Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Maternal heterozygosity of Slc6a19 causes metabolic perturbation and congenital NAD deficiency disorder in mice.

Nicotinamide adenine dinucleotide (NAD) is a key metabolite synthesised from vitamin B3 or tryptophan. Disruption of genes encoding NAD synthesis enzymes reduces NAD levels and causes congenital NAD deficiency disorder (CNDD), characterised by multiple congenital malformations. SLC6A19 (encoding B0AT1, a neutral amino acid transporter), represents the main transporter for free tryptophan in the intestine and kidney. Here, we tested whether Slc6a19 heterozygosity in mice limits the tryptophan available for NAD synthesis during pregnancy and causes adverse pregnancy outcomes. Pregnant Slc6a19+/- mice were fed diets depleted of vitamin B3, so that tryptophan was the source of NAD during gestation. This perturbed the NAD metabolome in pregnant Slc6a19+/- females, resulting in reduced NAD levels and increased rates of embryo loss. Surviving embryos were small and exhibited specific combinations of CNDD-associated malformations. Our results show that genes not directly involved in NAD synthesis can affect NAD metabolism and cause CNDD. They also suggest that human female carriers of a SLC6A19 loss-of-function allele might be susceptible to adverse pregnancy outcomes unless sufficient NAD precursor amounts are available during gestation. This article has an associated First Person interview with the first author of the paper.

Regulatory T cells regulate blastemal proliferation during zebrafish caudal fin regeneration.

The role of T cells in appendage regeneration remains unclear. In this study, we revealed an important role for regulatory T cells (Tregs), a subset of T cells that regulate tolerance and tissue repair, in the epimorphic regeneration of zebrafish caudal fin tissue. Upon amputation, fin tissue-resident Tregs infiltrate into the blastema, a population of progenitor cells that produce new fin tissues. Conditional genetic ablation of Tregs attenuates blastemal cell proliferation during fin regeneration. Blastema-infiltrating Tregs upregulate the expression of igf2a and igf2b, and pharmacological activation of IGF signaling restores blastemal proliferation in Treg-ablated zebrafish. These findings further extend our understandings of Treg function in tissue regeneration and repair.

Cardiac Resection Injury in Zebrafish.

The zebrafish (Danio rerio) possesses a spectacular capacity for cardiac regeneration. Zebrafish have been used in cardiac regeneration research for nearly two decades, contributing to the identification of signals and cellular mechanisms as potential targets for human heart repair. Investigations into cardiac regeneration in zebrafish have been facilitated by multiple methods of inducing cardiac tissue damage. Among the established methods, cardiac resection injury is a relatively simple, yet robust approach traditionally used to induce cardiac tissue damage in a reproducible manner. Here, we describe a detailed protocol to perform a cardiac resection injury in adult zebrafish and discuss potential complications for researchers who are new to this technique.

Krüppel-like factor 1 is a core cardiomyogenic trigger in zebrafish.

Cardiac regeneration requires dedifferentiation and proliferation of mature cardiomyocytes, but the mechanisms underlying this plasticity remain unclear. Here, we identify a potent cardiomyogenic role for Krüppel-like factor 1 (Klf1/Eklf), which is induced in adult zebrafish myocardium upon injury. Myocardial inhibition of Klf1 function does not affect heart development, but it severely impairs regeneration. Transient Klf1 activation is sufficient to expand mature myocardium in uninjured hearts. Klf1 directs epigenetic reprogramming of the cardiac transcription factor network, permitting coordinated cardiomyocyte dedifferentiation and proliferation. Myocardial expansion is supported by Klf1-induced rewiring of mitochondrial metabolism from oxidative respiration to anabolic pathways. Our findings establish Klf1 as a core transcriptional regulator of cardiomyocyte renewal in adult zebrafish hearts.

Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch.

Despite the extensive use of zebrafish as a model organism in developmental biology and regeneration research, genetic techniques enabling conditional analysis of gene function are limited. In this study, we generated Zwitch, a Cre-dependent invertible gene-trap cassette, enabling the establishment of conditional alleles in zebrafish by generating intronic insertions via in vivo homologous recombination. To demonstrate the utility of Zwitch, we generated a conditional sonic hedgehog a (shha) allele. Homozygous shha mutants developed normally; however, shha mutant embryos globally expressing Cre exhibited strong reductions in endogenous shha and shha target gene mRNA levels and developmental defects associated with null shha mutations. Analyzing a conditional shha mutant generated using an epicardium-specific inducible Cre driver revealed unique roles for epicardium-derived Shha in myocardial proliferation during heart development and regeneration. Zwitch will extend the utility of zebrafish in organ development and regeneration research and might be applicable to other model organisms.

Zebrafish FOXP3 is required for the maintenance of immune tolerance.

Regulatory T (Treg) cells play a central role in the suppression of excessive immune responses against both self and non-self antigens. The development and function of Treg cells are controlled by a master regulatory gene encoding the forkhead box P3 (FOXP3) protein in mammals. However, little is known regarding the functions of Treg cells and FOXP3 in non-mammalian vertebrates. In this study, we generated mutant zebrafish lacking a functional FOXP3 ortholog, and demonstrated a significant reduction in survival accompanied by a marked increase in inflammatory gene expression, mononuclear cell infiltration, and T cell proliferation in peripheral tissues. Our findings indicate that the zebrafish FOXP3 protein may have an evolutionally conserved role in the control of immune tolerance, illuminating the potential of the zebrafish as a novel model for investigating the development and functions of Treg cells.

Zebrafish Regulatory T Cells Mediate Organ-Specific Regenerative Programs.

The attenuation of ancestral pro-regenerative pathways may explain why humans do not efficiently regenerate damaged organs. Vertebrate lineages that exhibit robust regeneration, including the teleost zebrafish, provide insights into the maintenance of adult regenerative capacity. Using established models of spinal cord, heart, and retina regeneration, we discovered that zebrafish Treg-like (zTreg) cells rapidly homed to damaged organs. Conditional ablation of zTreg cells blocked organ regeneration by impairing precursor cell proliferation. In addition to modulating inflammation, infiltrating zTreg cells stimulated regeneration through interleukin-10-independent secretion of organ-specific regenerative factors (Ntf3: spinal cord; Nrg1: heart; Igf1: retina). Recombinant regeneration factors rescued the regeneration defects associated with zTreg cell depletion, whereas Foxp3a-deficient zTreg cells infiltrated damaged organs but failed to express regenerative factors. Our data delineate organ-specific roles for Treg cells in maintaining pro-regenerative capacity that could potentially be harnessed for diverse regenerative therapies.