2a (formerly "igfbp-2.1") expression was reported in skeletal muscle during refeeding after a period of restricted food intake, suggesting an inhibitory role on growth (90,92). Similarly, all three tested igfbp-2 paralogs (igfbp-2a, -2b1, and -2b1) were downregulated in Atlantic salmon liver upon post-fasting refeeding, again suggesting an inhibitory role on growth (16). However, such data have not been replicated in vitro where neither amino acid deprivation nor addition of Igf-I and amino acids to Atlantic salmon cultured myotubes modified the expression of the same paralogs (57).
Moreover, the regulation of igfbp-2 expression by Gh does not clearly support a growth inhibitory role common to teleosts. While a study in zebrafish embryos reported that Gh inhibits igfbp-2 expression (91), work in Atlantic salmon demonstrated an increase in circulating Igfbp-2b in response to Gh (48,81,93). By contrast, treatment with dexamethasone, known to enhance catabolism, led to an increase in igfbp-2a expression in salmon skeletal muscle myotubes (57). Despite not distinguishing teleost paralogs, recent work revealed upregulation of skeletal muscle igfbp-2 expression in fine flounder (Paralichthys adspersus), concomitant to a rise in blood cortisol (94). Differences in Igfbp-2 expression across studies suggest a complex role for this Igfbp family member in teleost growth, dependent on both physiological and species context.

igfbp-3: Divergent Physiological Regulation across Teleost Species
Igfbp-3 is the main carrier of circulating Igf in mammals, forming a tertiary structure with the acid-labile subunit (ALS) that increases Igf half-life and regulates Igf bioavailability (95). Igfbp-3 has anti-proliferative effects in many mammalian cell types, preventing the interaction of Igf-I and Igf-II with Igf-1R, and it also has Igf-independent roles (96). In this respect, Igfbp-3 directly interacts with two-cell surface receptors independently of Igf-I, Lrp1, and Tmem29, which mediates its anti-proliferative effects (97,98). However, mammalian Igfbp-3 can also enhance cellular proliferation in some conditions, through both Igf-dependent and Igf-independent mechanisms (99,100).
In contrast to mammals, teleost Igfbp-3 proteins are not considered major circulatory Igfbps (6). In fact, there exists no known association between Igfbp-3 proteins-or indeed any teleost Igfbp subtype-and ALS (6), highlighting fundamental differences in the way Igfs are regulated in circulation. In zebrafish, the single igfbp-3a (16) gene has important roles in early development, showing dorsalizing effects in embryos through an Igf-independent interaction with bone morphogenic protein 2b (101), one of few studies demonstrating an Igf-independent role for a teleost Igfbp. The four distinct igfbp-3 paralogs of salmonids (igfbp-3a1, -3a2, -3b1, and -3b2) were lowly expressed in 11 tested adult Atlantic salmon tissues (and each absent in liver), although igfbp-3a1 was among the most abundant of all Igfbp family member genes in heart (16) and the only detected igfbp-3 gene in primary myotube culture (57). Conversely, igfbp-3b of adult fine flounder was reported as more highly expressed in liver (the main route for Igfbp to circulation) than several other tested tissues, while igfbp-3a was not considered in the same study (88).
Studies in zebrafish, flounder, and yellowtail reported a significant increase in the expression of igfbp-3 genes in liver and/ or muscle in response to fasting (55,88,102), which may act to restrict Igf signaling. However, on the other hand, studies in salmonids have reported no changes in muscle igfbp-3 gene expression in response to food deprivation (57,89) with an increase in igfbp-3a1 expression in liver during post-fasting refeeding, more consistent with a growth-promoting function (16). Similarly, in coho salmon (Oncorhynchus kisutch), igfbp-3a1 muscle expression was increased by Gh transgenesis (103), again supporting a growth-promoting role. However, stress caused a downregulation of igfbp-3b gene expression in skeletal muscle of fine flounder (94), which is inconsistent with a role in growth inhibition.
Overall, there is a relatively limited body of research on teleost Igfbp-3 genes, leaving their roles unclear in many species, with the available evidence suggesting functional divergence among different lineages.

igfbp-4: Growth-Promoting Role in Some Teleosts?
In mammals, Igfbp-4 is expressed in many cell types and tissues, where it is often considered to inhibit Igf action (104,105). However, it is also considered to have growth-promoting roles during early embryogenesis, where it enhances Igf-II activity (106). Some studies have reported Igf-independent actions for Igfbp-4, including in relation to the inhibition of apoptosis (104,105) and cardiogenesis (107).
In teleosts, igfbp-4 was expressed in most tissues for each species investigated, including Atlantic salmon (16), tiger pufferfish (Takifugu rubripes) (108) and fine flounder (88). Moreover, in Atlantic salmon, igfbp-4 was described as showing high abundance in gill (108). Atlantic salmon was recently shown to retain two highly similar Igfbp-4 paralogs (see Figures 1 and 2), which show conserved regulation across tissues (24). In tiger pufferfish, fasting caused upregulation of igfbp-4 expression in several tissues, consistent with an inhibitory role on growth (108). In addition, the expression of recombinant pufferfish Igfbp-4 in zebrafish embryos resulted in significant growth retardation (108). However, these findings contrast studies of several species (including salmonids and fine flounder), where igfbp-4 expression in response to nutritional status manipulation suggested a growth-promoting role. Such work revealed no change in igfbp-4 expression during fasting (57,65,(88)(89)(90)92), but upregulation in response to subsequent refeeding (57, 65, 88-90, 92, 109-111). A study of Arctic charr (Salvelinus alpinus) showed that dwarf populations with highly restricted growth had lower constitutive igfbp-4 expression in muscle than populations reaching larger body size (112). A growth-promoting role for igfbp-4 in salmonids was also supported by a strong positive correlation between igfbp-4 and several pro-myogenic gene markers during in vitro myogenesis in Atlantic salmon (110). Studies of Igfbp-4 expression in response to stress also suggest a growth-promoting role. For instance, addition of dexamethasone to Atlantic salmon myotubes (57), and stress confinement in fine flounder (94) induced a significant reduction in igfbp-4 expression. Conversely, an increase in igfbp-4 expression was reported in skeletal muscle during maturation-induced atrophy in rainbow trout (113).
Overall, the available evidence suggests that the physiological role of Igfbp-4, when conserved, differs across species and physiological contexts, though for some lineages, particularly salmonids, a growth-promoting function is implicated.

igfbp-5: Conserved Roles in Muscle Growth
Igfbp-5 is the most conserved Igfbp family member. In mammals, it forms a ternary complex with ALS, similar to Igfbp-3, although much more circulating Igf is carried by Igfbp-3-ALS (114). Igfbp-5 represents an essential regulator of many processes in mammalian bone, kidney, mammary gland, and skeletal muscle (114) and can assert both stimulatory and inhibitory effects (depending on cell type) through Igf-dependent or Igf-independent routes. For instance, it has growth factor-like actions, stimulating bone growth in Igf-I knockout mice (115), and smooth muscle cell migration (116). There is also evidence that Igfbp-5 can translocate into the nucleus (117) and have nuclear functions (118). It is thought that Igfbp-5 cellular internalization is achieved by interaction with membrane proteins such as heparin sulfate proteoglycans, and that the Igfbp-5 N-terminal region has an Igfindependent transcriptional activity (118). Furthermore, Igfbp-5 can interact with transcription co-activators such as the four and half Lim domains 2 (119).
In zebrafish and grass carp embryos, igfbp-5a and igfbp-5b have distinctive patterns of expression during early development, suggesting evolutionary divergence in regulation (19,120), which has also been demonstrated at the functional level (19). In adult zebrafish, igfbp-5a was expressed at high levels in brain and gill, and lower levels in several other tissues, but was absent in liver or skeletal muscle; while igfbp-5b was ubiquitously expressed. Similarly, in other studied teleost species igfbp-5 genes were reported to show a broad tissue distribution, with differences noted among species and paralogs (51,52,55,63,120), including three paralogs in Atlantic salmon (16,24).
The importance of igfbp-5 genes for muscle differentiation and growth in teleosts is well established. Igfbp-5 expression has been studied across in vitro myogenesis, with both teleost paralogs (when distinguished) detected from early stages (i.e., myogenic progenitor cells) through to fully differentiated myotubes (57,110,111). In Atlantic salmon, both igfbp-5a (formerly: igfbp5.1) and igfbp-5b (formerly: igfbp5.2) showed highest expression in early-stage myoblasts, which decreased during myogenic differentiation (110). Using the same in vitro models, it was observed that pro-growth stimuli such as amino acids and Igfs increase igfbp-5 gene expression (111,113), including both igfbp-5a and igfbp-5b paralogs distinguished in salmonids (57,110). However, igfbp-5 paralogs appear to have different patterns of expression in response to catabolic signals. For instance, while amino acid deprivation had no effect on the regulation of any igfbp-5 paralog in Atlantic salmon myotubes (57,113), dexamethasone reduced igfbp-5a expression, while simultaneously increasing igfbp-5b1 (57). A past study of rainbow trout skeletal muscle recorded no change in igfbp-5 gene expression in response to fasting or re-feeding (89), though it was unclear which paralog was measured. Similarly, igfbp-5a and igfbp-5b muscle expression did not change in response to short-or long-term fasting in Atlantic salmon (90,92). However, in grass carp, igfbp-5a and igfbp-5b expression decreased in skeletal muscle during fasting, while both paralogs were upregulated in liver, and upon injection of Gh in both tissues (120). In Gh transgenic coho salmon, igfbp-5b1 was significantly upregulated (103).
There is also emerging evidence that igfbp-5 genes play a role in ionic homeostasis and Igf regulation in zebrafish (121), stickleback [e.g., Ref. (122)] and Atlantic salmon gills (123) with Igfbp-5a acting to regulate calcium influx in zebrafish gills (121) and being differentially expressed and under divergent selective pressures in marine vs. freshwater sticklebacks (122,124).
To sum up, the available evidence suggests that Igfbp-5 genes play conserved functions in multiple aspects of teleost biology, with roles most clearly demonstrated in myogenesis, muscle growth, and gill function. There is also considerable evidence that both teleost and salmonid-specific Igfbp-5 paralogs have evolved divergent roles.

igfbp-6: A Growth inhibitor with emerging Roles
Igfbp-6 represents a special case among the Igfbp family. In mammals, it has a 50-fold binding preference for Igf-II over Igf-I (125, 126) (a unique feature among Igfbps), but also shows differences in key protein motifs, with three disulfide bonds in the N-terminal region instead of the 4 found in Igfbp-1 to 5 (127). Igfbp-6 is a relatively specific inhibitor of Igf-II actions and, therefore, regulates processes where Igf-II is involved such as proliferation, survival, migration, and differentiation (125,126). Igfbp-6 also has known Igf-independent actions (125,128), including the inhibition of fibroblast proliferation (129), cancer cell migration (130), and apoptosis (131). The gene has a broad tissue expression distribution in mammals, including lung, liver, and the gastrointestinal tract.
While differences in the affinity of Igfbp-6 proteins for Igf-II and Igf-I are yet to be confirmed in teleosts, the main underlying structural features are conserved (16). In zebrafish adults, igfbp-6a was highly expressed in muscle, and almost undetectable in other tissues, while igfbp-6b was only abundant in brain, heart, and muscle (20). In adult fine flounder, igfbp-6b was most highly expressed in heart, gills, and the gastrointestinal tract (88). In adult Atlantic salmon, neither igfbp-6a1 nor -6a2 were notably expressed across a panel of 11 tissues, while igfbp-6b1 and 6b2 were each broadly expressed, with the latter being especially highly expressed in gill, brain, and spleen (16). Both igfbp-6b1 and 6b2 were also reported as being highly expressed in Atlantic salmon gills, where they were dynamically regulated during smoltification (123).
The overexpression of either zebrafish igfbp-6 paralog caused a significant reduction of embryonic growth (20), suggesting a role in growth inhibition. Studies of igfbp-6 gene regulation in skeletal muscle support a similar role in other species, though some conflicting data exist. For example, a study in Atlantic salmon reported no change in igfbp-6b expression in response to food intake manipulation (92), while another reported downregulation of igfbp-6b in tilapia skeletal muscle in response to feeding after a period of fasting (53). Similar results were observed in fine flounder skeletal muscle, where igfbp-6b expression decreased in response to feeding immediately postfasting, although expression then increased during long-term refeeding (88). However, igfbp-6b was repressed in fine flounder skeletal muscle in response to stress (94), which is less consistent with a negative role in growth. Conversely, in Atlantic salmon myotubes treated with dexamethasone, igfbp-6a1 was downregulated, while igfbp-6b2 was upregulated, highlighting complex functions that cannot be easily interpreted without functional data (57).
Recent work also implies a novel role for igfbp-6 genes in linking growth and immune regulation in teleosts. Alzaid et al. observed a significant increase of igfbp-6a2 in primary immune tissues of rainbow trout following a bacterial infection, and provided evidence that immune-responsive igfbp-6a2 upregulation was stimulated by immune signaling pathways driven by proinflammatory cytokines (27). Past work in salmonids has also shown that pro-inflammatory cytokines can promote the expression of igfbp-6 genes in skeletal muscle cell cultures (132) and in vivo (103), which may be linked to the balancing of energetic allocation toward effective immune function.
In summary, Igfbp-6 genes of teleosts are rather understudied, and it is difficult to draw overarching conclusions about their roles and functions at this time.

PeRSPeCTiveS AND FUTURe wORK
Our current understanding of the Igfbp repertoire of different teleosts has benefited greatly from recent expansions to genomic resources. We can now be confident in the existence of many teleost paralogs, which are expressed and presumably functional. However, our understanding of the functions and regulatory control of these genes remains highly fragmented across teleosts as a group and remains highly underdeveloped compared to mammals. It is becoming increasingly clear-perhaps with the exception of Igfbp-1-that teleost and mammalian Igfbp orthologs have evolved distinct expression-level regulation. This points to distinct functional roles in the regulation of growth in teleosts compared to mammals, which may be related to differences in growth dynamics, for example, indeterminate growth in teleosts. Moreover, there is also evidence that Igfbp orthologs from different species have evolved distinct regulation and hence, potentially functions, during teleost evolution. This can be speculatively linked to the additional functional flexibility or redundancy linked to Igfbp duplication events, which has allowed divergent regulation of paralogs to evolve under different physiological contexts.
It is also clear that differences in the expression of homologous Igfbp genes across teleost species are often difficult to interpret. In many cases, this may be linked to the historic ignorance of paralogous genes, either by considering one paralog in pair, or detecting signals from both paralogs in gene expression analyses. Hence, a fuller understanding of Igfbp genes will be possible in the presence of high-quality reference genomes, where all genes are properly annotated and can then be distinguished experimentally. The evidence for divergent regulation of Igfbp paralog expression is overwhelming, even for genes with very similar coding sequences (24), suggesting gene expression studies should make every effort to distinguish Igfbp paralogs.
An additional priority for future research should be to characterize the individual protein-level functions of all teleost Igfbp paralogs in multiple species extending beyond model organisms. While it has classically been challenging to perform functional analyses in non-model teleosts, the research landscape is rapidly changing. For example, genome editing using engineered CRIPSR/Cas9 systems has been demonstrated in vivo for various large commercial species, including salmonids (133) and catfishes (134), as well as in teleost cell culture (135). Hence, while even 5 years ago, the full repertoire of Igfbp genes was not even recognized in many teleosts, we can look forward to a future where every paralog within a species has its function cataloged by such approaches, even in lineages with hugely expanded Igfbp gene families. This will open the door for associating protein-level functional divergence in Igfbp paralogs with evolutionary changes in gene expression regulation, generating a fuller picture of the biological roles of this fascinating gene family in teleosts.

AUTHOR CONTRiBUTiONS
DGS and DJM wrote the manuscript and prepared the figures. DM built the phylogenetic tree showed in the manuscript.

FUNDiNG
DGS would like to thank the Marine Alliance Science Technology Scotland (MASTS) for their support. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions.