Proteins are molecules made up of long chains of building blocks called amino acids. When a mutation changes one of these amino acids, it can lead to the protein malfunctioning, which can have many effects at the cell and tissue level. Given that human proteins are made up of 20 different amino acids, each building block in a protein could mutate to any of the other 19 amino acids, and each mutations could have different effects.

Tubulins are proteins that form microtubules, thin tubes that help give cells their shape and allow them to migrate. These proteins are added or removed to microtubules depending on the cell’s needs, meaning that microtubules can grow or shrink depending on the situation. Mutations in the tubulin proteins have been linked to malformations of varying severities involving the formation of ridges and folds on the surface of the brain, including lissencephaly, pachygyria or polymicrogyria.

Hoff et al. wanted to establish links between tubulin mutations and the effects observed at both cell and tissue level in the brain. They focused on two mutations in the tubulin protein TUBA1A that affect the amino acid in position 409 in the protein, which is normally a valine. One of the mutations turns this valine into an amino acid called isoleucine. This mutation is associated with pachygyria, which leads to the brain developing few ridges that are broad and flat. The second mutation turns the valine into an alanine, and is linked to lissencephaly, a more severe condition in which the brain develops no ridges, appearing smooth.

Hoff et al. found that both mutations interfere with the development of the brain by stopping neurons from migrating properly, which prevents them from forming the folds in the brain correctly. At the cellular level, the mutations lead to tubulins becoming harder to remove from microtubules, making microtubules more stable than usual. This results in longer microtubules that are harder for the cell to shorten or destroy as needed. Additionally, Hoff et al. showed that the mutant versions of TUBA1A have weaker interactions with a protein called XMAP215, which controls the addition of tubulin to microtubules. This causes the microtubules to grow uncontrollably.

Hoff et al. also established that the magnitude of the effects of each mutation on microtubule growth scale with the severity of the disorder they cause. Specifically, cells in which TUBA1A is not mutated have microtubules that grow at a normal rate, and lead to typical brain development. Meanwhile, cells carrying the mutation that turns a valine into an alanine, which is linked to the more severe condition lissencephaly, have microtubules that grow very fast. Finally, cells in which the valine is mutated to an isoleucine – the mutation associated with the less severe malformation pachygyria – have microtubules that grow at an intermediate rate.

These findings provide a link between mutations in tubulin proteins and larger effects on cell movement that lead to brain malformations. Additionally, they also link the severity of the malformation to the severity of the microtubule defect caused by each mutation. Further work could examine whether microtubule stabilization is also seen in other similar diseases, which, in the long term, could reveal ways to detect and treat these illnesses.

Brain development requires cargo transport, force generation, and structural reinforcement by the microtubule cytoskeleton. The regulation of microtubules must be finely tuned to meet specific demands for different cell types, time points in development, and even different locations within a cell. Particularly in neurons, the spatial and temporal regulation of microtubules establishes cargo transport networks, facilitates efficient signal transduction, and supports the extension and retraction of neurites (Dent and Baas, 2014; Dent and Kalil, 2001; He et al., 2002; Lin et al., 2012; Witte et al., 2008). Accordingly, defects in microtubule regulation in neurons have been linked to brain malformations such as lissencephaly, microcephaly, and autism spectrum disorders, among others (Bahi-Buisson et al., 2014; Chakraborti et al., 2016; Srivastava and Schwartz, 2014).

Brain malformations such as lissencephaly, pachygyria, and polymicrogyria are associated with defects in neuronal migration. During neuronal migration, neurons that are born at the neuroepithelium must migrate out of the ventricular zone along radial glia cells to reach their destination at the cortical plate (Barkovich et al., 2012; Barkovich et al., 2005). Throughout the course of radial migration, cortical neurons must transition through various morphologies, first by transitioning from a bipolar to a multipolar state, then back to a bipolar state to complete migration (Nadarajah et al., 2001; Noctor et al., 2004; Tabata and Nakajima, 2003). After arriving at the cortical plate, the neurons are correctly positioned and polarized to connect to each other and send and receive information. During this dynamic process of neuronal morphogenesis, microtubules generate force and provide structural support. Therefore, microtubule networks must be acutely regulated during these transitions (Chesta et al., 2014; Dehmelt et al., 2006; Dent et al., 2011; He et al., 2002; Lu et al., 2013; Sainath and Gallo, 2015; Winding et al., 2016). For example, the microtubule motor, kinesin-6, is important for establishing a leading process that is required for the transition from a multipolar to bipolar state and subsequent neuronal migration (Falnikar et al., 2013). Together this suggests the critical importance of regulating microtubules in the right place and at the right time during the morphological transitions neurons must undergo for migration and, ultimately, proper brain development.

‘Tubulinopathies’ encompass a wide range of heterozygous, missense mutations in the genes encoding α- and β-tubulins that are associated with a spectrum of brain malformations (Bahi-Buisson et al., 2014; Fallet-Bianco et al., 2014). α- and β-tubulins are encoded by a number of different genes, known as isotypes, that differ in amino acid sequence. The expression of different isotypes varies across different cell types as well as during different stages of development. Tubulinopathy mutations have been identified in three of the eight β-tubulin isotypes, and in one of the seven α-tubulin isotypes, a gene known as TUBA1A (Bahi-Buisson et al., 2014; Ludueña and Banerjee, 2008). TUBA1A is the most highly expressed α-tubulin isotype in post-mitotic neurons in the developing brain (Buscaglia et al., 2020b; Gloster et al., 1994; Gloster et al., 1999). To date, a total of 121 heterozygous, missense mutations have been identified in TUBA1A and are associated with neurodevelopment disorders (Hebebrand et al., 2019). It remains unclear whether the different severities of malformations observed in these patients are a result of differences in genetic background or are a result of a specific functional difference in the mutant tubulin. A few of these TUBA1A mutants have been identified as loss-of-function mutants that are unable to properly assemble into microtubule polymer and thus result in an undersupply of tubulin in the cell (Belvindrah et al., 2017; Keays et al., 2007). However, other TUBA1A mutants appear to be gain-of-function, as they are capable of microtubule assembly and act dominantly to perturb microtubule function in migrating neurons (Aiken et al., 2020; Aiken et al., 2019). If haploinsufficiency does not explain all TUBA1A tubulinopathies, then it is necessary to define clear mechanistic models for how different mutations in α-tubulin can result in different developmental outcomes. Only a small fraction of TUBA1A mutants have been studied, and more continue to be identified in the clinic. Therefore, it is important to establish clear mechanistic themes that will be useful in predicting the molecular, cellular, and tissue-level phenotypes of specific mutations.

The fundamental role of α-tubulin is to complex with β-tubulin into heterodimers and form microtubule polymers that are dynamic and highly regulated via both intrinsic and extrinsic factors (Bodakuntla et al., 2019; Borys et al., 2020; Goodson and Jonasson, 2018; Manka and Moores, 2018; Mitchison and Kirschner, 1984). Purified tubulin assembles into dynamic microtubules in the absence of extrinsic microtubule-associated proteins (MAPs) that are found in cells (Horio and Hotani, 1986; Mitchison and Kirschner, 1984; Walker et al., 1988). This intrinsic activity is in part regulated by the series of conformational states that tubulin undergoes as it transitions from free heterodimer into microtubule polymer (Chrétien et al., 1995; Mandelkow et al., 1991). In recent years, studies using cryo-electron microscopy and recombinant tubulin approaches have advanced the field’s understanding of the transitions that accompany GTP hydrolysis as free tubulin assembles into microtubule lattice (Geyer et al., 2018; Manka and Moores, 2018; Roostalu et al., 2020; Zheng et al., 1995). In addition to nucleotide-dependent conformational changes, tubulin must transition from a ‘curved’ free heterodimer to a straight conformation in the microtubule lattice (Buey et al., 2006; Jánosi et al., 1998; Nawrotek et al., 2011; Nogales and Wang, 2006; Ravelli et al., 2004; Rice et al., 2008). Tubulin domains that control the curved-to-straight transition are not completely understood. The ability of tubulin to adopt a range of conformational states not only impacts its intrinsic microtubule dynamics, but also tubulin’s interactions with a wide spectrum of MAPs. For example, doublecortin (DC) is predominantly expressed in the developing brain and its N-terminal DC-like repeat domain binds at the corner of four tubulin subunits in a straight microtubule lattice (Fourniol et al., 2010; Manka and Moores, 2020; Moores et al., 2006). On the other hand, members of the XMAP215 protein family possess a variable number of TOG (tumor overexpressed gene) domains, some of which preferentially bind to curved tubulin (Ayaz et al., 2014; Ayaz et al., 2012; Slep and Vale, 2007). XMAP215 proteins use this activity to either pull curved tubulin toward the microtubule plus end for polymerization or drive lattice-bound tubulin into a curved state and promote catastrophe (Ayaz et al., 2014; Brouhard et al., 2008; Farmer et al., 2021; Geyer et al., 2018). Therefore, understanding the effects of tubulin conformations on both intrinsic activity and extrinsic regulatory mechanisms are essential for elucidating the regulation of microtubule dynamics.

In this study we investigate the molecular mechanism of two previously identified tubulinopathy-associated mutations at the valine 409 residue of TUBA1A, V409I, and V409A. The patient harboring the V409A mutation exhibited lethal lissencephaly, whereas the patient with the V409I mutation presents with a milder malformation known as pachygyria (Bahi-Buisson et al., 2014; Fallet-Bianco et al., 2014). Studying these two mutations that occur at the same residue, yet are associated with varying degrees of brain malformations in patients, has potential to provide insight into how perturbing tubulin activity and the microtubule cytoskeleton in slightly different ways ultimately impacts brain development. We find that the expression of V409I or V409A dominantly disrupts neuron migration and morphology in mice and neuron branching during in vitro development. Additionally, V409I/A mutants increase microtubule acetylation and polymerization rates in neuronal cultures. By modeling the V409I/A mutants at the corresponding residue in budding yeast, V410, we find that the mutants increase microtubule polymerization and depolymerization rates in cells and disrupt XMAP215/Stu2 activity. Furthermore, the V410A mutation promotes faster microtubule polymerization in reconstitution experiments with purified tubulin, indicating a change in tubulin’s intrinsic polymerization activity. We propose that these results may be reconciled by the V410I/A mutants adopting a persistently straightened state as compared to wild-type (WT) tubulin, and this drives intrinsically faster microtubule polymerization rates in vitro. Importantly, we find that the severity of the V409I and V409A mutations scale from protein activity, to microtubule dynamics in cells, to neuron morphology and migration. Together, our results suggest that the TUBA1A-V409I/A mutations cause brain malformations by subverting dynamic regulation typically conferred by XMAP215.

The missense, heterozygous mutations identified in human tubulin genes and associated with brain malformations are numerous and varied (Bahi-Buisson et al., 2014; Fallet-Bianco et al., 2014). To date, we do not have a good understanding of how different mutations in tubulin genes have such a wide spectrum of detrimental effects on brain development. Investigating the molecular impact of different tubulinopathy mutations and how these mutants affect greater tissue level development is critical to advancing our understanding of the role of the microtubule cytoskeleton during neurodevelopment and disease. In this study, we aim to determine the mechanism of two different substitutions at the valine 409 residue of TUBA1A that are associated with different severities of brain malformations. We find that ectopically expressed TUBA1A-V409I/A mutants assemble into microtubules in neuron cultures and dominantly disrupt radial neuron migration, and most significantly in cells expressing TUBA1A-V409A (Figure 1). Our work indicates that the mutation associated with the most severe brain phenotype, TUBA1A-V409A, also has the most severe impact on neurite branching and microtubule dynamics, while the milder -V409I mutant has intermediate phenotypes. These findings provide evidence that the impact of tubulinopathy mutations scale up from altered microtubule polymerization activity and loss of regulation by MAPs to cellular and tissue defects.

As neurons migrate from the ventricular zone to the cortical plate, they must transition through different polarization states (Nadarajah et al., 2001; Noctor et al., 2004; Tabata and Nakajima, 2003). The overexpression or depletion of a wide variety of signaling molecules and cytoskeletal proteins delay or inhibit neuron morphology transitions that impair subsequent migration (reviewed in Cooper, 2014). Several cases of brain malformations similar to those investigated in this study have been linked to mutations that disrupt radial migration by inhibiting the multipolar-to-bipolar transition (Ohshima et al., 2007). While there are numerous factors that are required for proper neuron migration, our data support a hypothesis that TUBA1A-V409I/A mutants disrupt and/or delay proper morphology transitions. At the cellular level, we find that neurons ectopically expressing TUBA1A-V409I and -V409A disrupt neuron morphogenesis by promoting excessive branch stabilization (Figures 1F and 2A–E). We find that TUBA1A-WT and TUBA1A-V409I/A expressing neurons extend and retract neurite branches at similar rates, however the mutant-expressing cells have neurites that retract less often and spend more time in a paused state (Figure 2D–E). In the context of a developing cortex, neurons need to efficiently extend and retract neurites to sample the surrounding space, respond to developmental cues, and ultimately polarize to form a bipolar neuron. Our data suggests that the TUBA1A-V409I/A mutant-expressing cells have a more elaborate yet static morphology, with neurites that spend less time sampling space as compared to WT neurons. Our findings present the question of whether TUBA1A-V409I/A mutants completely fail to migrate to the cortical plate, or whether migration is merely delayed. Future studies addressing this question, potentially by monitoring the migration of neurons at later developmental time points or via in vitro slice cultures, will provide insight into whether neuronal migration is completely impaired or simply delayed. To the best of our knowledge, there are no tubulinopathy mutations to date that disrupt neuron migration via impaired neuron morphology transitions. Therefore, future work will be required to determine whether this is a common mechanism for particular types of brain malformations.

Our findings support the idea that microtubule stability plays a determining role in neuron branching and morphogenesis. Previous studies have identified that taxol-treated neurons have an increase in neurite extension, axon formation, and branching, while nocodazole treatments result in diminished neurite extension and increased retraction (He et al., 2002; Witte et al., 2008). The altered expression of, or mutations in, MAPs also alter neuron morphogenesis (reviewed in Lasser et al., 2018). For example, the MAP SSNA1 promotes axonal branching by mediating microtubule nucleation and branching, further highlighting the role of microtubule dynamics in neuron morphogenesis (Basnet et al., 2018). Neurons must tightly control both microtubule turnover and nucleation to control neurite branching (Basnet et al., 2018; Witte et al., 2008). Both taxol treatment and SSNA1 overexpression result in increased neurite branching, yet each drives this phenotype via separate mechanisms; Taxol suppresses microtubule turnover, whereas overexpression of SSNA1 induces microtubule nucleation and branching. We find that DIV2 neurons ectopically expressing TUBA1A-V409I or -V409A are resistant to cold-induced neurite loss and have increased levels of microtubule acetylation (Figures 2F–H ,–3C). Our analysis of microtubule dynamics in neurons reveals a similar number of polymerizing microtubules, but faster polymerization rates when V409I and V409A is expressed, compared to WT (Figure 3F and G). We also find that the duration of polymerization events is shorter when V409I and V409A is expressed, compared to WT (Figure 3H). Whether these shorter polymerization events are followed by transitions to depolymerization (i.e., catastrophe) or to a stable, non-polymerizing state is unclear. Our data suggest that the V409 mutants do not promote increased nucleation as compared to WT, but rather V409I, and more strongly V409A, increase the content of stable microtubule polymer in neurons. This may be due in part to increased polymerization rates; however, we cannot rule out changes in transition frequencies or depolymerization rate, neither of which are accessible in our dynamics data.

Our results from modeling V409 mutants in budding yeast further reveal the mechanistic origins of the highly stable microtubules in neurons. Similar to our results in neurons we find that, compared to WT, tub1/tub3-V410I/A microtubules have faster polymerization rates (Figure 4D). In yeast we are able to analyze additional microtubule dynamics parameters, and we find that tub1/tub3-V410I/A microtubules also have increased depolymerization rates, decreased catastrophe frequencies, and increased dynamicity (Figure 4E, F,I). Dynamicity is calculated as the total change in microtubule length (taking into account states of growth and shrinkage) divided by the change in time (Jordan et al., 1993). It is interesting that despite the tub1/tub3-V410I/A microtubules undergoing catastrophe less often than WT, they have increased dynamicity values. This suggests that the increase in dynamicity is driven by increased polymerization and depolymerization rates, as opposed to frequent transitions between the states of polymerizing and depolymerizing. Accordingly, V410I/A microtubules reach longer lengths and are longer lived. Assuming these dynamic parameters hold in neurons (as we find the polymerization rates do), longer microtubules that catastrophe less often could also contribute to the increase in microtubule acetylation that we observe in neurons expressing TUBA1A-V409I/A. Our data support a model in which the V409I/A mutants promote excessive neurite branching similar to taxol-treated neurons, but alter microtubule dynamics in a way that is distinct from the mechanisms of taxol.

Microtubule dynamics in cells are reliant on both extrinsic factors that interact with microtubules and intrinsic factors conferred by the tubulin heterodimer (Bodakuntla et al., 2019; Borys et al., 2020; Goodson and Jonasson, 2018; Manka and Moores, 2018; Mitchison and Kirschner, 1984). One way in which tubulin heterodimers intrinsically control microtubule dynamics is by adopting a series of conformations as microtubules polymerize and depolymerize (Chrétien et al., 1995; Mandelkow et al., 1991). Free heterodimer exists in a kinked conformation, which straightens out as the heterodimer is assembled into the microtubule lattice (Buey et al., 2006; Jánosi et al., 1998; Nawrotek et al., 2011; Nogales and Wang, 2006; Ravelli et al., 2004; Rice et al., 2008). XMAP215/Stu2 is thought to play an important role in driving microtubule polymerization by binding free tubulin near microtubule plus ends and facilitating the transition from the kinked, free heterodimer to the straight heterodimer in the microtubule lattice (Figure 7). We find that V410I/A tubulins have weaker affinity for Stu2 TOG1/TOG2 domains in our in vitro binding assays, and that V410I microtubules, and more significantly -V410A microtubules, have less Stu2 at their plus ends in yeast cells (Figure 5C and D). Our finding that V409I, and more so V409A, have decreased XMAP215/Stu2 activity and faster microtubule polymerization rates seem paradoxical.

Figure 7

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Figure 7 depicts two models that could reconcile weak TOG binding with faster polymerization: (1) V410I/A mutant tubulin unbinds faster from TOG domains and increases the concentration of free tubulin available to polymerize into microtubules, and (2) V410I/A alters the intrinsic tubulin conformation to constitutively adopt a straighter, lattice-favoring state. These two models are not mutually exclusive as it is common for MAPs to influence tubulin conformation states and vice versa. However, these two models can be distinguished by their dependence on TOG binding. The first model posits that V410I/A tubulin exhibits faster polymerization than WT tubulin because WT tubulin visits a slow step in the microtubule assembly pathway when it is bound to the TOG domains of XMAP215/Stu2. In the second model, V410I/A tubulin exhibits faster polymerization regardless of whether XMAP215/Stu2 is present. Our finding that V410A increases the rate of tubulin polymerization in the absence of extrinsic MAPs favors the second model and is consistent with an effect on tubulin’s intrinsic conformation (Figure 6C). Additionally, when Stu2 is conditionally depleted in cells, tub1/tub3-V410I/A microtubules are maintained while WT microtubules are lost (Figure 5G). Further experiments are needed to directly test whether V410 mutants limit the range of heterodimer conformations to adopt an intrinsically straighter state. At the cellular level, this model predicts that limiting tubulin conformational states could diminish regulation that is typically conferred by Stu2.

Tight control over microtubule dynamics is essential for cells, and particularly in neuron morphogenesis and migration as the cytoskeletal must nimbly respond to cues throughout the migratory process (Kapitein and Hoogenraad, 2015). XMAP215/Stu2 works in cells as an extrinsic regulator to finely tune microtubule dynamics (Brouhard et al., 2008; Hahn et al., 2021; Lee et al., 2001; van der Vaart et al., 2011; Vasquez et al., 1994; Zanic et al., 2013). XMAP215’s polymerase activity is required for axon outgrowth and microtubule bundling, and in the growth cone XMAP215 plays a role in linking microtubules and F-actin (Hahn et al., 2021; Lowery et al., 2013; Slater et al., 2019). When XMAP215 is depleted in neurons, the number of polymerizing microtubules and the rate of polymerization decrease, and axons are shorter due to slower axon growth rates (Hahn et al., 2021; Lowery et al., 2013). As most of the role of XMAP215 in neurons has been explored primarily in the axon and growth cone, future studies should focus on the role XMAP215 plays in neuron morphogenesis and migration, including in establishing neuron polarity and branching. Beyond XMAP215/Stu2, which preferentially binds to the kinked conformation of tubulin, there are numerous other MAPs that bind to a range of particular tubulin conformations (Brouhard and Rice, 2018). Therefore, we predict that the activity of multiple extrinsic regulators would be impacted by mutations that alter the conformational state of the tubulin heterodimer. How these regulatory mechanisms are impacted by tubulin conformational states and the subsequent effects of these changes on microtubule networks in cells will be a major focus of future studies.

Our results, particularly for the V410A mutant, highlight how tubulin mutants can create unexpected dissonance between extrinsic and intrinsic factors. Compared to WT tubulin, V410A mutant tubulin promotes faster polymerization rates in cells and in vitro (Figures 3G, 4D and 6C). It is interesting that in our in vitro system, we were unable to identify a concentration at which homogeneous V410A polymerizes from the GMPCPP-stabilized porcine tubulin seeds. However, blending V410A with WT tubulin in a one-to-one ratio supports the assembly of microtubules with significantly increased polymerization rates as compared to WT at the same concentrations of total tubulin. This suggests that V410A may be unable to polymerize from GMPCPP seeds on its own, but it drives fast polymerization when mixed with WT tubulin. Based on our data acquired from neuron cultures, and particularly from budding yeast in which all the α-tubulin in the cell is V410A, we know that this mutant assembles into microtubules that polymerize faster and are longer lived in cells. Why then is V410A able to assemble into microtubules when it is the only source of α-tubulin in the cell, but not when it is the only source of α-tubulin in vitro? Beyond V410A, how synergistic interactions between different tubulin heterodimers occur (such as different mutants or isotypes) is an important question to address both in regard to tubulinopathy disease as well as normal tubulin biology. In tubulinopathy patients that harbor heterozygous mutations, the mutant tubulin comprises at most 50% of either the α- or β-tubulin pool. This underscores the necessity of future work to (1) determine the relative protein constituency of different tubulin isotypes in different cell types and (2) use blending experiments in vitro to recapitulate these relative proportions. Additionally, this is an important consideration for furthering our understanding of how normal tubulin biology works in cells. Using mutants that affect particular intrinsic properties of tubulin (e.g., conformation, GTPase activity, etc.) in various blends with WT tubulin will provide insight into how these different properties work in trans to alter microtubule dynamics, and whether some mutants or isotypes might elicit outsized effects.

We propose a model in which the V409I/A mutants are intrinsically altered in a way that perturbs XMAP215/Stu2 regulation and ultimately drive faster microtubule dynamics that perturb neuron morphologies and migration (Figure 7). An isoleucine or alanine substitution at the valine 409 residue of TUBA1A seems relatively insignificant. However, these subtle changes lead to drastic consequences that scale from altered microtubule dynamics to an underdeveloped neocortex. Strikingly, TUBA1A-V409I presents an intermediate phenotype between WT and TUBA1A-V409A in each assay we tested, tracking with the observed brain malformations observed in these patients. To date, there are numerous missense, heterozygous mutations in human tubulin genes that are associated with brain malformations, yet the molecular mechanisms driving these tissue level defects remain unknown. Based off our work and that of others, there does not appear to be a consistent mechanism for how TUBA1A tubulinopathy mutations affect microtubule networks and neuron cell biology (Aiken et al., 2019; Belvindrah et al., 2017; Gartz Hanson et al., 2016; Keays et al., 2007). The well-established conformational dynamics of tubulin raise the possibility that mutations altering one region of the heterodimer could allosterically affect distant regions, and thus disrupt the normal conformational transitions that heterodimers undergo during assembly and disassembly. Therefore, it is crucial to use quality genetic models to further our understanding of the molecular mechanism of these patient-associated mutations. Additionally, our work highlights the need for more detailed studies into how different regions of the tubulin heterodimer impact tubulin conformational states, and how this affects extrinsic regulation that ultimately result in altered microtubule dynamics. Investigating how subtle alterations in specific regions of the heterodimer can have long ranging effects across microtubule networks will be a primary focus of future work.

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided.

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Author details

1. Katelyn J Hoff

   Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2678-1859

2. Jayne E Aiken

   1. Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, United States
   2. Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Mark A Gutierrez

   Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, United States

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Santos J Franco

   Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, United States

   Contribution

   Methodology, Project administration, Resources, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jeffrey K Moore

   Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing - original draft, Writing - review and editing

   For correspondence

   Jeffrey.moore@cuanschutz.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3262-3248

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