Matters Arising Paper Research Articles

Polyphosphate attachment to lysine repeats is a non-covalent protein modification

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo etal. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo etal. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo etal. (2024), published in this issue.

Intercellular genetic tracing of cardiac endothelium in the developing heart

Cardiac resident macrophages play vital roles in heart development, homeostasis, repair, and regeneration. Recent studies documented the hematopoietic potential of cardiac endothelium that supports the generation of cardiac macrophages and peripheral blood cells in mice. However, the conclusion was not strongly supported by previous genetic tracing studies, given the non-specific nature of conventional Cre-loxP tracing tools. Here, we develop an intercellular genetic labeling system that can permanently trace heart-specific endothelial cells based on cell-cell interaction in mice. Results from cell-cell contact-mediated genetic fate mapping demonstrate that cardiac endothelial cells do not exhibit hemogenic potential and do not contribute to cardiac macrophages or other circulating blood cells. This Matters Arising paper is in response to Shigeta etal. (2019), published in Developmental Cell. See also the response by Liu and Nakano (2023), published in this issue.

Challenges of accurately estimating sex-biased admixture from X chromosomal and autosomal ancestry proportions

Sex-biased admixture can be inferred from ancestry-specific proportions of X chromosome and autosomes. In a paper published in the American Journal of Human Genetics, Micheletti etal.1 used this approach to quantify male and female contributions following the transatlantic slave trade. Using a large dataset from 23andMe, they concluded that African and European contributions to gene pools in the Americas were much more sex biased than previously thought. We show that the reported extreme sex-specific contributions can be attributed to unassigned genetic ancestry as well as the limitations of simple models of sex-biased admixture. Unassigned ancestry proportions in the study by Micheletti etal. ranged from ∼1% to 21%, depending on the type of chromosome and geographic region. A sensitivity analysis illustrates how this unassigned ancestry can create false patterns of sex bias and that mathematical models are highly sensitive to slight sampling errors when inferring mean ancestry proportions, making confidence intervals necessary. Thus, unassigned ancestry and the sensitivity of the models effectively prohibit the interpretation of estimated sex biases for many geographic regions in Micheletti etal. Furthermore, Micheletti etal. assumed models of a single admixture event. Using simulations, we find that violations of demographic assumptions, such as subsequent gene flow and/or sex-specific assortative mating, may have confounded the analyses of Micheletti etal., but unassigned ancestry was likely the more important confounding factor. Our findings underscore the importance of using complete ancestry information, sufficiently large sample sizes, and appropriate models when inferring sex-biased patterns of demography. This Matters Arising paper is in response to Micheletti etal.,1 published in American Journal of Human Genetics. See also the response by Micheletti etal.,2 published in this issue.

Low disease risk and penetrance in Leber hereditary optic neuropathy

The risk of Leber hereditary optic neuropathy (LHON) has largely been extrapolated from disease cohorts, which underestimate the population prevalence of pathogenic primary LHON variants as a result of incomplete disease penetrance. Understanding the true population prevalence of primary LHON variants, alongside the rate of clinical disease, provides a better understanding of disease risk and variant penetrance. We identified pathogenic primary LHON variants in whole-genome sequencing data of a well-characterized population-based control cohort and found that the prevalence is far greater than previously estimated, as it occurs in approximately 1 in 800 individuals. Accordingly, we were able to more accurately estimate population risk and disease penetrance in LHON variant carriers, validating our findings by using other large control datasets. These findings will inform accurate counseling in relation to the risk of vision loss in LHON variant carriers and disease manifestation in their family. This Matters Arising paper is in response to Lopez Sanchez etal. (2021), published in The American Journal of Human Genetics. See also the response by Mackey etal. (2022), published in this issue.

Mouse primordial germ-cell-like cells lack piRNAs.

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse primordial germ cell (mPGC) specification around E6.25, fetal piRNAs emerge in male gonocytes from E13.5 onward. The invitro differentiation of mPGC-like cells (mPGCLCs) has raised the possibility of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not fully expressed in Day 6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of Day 6 mPGCLC lines using small RNA-seq. Our combined efforts highlight that invitro differentiated Day 6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active. This Matters Arising paper is in response to von Meyenn etal. (2016), published in Developmental Cell. See also the correction by von Meyenn etal. published in this issue.

Mouse placenta fetal macrophages arise from endothelial cells outside the placenta.

Placental fetal macrophages (fMacs) are the only immune cells on the fetal side of the placental barrier. Mouse models have not been used to test their function because they have previously been found to have distinct cellular origins and functions in mice and humans. Here, we test the ontogeny of mouse placental fMacs. Using a new Hoxa13Cre allele that labels all placental endothelial cells (ECs), we demonstrate that mouse placenta fMacs do not arise from placental endothelium. Instead, lineage tracing studies using Tie2-Cre and Cx3cr1CreERT2 alleles demonstrate that mouse placental fMacs arise from yolk sac endothelium. Administration of blocking antibodies against CSF1R at E6.5 and E7.5 results in depletion of placental fMacs throughout pregnancy, and this suggests a yolk sac origin, similar to that in human fMacs. This Matters Arising paper is in response to Liang etal., published in Developmental Cell. A response by Liang and Liu is published in this issue.

Response to Wyckelsma et al.: Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation

The common loss-of-function mutation R577X in the structural muscle protein ACTN3 emerged as a potential target of positive selection from early studies and has been the focus of insightful physiological work suggesting a significant impact on muscle metabolism. Adaptation to cold climates has been proposed as a key adaptive mechanism explaining its global allele frequency patterns. Here, we re-examine this hypothesis analyzing modern (n= 3,626) and ancient (n= 1,651) genomic data by using allele-frequency as well as haplotype homozygosity-based methods. The presented results are more consistent with genetic drift rather than selection in cold climates as the main driver of the ACTN3 R577X frequency distribution in human populations across the world. This Matters Arising paper is in response to Wyckelsma etal. (2021),1 published in The American Journal of Human Genetics. See also the response by Wyckelsma etal. (2022),2 published in this issue.

Do catecholaminergic TrkC DRG neurons represent a class of cardiovascular enteroceptor?

In a recent issue of Cell Reports, Morelli etal. (2021) identify a subpopulation of mechanosensitive peripheral sensory neurons that coexpress tyrosine hydroxylase (TH) and tropomyosin receptor kinase C (TrkC) and innervate cutaneous arterioles. They show that activation of TrkC sensory neurons causes cutaneous vasoconstriction and, most remarkably, that their lesion is associated with sudden death of an undetermined cause, preceded by a progressive drop in blood pressure, and conclude that TrkC+ TH+ neurons represent a baroreceptor class of homeostatic enteroceptor. This represents a radical departure from current consensus models for the central control of blood pressure. Here, we offer an alternative perspective on their findings and suggest priorities for further investigation. This Matters Arising paper is in response to Morelli etal. (2021), published in Cell Reports. See also the response by Heppenstall etal. (2022), published in this issue.

Mice hypomorphic for Pitx3 show robust entrainment of circadian behavioral and metabolic rhythms to scheduled feeding.

Pitx3ak mice lack a functioning retina and develop fewer than 10% of dopamine neurons in the substantia nigra. Del Río-Martín etal. (2019) reported that entrainment of circadian rhythms to daily light-dark (LD) cycles is absent in these mice, and that rhythms of locomotor activity, energy expenditure, and other metabolic variables are disrupted with food available ad libitum and fail to entrain to a daily feeding. The authors propose that retinal innervation of the suprachiasmatic nucleus is required for development of cyclic metabolic homeostasis, but methodological issues limit interpretation of the results. Using standardized feeding schedules and procedures for distinguishing free-running from entrained circadian rhythms, we confirm that behavioral and metabolic rhythms in Pitx3ak mice do not entrain to LD cycles, but we find no impairment in circadian organization of metabolism with food available ad libitum and no impairment in entrainment of metabolic or behavioral rhythms by daily feeding schedules. This Matters Arising paper is in response to Del Río-Martín etal. (2019), published in Cell Reports. See also the response by Fernandez-Perez etal. (2022), published in this issue.

NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming

The regenerative capacity of neurons is limited in the central nervous system (CNS), with irreversible neuronal loss upon insult. In contrast, microglia exhibit extraordinary capacity for repopulation. Matsuda etal. (2019) recently reported NeuroD1-induced microglia-to-neuron conversion, aiming to provide an "unlimited" source to regenerate neurons. However, the extent to which NeuroD1 can exert cross-lineage reprogramming of microglia (myeloid lineage) to neurons (neuroectodermal lineage) is unclear. In this study, we unexpectedly found that NeuroD1 cannot convert microglia to neurons in mice. Instead, NeuroD1 expression induces microglial cell death. Moreover, lineage tracing reveals non-specific leakage of similar lentiviruses as previously used for microglia-to-neuron conversion, which confounds the microglia-to-neuron observation. In summary, we demonstrated that NeuroD1 cannot induce microglia-to-neuron cross-lineage reprogramming. We here propose rigid principles for verifying glia-to-neuron conversion. This Matters Arising paper is in response to Matsuda etal. (2019), published in Neuron.

PSMB11 regulates gene expression in cortical thymic epithelial cells.

The PSMB11 proteasomal subunit, expressed only in cortical thymic epithelial cells (cTECs), is essential for the development of functional CD8+ Tcells. An attractive yet unproven theory holds that PSMB11 generates unique major histocompatibility complex class I (MHC I)-associated peptides required for positive selection. We recently reported that PSMB11 regulates the expression of hundreds of genes in cTECs, mainly by differential proteolysis of transcription factors. Thereby, PSMB11 maintains the distinctness of cTECs relative to medullary TECs (mTECs) and promotes cortex-to-medulla migration of developing thymocytes. These conclusions have been challenged by Ohigashi and colleagues, who suggest that their data show that PSMB11 uniquely controls antigen presentation without affecting cTEC biology. Here, we perform a comprehensive reanalysis of transcriptomic and proteomic data from the Ohigashi lab and confirm our original conclusions. This Matters Arising paper is in response to Ohigashi etal. (2019), published in Cell Reports. See also the response by Ohigashi and Takahama (2021), published in this issue of Cell Reports.

TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and Tcell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed etal. (2019), published in Cell. See also the response by Ahmed etal. (2021), published in this issue.

So many Nigerians: why is Nigeria overrepresented as the ancestral genetic homeland of Legacy African North Americans?

The genetics of African North Americans are complex amalgamations of various West and Central African peoples with modest gene flow from specific European and Amerindian peoples. A comprehensive understanding of African North American biohistory is a prerequisite for accurate interpretations of the ancestral genetics of this population. Too often, genetic interpretations falter with ahistorical reconstructions. The recently reported overrepresentation of Nigerian lineages in African North Americans reflects pronounced limitations in the African genomic database, the artificiality of the colonial maps of Africa, the contributions of multiple African empires and kingdoms into the transatlantic trade in enslaved Africans, and the overrepresentation of Yoruba peoples in the existing limited representation of West Africans in public genomic databases. This Matters Arising paper is in response to Micheletti etal. (2020), published in The American Journal of Human Genetics. See also the response by Micheletti etal. (2020), published in this issue.

Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice

A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu etal. (2016), published in Neuron. See also the response by Nguyen etal. (2020), published in this issue.

Gain-of-Function Claims for Type-2-Diabetes-Associated Coding Variants in SLC16A11 Are Not Supported by the Experimental Data

Human genetic variants in SLC16A11 are associated with increased risk of type 2 diabetes (T2D). We previously identified two distinct mechanisms through which co-inherited T2D-risk coding and non-coding variants disrupt SLC16A11 expression and activity, thus implicating reduced SLC16A11 function as the disease-relevant direction of effect. In a recent publication, Zhao etal. (2019a) argue that human SLC16A11 coding variants confer gain of function, basing their conclusions on phenotypic changes observed following overexpression of mutant murine Slc16a11. However, data necessary to demonstrate gain-of-function activity are not reported. Furthermore, several fundamental flaws in their experimental system-including inaccurate modeling of the human variant haplotype and expression conditions that are not physiologically relevant-prevent conclusions about T2D-risk variant effects on human physiology. This Matters Arising paper is in response to Zhao etal. (2019a), published in Cell Reports. See also the response by Zhao etal. (2019b) in this issue of Cell Reports.

Meta-analysis of Chromatin Programming by Steroid Receptors

Transcription factors (TFs) must access chromatin to bind to their response elements and regulate gene expression. A widely accepted model proposes that only a special subset of TFs, pioneer factors, can associate with condensed chromatin and initiate chromatin opening. We previously reported that steroid receptors (SRs), not considered pioneer factors, can assist the binding of an archetypal pioneer, the forkhead box protein 1 (FOXA1), at a subset of receptor-activated enhancers. These findings have been challenged recently, with the suggestion that newly acquired data fully support the prevailing pioneer model. Here, we reexamine our results and confirm the original conclusions. We also analyze and discuss a number of available datasets relevant to chromatin penetration by SRs and find a general consensus supporting our original observations. Hence, we propose that chromatin opening at some sites can be initiated by SRs, with a parallel recruitment of factors often treated as having a unique pioneer function. This Matters Arising paper is in response to Glont etal. (2019), published in Cell Reports.

Integrating Results across Methodologies Is Essential for Producing Robust Neuronal Taxonomies

Elucidating the diversity and spatial organization of cell types in the brain is an essential goal of neuroscience, with many emerging technologies helping to advance this endeavor. Using a new insitu hybridization method that can measure the expression of hundreds of genes in a given mouse brain section (amplified seqFISH), Shah etal. (2016) describe a spatial organization of hippocampal cell types that differs from previous reports. In seeking to understand this discrepancy, we find that many of the barcoded genes used by seqFISH to characterize this spatial organization, when cross-validated by other sensitive methodologies, exhibit negligible expression in the hippocampus. Additionally, the results of Shah etal. (2016) do not recapitulate canonical cellular hierarchies and improperly classify major neuronal cell types. We suggest that, when describing the spatial organization of brain regions, cross-validation using multiple techniques should be used to yield robust and informative cellular classification. This Matters Arising paper is in response to Shah etal. (2016), published in Neuron. See also the response by Shah etal. (2017), published in this issue.

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression ofcandidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong etal., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao etal., 2016). When we attempted to replicate these results, we found each construct conferred modest MA currents in a small fraction of naive HEK cells similar to thepublished work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9 inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins. This Matters Arising paper is in response to Hong etal. (2016) and Zhao etal. (2016) in Neuron. See also the response papers by Hong etal. (2017) and Zhao etal. (2017) published concurrently with this Matters Arising.

Clonally Related GABAergic Interneurons Do Not Randomly Disperse but Frequently Form Local Clusters in the Forebrain

Progenitor cells in the medial ganglionic eminence (MGE) and preoptic area (PoA) give rise to GABAergic inhibitory interneurons that are distributed in the forebrain, largely in the cortex, hippocampus, and striatum. Two previous studies suggest that clonally related interneurons originating from individual MGE/PoA progenitors frequently form local clusters in the cortex. However, Mayer etal. and Harwell etal. recently argued that MGE/PoA-derived interneuron clones disperse widely and populate different forebrain structures. Here, we report further analysis of the spatial distribution of clonally related interneurons and demonstrate that interneuron clones do not non-specifically disperse in the forebrain. Around 70% of clones are restricted to one brain structure, predominantly the cortex. Moreover, the regional distribution of clonally related interneurons exhibits a clear clustering feature, which cannot occur by chance from a random diffusion. These results confirm that lineage relationship influences the spatial distribution of inhibitory interneurons in the forebrain. This Matters Arising paper is in response to Harwell etal. (2015) and Mayer etal. (2015), published in Neuron. See also the response by Turrero García etal. (2016) and Mayer etal. (2016), published in this issue.

NR1H3 p.Arg415Gln Is Not Associated to Multiple Sclerosis Risk

A recent study by Wang etal. (2016a) claims that the low-frequency variant NR1H3 p.Arg415Gln is sufficient to cause multiple sclerosis in certain individuals and determines a patient's likelihood of primary progressive disease. We sought to replicate this finding in the International MS Genetics Consortium (IMSGC) patient collection, which is 13-fold larger than the collection of Wang etal. (2016a), but we find no evidence that this variant is associated with either MS or disease subtype. Wang etal. (2016a) also report a common variant association in the region, which we show captures the association the IMSGC reported in 2013. Therefore, we conclude that the reported low-frequency association is a false positive, likely generated by insufficient sample size. The claim of NR1H3 mutations describing a Mendelian form of MS-of which no examples exist-can therefore not be substantiated by data. This Matters Arising paper is in response to Wang etal. (2016a), published in Neuron. See also the related Matters Arising paper by Minikel and MacArthur (2016) and the response by Wang etal. (2016b), published in this issue.