Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections.

The balance of contralateral and ipsilateral retinogeniculate projections is critical for binocular vision, but the transcriptional programs regulating this process remain ill defined. Here we show that the Pou class homeobox protein POU3F1 is expressed in nascent mouse contralateral retinal ganglion cells (cRGCs) but not ipsilateral RGCs (iRGCs). Upon Pou3f1 inactivation, the proportion of cRGCs is reduced in favor of iRGCs, leading to abnormal projection ratios at the optic chiasm. Conversely, misexpression of Pou3f1 in progenitors increases the production of cRGCs. Using CUT&RUN and RNA sequencing in gain- and loss-of-function assays, we demonstrate that POU3F1 regulates expression of several key members of the cRGC gene regulatory network. Finally, we report that POU3F1 is sufficient to induce RGC-like cell production, even in late-stage retinal progenitors of Atoh7 knockout mice. This work uncovers POU3F1 as a regulator of the cRGC transcriptional program, opening possibilities for optic nerve regenerative therapies.

Numb regulates Tau levels and prevents neurodegeneration in tauopathy mouse models.

Accumulation of the microtubule-associated protein Tau is linked to neuronal cell death in tauopathies, but how intraneuronal Tau levels are regulated in health and disease remains unclear. Here, we show that conditional inactivation of the trafficking adaptor protein Numb in retinal ganglion cells (RGCs) increases Tau levels and leads to axonal blebbing, which is followed by neuronal cell loss in aged mice. In the TauP301S mouse model of tauopathy, conditional inactivation of Numb in RGCs and spinal motoneurons accelerates neurodegeneration, and loss of Numb in motoneurons also leads to precocious hindlimb paralysis. Conversely, overexpression of the long isoform of Numb (Numb-72) decreases intracellular Tau levels and reduces axonal blebbing in TauP301S RGCs, leading to improved electrical activity in cultured neurons and improves performance in a visually guided behavior test in vivo. These results uncover Numb as a key regulator of intracellular Tau levels and identify Numb-72 as a potential therapeutic factor for tauopathies.

Mutations in BCOR, a co-repressor of CRX/OTX2, are associated with early-onset retinal degeneration.

Many transcription factors regulating the production, survival, and function of photoreceptor cells have been identified, but little is known about transcriptional co-regulators in retinal health and disease. Here, we show that BCL6 co-repressor (BCOR), a Polycomb repressive complex 1 factor mutated in various cancers, is involved in photoreceptor degenerative diseases. Using proteomics and transcription assays, we report that BCOR interacts with the transcription factors CRX and OTX2 and reduces their ability to activate the promoters of photoreceptor-specific genes. CUT&RUN sequencing further shows that BCOR shares genome-wide binding profiles with CRX/OTX2, consistent with a general co-repression activity. We also identify missense mutations in human BCOR in five families that have no evidence of cancer but present severe early-onset X-linked retinal degeneration. Last, we show that the human BCOR mutants cause degeneration when expressed in the mouse retina and have enhanced repressive activity on OTX2. These results uncover a role for BCOR in photoreceptors in both health and disease.

A Casz1-NuRD complex regulates temporal identity transitions in neural progenitors.

Neural progenitor cells undergo identity transitions during development to ensure the generation different types of neurons and glia in the correct sequence and proportions. A number of temporal identity factors that control these transitions in progenitor competence have been identified, but the molecular mechanisms underlying their function remain unclear. Here, we asked how Casz1, the mammalian orthologue of Drosophila castor, regulates competence during retinal development. We show that Casz1 is required to control the transition between neurogenesis and gliogenesis. Using BioID proteomics, we reveal that Casz1 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in retinal cells. Finally, we show that both the NuRD and the polycomb repressor complexes are required for Casz1 to promote the rod fate and suppress gliogenesis. As additional temporal identity factors have been found to interact with the NuRD complex in other contexts, we propose that these factors might act through this common biochemical process to regulate neurogenesis.

Boc Acts via Numb as a Shh-Dependent Endocytic Platform for Ptch1 Internalization and Shh-Mediated Axon Guidance.

During development, Shh attracts commissural axons toward the floor plate through a non-canonical, transcription-independent signaling pathway that requires the receptor Boc. Here, we find that Shh induces Boc internalization into early endosomes and that endocytosis is required for Shh-mediated growth-cone turning. Numb, an endocytic adaptor, binds to Boc and is required for Boc internalization, Shh-mediated growth-cone turning in vitro, and commissural axon guidance in vivo. Similar to Boc, Ptch1 is also internalized by Shh in a Numb-dependent manner; however, the binding of Shh to Ptch1 alone is not sufficient to induce Ptch1 internalization nor growth-cone turning. Therefore, the binding of Shh to Boc is required for Ptch1 internalization and growth-cone turning. Our data support a model where Boc endocytosis via Numb is required for Ptch1 internalization and Shh signaling in axon guidance. Thus, Boc acts as a Shh-dependent endocytic platform gating Ptch1 internalization and Shh signaling.

Repeatedly washed semen stains: Optimal screening and sampling strategies for DNA analysis.

In many sexual assault cases, bedding and clothing are essential pieces of evidence that are screened for semen stains to gather DNA from the assailant. In some cases, these items have been washed before being seized and sent to the forensic lab. However, few data exist on the optimal methods for detecting and sampling semen stains on washed fabrics. In this paper, we used semen stains washed up to six times to evaluate the efficiency of commonly used screening methods for the detection of semen: alternate light source (ALS), acid phosphatase (AP), prostate specific antigen (PSA) and microscopy (sperm Hy-Liter™, SHL). We also assessed different washing conditions (detergents, washing machines, addition of bleach) and sampling methods (cutting and swabbing). The results show that some semen stain detection strategies, such as ALS, PSA, and SHL, are effective even when the item was washed multiple times. We also show that a complete genetic profile could be obtained from semen stains washed six times. Based on these findings, we present different strategies for the detection and sampling of semen stains depending on the circumstances of the case.

DNA transfer during laundering may yield complete genetic profiles.

In a number of child sexual abuse cases, the alleged perpetrator is a member of the nuclear family. In those cases, there is a possibility that the suspect's DNA was innocently deposited onto the child's clothing without acts of sexual assault ever occurring, for example via secondary transfer within the washing machine. To assess the quantity and quality of DNA that may be transferred among clothing during laundering, we conducted three series of experiments. First, we evaluated the level of spermatozoa that may be transferred by washing pristine pairs of underwear with bed sheets containing a varying number of ejaculates. Secondly, we explored whether current genetic methods may also detect the transfer of DNA from vaginal secretions during a machine wash. Finally, we analyzed the background levels of DNA on children's underwear collected from control families where sexual abuse never occurred. For both spermatozoa and vaginal secretions, we revealed that sufficient amounts of DNA may transfer onto laundered clothing to yield complete genetic profiles. Furthermore, DNA from relatives living within the same household was found in most cuttings taken from control children's underwear. Based on these findings, we present a framework for the handling and interpretation of intrafamilial sexual abuse cases. These suggestions should help determine whether DNA was deposited directly onto a fabric or merely transferred during a wash.

The planar cell polarity protein Vangl2 is required for retinal axon guidance.

Vangl2 plays a critical role in the establishment of planar cell polarity (PCP). Previously, we detected expression of Vangl2 in the developing retina during late embryogenesis, which led us to investigate the possible role of Vangl2-mediated PCP signaling in eye development. We have generated a Vangl2(BGeo) knock-in mouse allowing us to evaluate Vangl2 mRNA expression during retinal development, and used an isoform-specific antibody to examine Vangl2 protein expression in cryosections. To investigate the role of Vangl2 in retinal development, we examined eyes taken from embryos homozygous for independent alleles of Looptail (Lp, Lp(m1jus) ) mutant mice. We found that Vangl2 mRNA and protein are dynamically expressed in the developing embryonic and postnatal retina, with Vangl2 expression becoming progressively restricted to the ganglion cell layer and optic nerve as the retina matures. The expression pattern of Vangl2 transcript and protein is most prominent in retinal ganglion cells (RGC), and their axons. Additionally, we show that Vangl2 is required for retinal and optic nerve development as Vangl2 (Lp/Lp) mutant embryos display a significantly reduced eye size, marked thickening of the retina, and striking abnormalities in the morphology of the optic nerve (significant hypoplasia, and aberrant exit trajectory). Notably, we identified a salient intraretinal axon guidance defect in Vangl2 (Lp/Lp) mutant embryos through which axon bundles traverse the entire thickness of the retina and become trapped within the subretinal space. Our observations identify a new and essential role for Vangl2-dependent PCP signaling in the intraretinal path-finding of RGC axons.

Leading-edge forensic DNA analyses and the necessity of including crime scene investigators, police officers and technicians in a DNA elimination database.

In recent years, sophisticated technology has significantly increased the sensitivity and analytical power of genetic analyses so that very little starting material may now produce viable genetic profiles. This sensitivity however, has also increased the risk of detecting unknown genetic profiles assumed to be that of the perpetrator, yet originate from extraneous sources such as from crime scene workers. These contaminants may mislead investigations, keeping criminal cases active and unresolved for long spans of time. Voluntary submission of DNA samples from crime scene workers is fairly low, therefore we have created a promotional method for our staff elimination database that has resulted in a significant increase in voluntary samples since 2011. Our database enforces privacy safeguards and allows for optional anonymity to all staff members. We also offer information sessions at various police precincts to advise crime scene workers of the importance and success of our staff elimination database. This study, a pioneer in its field, has obtained 327 voluntary submissions from crime scene workers to date, of which 46 individual profiles (14%) have been matched to 58 criminal cases. By implementing our methods and respect for individual privacy, forensic laboratories everywhere may see similar growth and success in explaining unidentified genetic profiles in stagnate criminal cases.

Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia.

A molecular blueprint at the apical surface establishes planar asymmetry in cochlear hair cells.

Sound perception relies on the planar polarization of the mechanosensory hair cell apex, which develops a V-shaped stereocilia bundle pointing toward an eccentric kinocilium. It remains unknown how intrinsically asymmetric bundles arise and are concomitantly oriented in the tissue. We report here that mInsc, LGN, and Gαi proteins, which classically regulate mitotic spindle orientation, are polarized in a lateral microvilli-free region, or "bare zone," at the apical hair cell surface. By creating and extending the bare zone, these proteins trigger a relocalization of the eccentric kinocilium midway toward the cell center. aPKC is restrained medially by mInsc/LGN/Gαi, resulting in compartmentalization of the apical surface that imparts the V-shaped distribution of stereocilia and brings the asymmetric bundle in register with the relocalized kinocilium. Gαi is additionally required for lateral orientation of cochlear hair cells, providing a possible mechanism to couple the emergence of asymmetric stereocilia bundles with planar cell polarity.

Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina.

In the developing nervous system, cell diversification depends on the ability of neural progenitor cells to divide asymmetrically to generate daughter cells that acquire different identities. While much work has recently focused on the mechanisms controlling self-renewing asymmetric divisions producing a differentiating daughter and a progenitor, little is known about mechanisms regulating how distinct differentiating cell types are produced at terminal divisions. Here we study the role of the endocytic adaptor protein Numb in the developing mouse retina. Using clonal numb inactivation in retinal progenitor cells (RPCs), we show that Numb is required for normal cell-cycle progression at early stages, but is dispensable for the production of self-renewing asymmetric cell divisions. At late stages, however, Numb is no longer required for cell-cycle progression, but is critical for the production of terminal asymmetric cell divisions. In the absence of Numb, asymmetric terminal divisions that generate a photoreceptor and a non-photoreceptor cell are decreased in favor of symmetric terminal divisions generating two photoreceptors. Using live imaging in retinal explants, we show that a Numb fusion protein is asymmetrically inherited by the daughter cells of some late RPC divisions. Together with our finding that Numb antagonizes Notch signaling in late-stage RPCs, and that blocking Notch signaling in late RPCs almost completely abolishes the generation of terminal asymmetric divisions, these results suggest a model in which asymmetric inheritance of Numb in sister cells of terminal divisions might create unequal Notch activity, which in turn drives the production of terminal asymmetric divisions.

In vivo evidence for unbiased Ikaros retinal lineages using an Ikaros-Cre mouse line driving clonal recombination.

BACKGROUND: We showed previously that the transcription factor Ikaros is expressed in early but not late retinal progenitors cells (RPCs), and is necessary and sufficient for the production of early-born neurons. Preliminary evidence using retinal explant cultures qualitatively suggested that Ikaros-positive RPCs might share a common lineage with Ikaros-negative RPCs. RESULTS: To explore further this question in vivo in a quantitative manner, we generated BAC transgenic mouse lines expressing Cre recombinase under the regulatory elements of the Ikaros gene, and crossed them with Cre reporter lines. Different transgenic lines labeled a variable number of RPCs, resulting in either dense or sparse radial arrays of reporter-positive progenies. Analysis of over 800 isolated cell arrays, which are most likely clones, confirmed that Ikaros-expressing RPCs generate both early- and late-born cell types in the same lineage, and that the overall cell composition of the arrays closely resembles that of the population of the mature retina. Interestingly, another sparse line did not label arrays, but appeared to specifically reflect Ikaros postmitotic expression in amacrine and ganglion cells. CONCLUSIONS: These mouse lines confirm the unbiased potential of the Ikaros lineage in vivo and provide novel tools for clonal lineage tracing and single neuron tracking in the retina.

Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development.

Semaphorins and Plexins are cognate ligand-receptor families that regulate important steps during nervous system development. The Plexin-B2 receptor is critically involved in neural tube closure and cerebellar granule cell development, however, its specific ligands have only been suggested by in vitro studies. Here, we show by in vivo and in vitro analyses that the two Semaphorin-4 family members Sema4C and Sema4G are likely to be in vivo ligands of Plexin-B2. The Sema4C and Sema4G genes are expressed in the developing cerebellar cortex, and Sema4C and Sema4G proteins specifically bind to Plexin-B2 expressing cerebellar granule cells. To further elucidate their in vivo function, we have generated and analyzed Sema4C and Sema4G knockout mouse mutants. Like Plexin-B2-/- mutants, Sema4C-/- mutants reveal exencephaly and subsequent neonatal lethality with partial penetrance. Sema4C-/- mutants that bypass exencephaly are viable and fertile, but display distinctive defects of the cerebellar granule cell layer, including gaps in rostral lobules, fusions of caudal lobules, and ectopic granule cells in the molecular layer. In addition to neuronal defects, we observed in Sema4C-/- mutants also ventral skin pigmentation defects that are similar to those found in Plexin-B2-/- mutants. The Sema4G gene deletion causes no overt phenotype by itself, but combined deletion of Sema4C and Sema4G revealed an enhanced cerebellar phenotype. However, Sema4C/Sema4G double mutants showed overall less severe cerebellar phenotypes than Plexin-B2-/- mutants, indicating that further ligands of Plexin-B2 exist. In explant cultures of the developing cerebellar cortex, Sema4C promoted migration of cerebellar granule cell precursors in a Plexin-B2-dependent manner, supporting the model that a reduced migration rate of granule cell precursors is the basis for the cerebellar defects of Sema4C-/- and Sema4C/Sema4G mutants.

Ikaros confers early temporal competence to mouse retinal progenitor cells.

In the developing mouse retina, multipotent retinal progenitor cells (RPCs) give rise to specific retinal cell types at different times, but the molecular mechanisms regulating how RPCs change over time remain unclear. In the Drosophila neuroblast lineage, the zinc finger transcription factor Hunchback (Hb) is both necessary and sufficient to specify early-born neuronal identity. We show here that Ikaros, a mouse ortholog of Hb, is expressed in all early embryonic RPCs, which then give rise to Ikaros-negative RPCs at later stages in the lineage. Remarkably, misexpression of Ikaros in late RPCs is sufficient to confer competence to generate early-born neurons. Conversely, Ikaros mutant mice have reduced numbers of early-born cell types, whereas late-born cell types are not affected. These results suggest a model in which Ikaros expression is both necessary and sufficient to confer early temporal competence to RPCs and raise the possibility that a similar strategy might be used to control the sequential order of cell birth in other parts of the nervous system.

The polarity protein Par-3 directly interacts with p75NTR to regulate myelination.

Cell polarity is critical in various cellular processes ranging from cell migration to asymmetric cell division and axon and dendrite specification. Similarly, myelination by Schwann cells is polarized, but the mechanisms involved remain unclear. Here, we show that the polarity protein Par-3 localizes asymmetrically in Schwann cells at the axon-glial junction and that disruption of Par-3 localization, by overexpression and knockdown, inhibits myelination. Additionally, we show that Par-3 directly associates and recruits the p75 neurotrophin receptor to the axon-glial junction, forming a complex necessary for myelination. Together, these results point to a critical role in the establishment of cell polarity for myelination.

Generation and characterization of oligodendrocytes from lineage-selectable embryonic stem cells in vitro.

Oligodendrocytes develop from proliferating oligodendrocyte precursor cells (OPCs), which arise in germinal zones, migrate throughout the developing white matter and divide a limited number of times before they terminally differentiate. Thus far, it has been possible to purify OPCs only from the rat optic nerve, but the purified cells cannot be obtained in large enough numbers for conventional biochemical analyses. Moreover, the central nervous system stem cells that give rise to OPCs have not been purified, limiting the ability to study the earliest stages of commitment to the oligodendrocyte lineage. Pluripotent mouse embryonic stem (ES) cells can be propagated indefinitely in culture and induced to differentiate into various cell types. We describe protocols for culture conditions in which neural precursor cells, OPCs, and oligodendrocytes can be efficiently produced from genetically modified ES cells. This strategy should be useful for study of the intracellular and extracellular factors that direct central nervous system stem cells down the oligodendrocyte pathway and influence subsequent oligodendrocyte differentiation. It may also be useful for producing OPCs and oligodendrocytes from human ES cells for cell therapy and drug screening.

Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes.

A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximately 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.

PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates.

In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. Here we show that a mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. We also show that PTK7 is dynamically localized during hair cell polarization, and that the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates.