Hawkmoth and bee pollinators impact pollen dispersal at the landscape but not local scales in two species of Oenothera.

PREMISE: Animal pollinators play an important role in pollen dispersal. Here, we assessed differences in pollen and seed dispersal and the role of pollinator functional groups with different foraging behaviors in generating patterns of genetic diversity over similar geographic ranges for two closely related taxa. We focused on two members of Oenothera section Calylophus (Onagraceae) that co-occur on gypsum outcrops throughout the northern Chihuahuan Desert but differ in floral phenotype and primary pollinator: Oenothera gayleana (bee) and O. hartwegii subsp. filifolia (hawkmoth). METHODS: We measured breeding system and floral traits and studied gene flow and population differentiation at the local (<13 km; four populations) and landscape (60-440 km; five populations) scales using 10-11 nuclear (pollen dispersal) and three plastid (seed dispersal) microsatellite markers. RESULTS: Both taxa were self-incompatible and floral traits were consistent with expectations for different pollinators. Seed and pollen dispersal patterns were distinctly different for both species. We found no evidence of genetic structure at the local scale but did at the landscape scale; O. gayleana showed greater differentiation and significant isolation by distance than in O. hartwegii subsp. filifolia. The plastid data were consistent with gravity dispersal of seeds and suggest that pollen dispersal is the principal driver of genetic structure in both species. CONCLUSIONS: We demonstrated that pollinator functional groups can impact genetic differentiation in different and predictable ways. Hawkmoths, with larger foraging distances, can maintain gene flow across greater spatial scales than bees.

Epigenetic regulation during human cortical development: Seq-ing answers from the brain to the organoid.

Epigenetic regulation plays an important role in controlling gene expression during complex processes, such as development of the human brain. Mutations in genes encoding chromatin modifying proteins and in the non-protein coding sequences of the genome can potentially alter transcription factor binding or chromatin accessibility. Such mutations can frequently cause neurodevelopmental disorders, therefore understanding how epigenetic regulation shapes brain development is of particular interest. While epigenetic regulation of neural development has been extensively studied in murine models, significant species-specific differences in both the genome sequence and in brain development necessitate human models. However, access to human fetal material is limited and these tissues cannot be grown or experimentally manipulated ex vivo. Therefore, models that recapitulate particular aspects of human fetal brain development, such as the in vitro differentiation of human pluripotent stem cells (hPSCs), are instrumental for studying the epigenetic regulation of human neural development. Here, we examine recent studies that have defined changes in the epigenomic landscape during fetal brain development. We compare these studies with analogous data derived by in vitro differentiation of hPSCs into specific neuronal cell types or as three-dimensional cerebral organoids. Such comparisons can be informative regarding which aspects of fetal brain development are faithfully recapitulated by in vitro differentiation models and provide a foundation for using experimentally tractable in vitro models of human brain development to study neural gene regulation and the basis of its disruption to cause neurodevelopmental disorders.

Geminin is required for Hox gene regulation to pattern the developing limb.

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.

Cellular and molecular characterization of multiplex autism in human induced pluripotent stem cell-derived neurons.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with pronounced heritability in the general population. This is largely attributable to the effects of polygenic susceptibility, with inherited liability exhibiting distinct sex differences in phenotypic expression. Attempts to model ASD in human cellular systems have principally involved rare de novo mutations associated with ASD phenocopies. However, by definition, these models are not representative of polygenic liability, which accounts for the vast share of population-attributable risk. Methods: Here, we performed what is, to our knowledge, the first attempt to model multiplex autism using patient-derived induced pluripotent stem cells (iPSCs) in a family manifesting incremental degrees of phenotypic expression of inherited liability (absent, intermediate, severe). The family members share an inherited variant of uncertain significance (VUS) in GPD2, a gene that was previously associated with developmental disability but here is insufficient by itself to cause ASD. iPSCs from three first-degree relatives and an unrelated control were differentiated into both cortical excitatory (cExN) and cortical inhibitory (cIN) neurons, and cellular phenotyping and transcriptomic analysis were conducted. Results: cExN neurospheres from the two affected individuals were reduced in size, compared to those derived from unaffected related and unrelated individuals. This reduction was, at least in part, due to increased apoptosis of cells from affected individuals upon initiation of cExN neural induction. Likewise, cIN neural progenitor cells from affected individuals exhibited increased apoptosis, compared to both unaffected individuals. Transcriptomic analysis of both cExN and cIN neural progenitor cells revealed distinct molecular signatures associated with affectation, including the misregulation of suites of genes associated with neural development, neuronal function, and behavior, as well as altered expression of ASD risk-associated genes. Conclusions: We have provided evidence of morphological, physiological, and transcriptomic signatures of polygenic liability to ASD from an analysis of cellular models derived from a multiplex autism family. ASD is commonly inherited on the basis of additive genetic liability. Therefore, identifying convergent cellular and molecular phenotypes resulting from polygenic and monogenic susceptibility may provide a critical bridge for determining which of the disparate effects of rare highly deleterious mutations might also apply to common autistic syndromes.

Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development.

Cortical interneurons (cINs) modulate excitatory neuronal activity by providing local inhibition. During fetal development, several cIN subtypes derive from the medial ganglionic eminence (MGE), a transient ventral telencephalic structure. While altered cIN development contributes to neurodevelopmental disorders, the inaccessibility of human fetal brain tissue during development has hampered efforts to define molecular networks controlling this process. Here, we modified protocols for directed differentiation of human embryonic stem cells, obtaining efficient, accelerated production of MGE-like progenitors and MGE-derived cIN subtypes with the expected electrophysiological properties. We defined transcriptome changes accompanying this process and integrated these data with direct transcriptional targets of NKX2-1, a transcription factor controlling MGE specification. This analysis defined NKX2-1-associated genes with enriched expression during MGE specification and cIN differentiation, including known and previously unreported transcription factor targets with likely roles in MGE specification, and other target classes regulating cIN migration and function. NKX2-1-associated peaks were enriched for consensus binding motifs for NKX2-1, LHX, and SOX transcription factors, suggesting roles in coregulating MGE gene expression. Among the NKX2-1 direct target genes with cIN-enriched expression was CHD2, which encodes a chromatin remodeling protein mutated to cause human epilepsies. Accordingly, CHD2 deficiency impaired cIN specification and altered later electrophysiological function, while CHD2 coassociated with NKX2-1 at cis-regulatory elements and was required for their transactivation by NKX2-1 in MGE-like progenitors. This analysis identified several aspects of gene-regulatory networks underlying human MGE specification and suggested mechanisms by which NKX2-1 acts with chromatin remodeling activities to regulate gene expression programs underlying cIN development.

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors.

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Microsatellites for Oenothera gayleana and O. hartwegii subsp. filifolia (Onagraceae), and their utility in section Calylophus.

PREMISE OF THE STUDY: Eleven nuclear and four plastid microsatellite markers were screened for two gypsum endemic species, Oenothera gayleana and O. hartwegii subsp. filifolia, and tested for cross-amplification in the remaining 11 taxa within Oenothera sect. Calylophus (Onagraceae). METHODS AND RESULTS: Microsatellite markers were tested in two to three populations spanning the ranges of both O. gayleana and O. hartwegii subsp. filifolia. The nuclear microsatellite loci consisted of both di- and trinucleotide repeats with one to 17 alleles per population. Several loci showed significant deviation from Hardy-Weinberg equilibrium, which may be evidence of chromosomal rings. The plastid microsatellite markers identified one to seven haplotypes per population. The transferability of these markers was confirmed in all 11 taxa within Oenothera sect. Calylophus. CONCLUSIONS: The microsatellite loci characterized here are the first developed and tested in Oenothera sect. Calylophus. These markers will be used to assess whether pollinator foraging distance influences population genetic parameters in predictable ways.