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Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type., Author summary How individual cell fates become specified from multipotent progenitors is a fundamental question in developmental and stem cell biology. Body pigment cells derive from a multipotent progenitor, but while in zebrafish there are three types of pigment cells (melanocytes, iridophores and xanthophores), in medaka these progenitors form four (as zebrafish, plus leucophores). Here, we address whether mechanisms generating each cell-type are conserved between the two species. We focus on two key regulatory proteins, Sox5 and Sox10, which we previously showed were involved in pigment cell development in medaka and zebrafish, respectively. We compare experimentally how the two proteins interact in regulating development of each of the pigment cell lineages in these fish. We show that development of all pigment cells, except leucophores, is dependent on Sox10, and that Sox5 modulates Sox10 activity antagonistically in all pigment cells in zebrafish, and melanocytes and iridophores in medaka. Surprisingly, in medaka, Sox5 acts co-operatively with Sox10 to promote xanthophore fate and to repress leucophore fate. Our findings reveal surprising diversity how Sox5 and Sox10 interact to govern pigment cell development in medaka and zebrafish, and suggest that this likely relates to the evolution of the novel leucophore pigment cell type in medaka.

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context., Author Summary Males of various species, including humans, remain near a (potential) mating partner and repel their rival males (mate-guarding). Mate-guarding is mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. Here we show that the arginine-vasotocin (AVT), a non-mammalian homolog of arginine-vasopressin (AVP), system mediates the molecular mechanisms underlying how mate presence (intersexual relationship) affects male competitive motivation (intrasexual relationship) in mate-guarding (a triadic relationship). We first established a novel behavioral paradigm in which medaka robustly exhibit mate-guarding behavior, that allowed us to study the genetic/molecular mechanisms underlying mate-guarding. Behavioral analysis of courtship behaviors, aggressive behaviors and mate-guarding using medaka mutants in this paradigm suggested that the AVT system is involved in the process in which mate (female) presence drives sexual motivation of males, which may facilitate mate guarding in the triadic relationship. Our study provides genetic evidence that the AVT/AVP system regulates mate-guarding behaviors, suggesting that the role of this system in mate-guarding is evolutionarily conserved from teleosts to mammals.

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor)., Author Summary How individual cell fates are specified from multipotent progenitor cells is a fundamental question in developmental and stem cell biology. Accumulating evidence indicates that stem cells develop into each of their final, diverse cell-types after progression through one or more partially-restricted intermediates, but the molecular mechanisms underlying final fate choice are largely unknown. Neural crest cells (NCCs) give rise to diverse cell-types including multiple pigment cells and thus are a favored model for understanding the mechanism of fate specification. We have investigated how a specific fate choice is made from partially-restricted pigment cell progenitors in medaka. We show that Sry-related transcription factor Sox5 is required for fate determination between yellow xanthophore and white leucophore, and its loss causes excessive formation of leucophores and absence of xanthophores. We demonstrate that Sox5 functions cell-autonomously in the xanthophore lineage in medaka. Furthermore, pax7a is expressed in the partially-restricted progenitor cells shared with xanthophore and leucophore lineages, and Sox5 acts in some of these cells to promote xanthophore lineage. Our work reveals the role of Sox5 as a molecular switch determining xanthophore versus leucophore fate choice from the shared progenitor, and identifies an important mechanism regulating pigment cell fate choice from NCCs.

There was an error in affiliation 7 for author Martin F. Lavin. Affiliation 7 should be: Centre for Clinical Research, University of Queensland, Royal Brisbane Hospital, Herston, Australia.

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis., Author Summary ASCIZ is a DNA damage response protein that has been proposed to be a regulator and stabilizing co-factor of the ATM kinase, mutations of which lead to a syndrome involving neurological and immune dysfunctions, tumour predisposition, and X-ray hypersensitivity. To study Asciz function in vivo, we have generated a knockout mouse model lacking this gene. Here we show that ASCIZ has a specific role in mediating cell survival in response to DNA base damage, but it is not required for stabilization and regulation of ATM. Strikingly, Asciz knockout mice fail to survive to birth and have tissue-specific defects in embryonic development. In particular, Asciz null embryos fail to develop lungs and undergo an early arrest in tracheal development. The precursor cells that normally form the lung are present in our embryos, but they fail to segregate from the foregut. These observations indicate that ASCIZ plays an important and previously unrecognized developmental role that is most likely unrelated to its function in mediating responses to DNA damage. Our study delineates the function of ASCIZ in DNA damage survival and highlights an exciting new function of the protein in controlling the early stages of lung development.

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases., Author Summary An important class of chemotherapeutic drugs used in the treatment of cancer induces DNA damage that interferes with DNA replication. The resulting block to replication results in the formation of single-strand gaps in DNA. These gaps can be filled by specialized DNA polymerases, a process associated with the introduction of mutations or by recombination with an undamaged segment of DNA with an identical or similar sequence. Our work shows that diversification of the antibody genes in the chicken B cell line DT40, which is initiated by localized replication-stalling DNA damage, proceeds by formation of a single-strand intermediate. These gaps are generated by the action of a specific nuclease complex, comprising the Mre11, Rad50, and Nbs1 proteins, which have previously been implicated in the initiation of homologous recombination from double-strand breaks. However, in this context, their dysfunction can be reversed by the expression of a bacterial single-strand–specific nuclease, SbcB. Antibody diversification in DT40 thus provides an excellent model for studying the process of replication-stalling DNA damage and will allow a more detailed understanding of the mechanisms underlying gap repair and cellular tolerance of chemotherapeutic agents.