Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4. METHODS: We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines. RESULTS: We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4. INTERPRETATION: These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.

The DUX4 protein is a co-repressor of the progesterone and glucocorticoid nuclear receptors.

DUX4 is a transcription factor required during early embryonic development in placental mammals. In this work, we provide evidence that DUX4 is a co-repressor of nuclear receptors (NRs) of progesterone (PR) and glucocorticoids (GR). The DUX4 C-ter and N-ter regions, including the nuclear localization signals and homeodomain motifs, contribute to the co-repressor activity of DUX4 on PR and GR. Immunoprecipitation studies, using total protein extracts of cells expressing tagged versions of DUX4 and GR, support that these proteins are physically associated. Our studies suggest that DUX4 could modulate gene expression by co-regulating the activity of hormone NRs. This is the first report highlighting a potential endocrine role for DUX4.

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871-driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Meeting report: the 2021 FSHD International Research Congress.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the late teens/early adulthood [1]. Although the etiology of the disease for both FSHD type 1 and type 2 has been attributed to gain-of-toxic function stemming from aberrant DUX4 expression, the exact pathogenic mechanisms involved in muscle wasting have yet to be elucidated [2-4]. The 2021 FSHD International Research Congress, held virtually on June 24-25, convened over 350 researchers and clinicians to share the most recent advances in the understanding of the disease mechanism, discuss the proliferation of interventional strategies and refinement of clinical outcome measures, including results from the ReDUX4 trial, a phase 2b clinical trial of losmapimod in FSHD [NCT04003974].

Human miRNA miR-675 inhibits DUX4 expression and may be exploited as a potential treatment for Facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating myopathy caused by de-repression of the DUX4 gene in skeletal muscles. Effective therapies will likely involve DUX4 inhibition. RNA interference (RNAi) is one powerful approach to inhibit DUX4, and we previously described a RNAi gene therapy to achieve DUX4 silencing in FSHD cells and mice using engineered microRNAs. Here we report a strategy to direct RNAi against DUX4 using the natural microRNA miR-675, which is derived from the lncRNA H19. Human miR-675 inhibits DUX4 expression and associated outcomes in FSHD cell models. In addition, miR-675 delivery using gene therapy protects muscles from DUX4-associated death in mice. Finally, we show that three known miR-675-upregulating small molecules inhibit DUX4 and DUX4-activated FSHD biomarkers in FSHD patient-derived myotubes. To our knowledge, this is the first study demonstrating the use of small molecules to suppress a dominant disease gene using an RNAi mechanism.

A stromal progenitor and ILC2 niche promotes muscle eosinophilia and fibrosis-associated gene expression.

Despite the well-accepted view that chronic inflammation contributes to the pathogenesis of Duchenne muscular dystrophy (DMD), the function and regulation of eosinophils remain an unclear facet of type II innate immunity in dystrophic muscle. We report the observation that group 2 innate lymphoid cells (ILC2s) are present in skeletal muscle and are the principal regulators of muscle eosinophils during muscular dystrophy. Eosinophils were elevated in DMD patients and dystrophic mice along with interleukin (IL)-5, a major eosinophil survival factor that was predominantly expressed by muscle ILC2s. We also find that IL-33 was upregulated in dystrophic muscle and was predominantly produced by fibrogenic/adipogenic progenitors (FAPs). Exogenous IL-33 and IL-2 complex (IL-2c) expanded muscle ILC2s and eosinophils, decreased the cross-sectional area (CSA) of regenerating myofibers, and increased the expression of genes associated with muscle fibrosis. The deletion of ILC2s in dystrophic mice mitigated muscle eosinophilia and impaired the induction of IL-5 and fibrosis-associated genes. Our findings highlight a FAP/ILC2/eosinophil axis that promotes type II innate immunity, which influences the balance between regenerative and fibrotic responses during muscular dystrophy.

Is Upregulation of Sarcolipin Beneficial or Detrimental to Muscle Function?

Sarcolipin (SLN) is a regulator of sarco/endo plasmic reticulum Ca2+-ATPase (SERCA) pump and has been shown to be involved in muscle nonshivering thermogenesis (NST) and energy metabolism. Interestingly, SLN expression is significantly upregulated both during muscle development and in several disease states. However, the significance of altered SLN expression in muscle patho-physiology is not completely understood. We have previously shown that transgenic over-expression of SLN in skeletal muscle is not detrimental, and can promote oxidative metabolism and exercise capacity. In contrast, some studies have suggested that SLN upregulation in disease states is deleterious for muscle function and ablation of SLN can be beneficial. In this perspective article, we critically examine both published and some new data to determine the relevance of SLN expression to disease pathology. The new data presented in this paper show that SLN levels are induced in muscle during systemic bacterial (Salmonella) infection or lipopolysaccharides (LPS) treatment. We also present data showing that SLN expression is significantly upregulated in different types of muscular dystrophies including myotubular myopathy. These data taken together reveal that upregulation of SLN expression in muscle disease is progressive and increases with severity. Therefore, we suggest that increased SLN expression should not be viewed as the cause of the disease; rather, it is a compensatory response to meet the higher energy demand of the muscle. We interpret that higher SLN/SERCA ratio positively modulate cytosolic Ca2+ signaling pathways to promote mitochondrial biogenesis and oxidative metabolism to meet higher energy demand in muscle.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes.

Facioscapulohumeral muscular dystrophy (FSHD) arises from epigenetic changes that de-repress the DUX4 gene in muscle. The full-length DUX4 protein causes cell death and muscle toxicity, and therefore we hypothesize that FSHD therapies should center on inhibiting full-length DUX4 expression. In this study, we developed a strategy to accomplish DUX4 inhibition using U7-small nuclear RNA (snRNA) antisense expression cassettes (called U7-asDUX4). These non-coding RNAs were designed to inhibit production or maturation of the full-length DUX4 pre-mRNA by masking the DUX4 start codon, splice sites, or polyadenylation signal. In so doing, U7-asDUX4 snRNAs operate similarly to antisense oligonucleotides. However, in contrast to oligonucleotides, which are limited by poor uptake in muscle and a requirement for lifelong repeated dosing, U7-asDUX4 snRNAs can be packaged within myotropic gene therapy vectors and may require only a single administration when delivered to post-mitotic cells in vivo. We tested several U7-asDUX4s that reduced DUX4 expression in vitro and improved DUX4-associated outcomes. Inhibition of DUX4 expression via U7-snRNAs could be a new prospective gene therapy approach for FSHD or be used in combination with other strategies, like RNAi therapy, to maximize DUX4 silencing in individuals with FSHD.

RNAi-Based Gene Therapy Rescues Developmental and Epileptic Encephalopathy in a Genetic Mouse Model.

Developmental and epileptic encephalopathy (DEE) associated with de novo variants in the gene encoding dynamin-1 (DNM1) is a severe debilitating disease with no pharmacological remedy. Like most genetic DEEs, the majority of DNM1 patients suffer from therapy-resistant seizures and comorbidities such as intellectual disability, developmental delay, and hypotonia. We tested RNAi gene therapy in the Dnm1 fitful mouse model of DEE using a Dnm1-targeted therapeutic microRNA delivered by a self-complementary adeno-associated virus vector. Untreated or control-injected fitful mice have growth delay, severe ataxia, and lethal tonic-clonic seizures by 3 weeks of age. These major impairments are mitigated following a single treatment in newborn mice, along with key underlying cellular features including gliosis, cell death, and aberrant neuronal metabolic activity typically associated with recurrent seizures. Our results underscore the potential for RNAi gene therapy to treat DNM1 disease and other genetic DEEs where treatment would require inhibition of the pathogenic gene product.

Gene therapies for axonal neuropathies: Available strategies, successes to date, and what to target next.

Nearly one-hundred loci in the human genome have been associated with different forms of Charcot-Marie-Tooth disease (CMT) and related inherited neuropathies. Despite this wealth of gene targets, treatment options are still extremely limited, and clear "druggable" pathways are not obvious for many of these mutations. However, recent advances in gene therapies are beginning to circumvent this challenge. Each type of CMT is a monogenic disorder, and the cellular targets are usually well-defined and typically include peripheral neurons or Schwann cells. In addition, the genetic mechanism is often also clear, with loss-of-function mutations requiring restoration of gene expression, and gain-of-function or dominant-negative mutations requiring silencing of the mutant allele. These factors combine to make CMT a good target for developing genetic therapies. Here we will review the state of relatively established gene therapy approaches, including viral vector-mediated gene replacement and antisense oligonucleotides for exon skipping, altering splicing, and gene knockdown. We will also describe earlier stage approaches for allele-specific knockdown and CRIPSR/Cas9 gene editing. We will next describe how these various approaches have been deployed in clinical and preclinical studies. Finally, we will evaluate various forms of CMT as candidates for gene therapy based on the current understanding of their genetics, cellular/tissue targets, validated animal models, and availability of patient populations and natural history data.

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.

RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. Therefore, developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed DUX4 mRNA in transfected HEK293 cells and endogenous DUX4 mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in DUX4 mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based DUX4 expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD.

RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s). We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD).

FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD.

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

Mouse Dux is myotoxic and shares partial functional homology with its human paralog DUX4.

D4Z4 repeats are present in at least 11 different mammalian species, including humans and mice. Each repeat contains an open reading frame encoding a double homeodomain (DUX) family transcription factor. Aberrant expression of the D4Z4 ORF called DUX4 is associated with the pathogenesis of Facioscapulohumeral muscular dystrophy (FSHD). DUX4 is toxic to numerous cell types of different species, and over-expression caused dysmorphism and developmental arrest in frogs and zebrafish, embryonic lethality in transgenic mice, and lesions in mouse muscle. Because DUX4 is a primate-specific gene, questions have been raised about the biological relevance of over-expressing it in non-primate models, as DUX4 toxicity could be related to non-specific cellular stress induced by over-expressing a DUX family transcription factor in organisms that did not co-evolve its regulated transcriptional networks. We assessed toxic phenotypes of DUX family genes, including DUX4, DUX1, DUX5, DUXA, DUX4-s, Dux-bl and mouse Dux. We found that DUX proteins were not universally toxic, and only the mouse Dux gene caused similar toxic phenotypes as human DUX4. Using RNA-seq, we found that 80% of genes upregulated by Dux were similarly increased in DUX4-expressing cells. Moreover, 43% of Dux-responsive genes contained ChIP-seq binding sites for both Dux and DUX4, and both proteins had similar consensus binding site sequences. These results suggested DUX4 and Dux may regulate some common pathways, and despite diverging from a common progenitor under different selective pressures for millions of years, the two genes maintain partial functional homology.