Scott E. Williams

Ephrin-B2 and EphB1 Mediate Retinal Axon Divergence at the Optic Chiasm

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Here, we show that ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2.

Asymmetric cell divisions promote Notch-dependent epidermal differentiation

Stem and progenitor cells use asymmetric cell divisions to balance proliferation and differentiation. Evidence from invertebrates shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: Par3–Par6–aPKC, which confer polarity, and Gαi–LGN/AGS3–NuMA–dynein/dynactin, which govern spindle positioning. Here we focus on developing mouse skin, where progenitor cells execute a switch from symmetric to predominantly asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNA interference, we investigate spindle orientation regulation and provide direct evidence that LGN (also called Gpsm2), NuMA and dynactin (Dctn1) are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNA interference in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.

Retinal Ganglion Cell Axon Guidance in the Mouse Optic Chiasm: Expression and Function of Robos and Slits

The ventral midline of the nervous system is an important choice point at which growing axons decide whether to cross and project contralaterally or remain on the same side of the brain. InDrosophila, the decision to cross or avoid the CNS midline is controlled, at least in part, by the Roundabout (Robo) receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of these molecules have been cloned and have also been implicated in regulating axon guidance. Using in situ hybridization, we have determined the expression patterns of robo1,2and slit1,2,3 in the mouse retina and in the region of the developing optic chiasm, a ventral midline structure in which retinal ganglion cell (RGC) axons diverge to either side of the brain. The receptors and ligands are expressed at the appropriate time and place, in both the retina and the ventral diencephalon, to be able to influence RGC axon guidance. In vitro,slit2 is inhibitory to RGC axons, with outgrowth of both ipsilaterally and contralaterally projecting axons being strongly affected. Overall, these results indicate that Robos and Slits alone do not directly control RGC axon divergence at the optic chiasm and may additionally function as a general inhibitory guidance system involved in determining the relative position of the optic chiasm at the ventral midline of the developing hypothalamus.

Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase

The haematopoietic protein tyrosine phosphatase (HePTP) is a negative regulator of the MAP kinases Erk1, Erk2 and p38. HePTP binds to these kinases through a kinase-interaction motif (KIM) in its non-catalytic amino terminus and inactivates them by dephosphorylating the critical phosphorylated tyrosine residue in their activation loop. Here we show that cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine residue 23 in the KIM of HePTP in vitro and in intact cells. This modification reduces binding of MAP kinases to the KIM, an effect that is prevented by mutation of serine 23 to alanine. The PKA-mediated release of MAP kinase from HePTP is sufficient to activate the kinase and to induce transcription from the c-fos promoter. Expression of a HePTP serine-23-to-alanine mutant inhibits MAP-kinase dissociation and activation and induction of transcription from the c-fos promoter. We conclude that HePTP not only controls the activity of MAP kinases, but also mediates crosstalk between the cAMP system and the MAP-kinase cascade.

Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos

Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using α1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

Loss of p120 catenin and links to mitotic alterations, inflammation, and skin cancer

Tumor formation involves epigenetic modifications and microenvironmental changes as well as cumulative genetic alterations encompassing somatic mutations, loss of heterozygosity, and aneuploidy. Here, we show that conditional targeting of p120 catenin in mice leads to progressive development of skin neoplasias associated with intrinsic NF-κB activation. We find that, similarly, squamous cell carcinomas in humans display altered p120 and activated NF-κB. We show that epidermal hyperproliferation arising from p120 loss can be abrogated by IκB kinase 2 inhibitors. Although this underscores the importance of this pathway, the role of NF-κB in hyperproliferation appears rooted in its impact on epidermal microenvironment because as p120-null keratinocytes display a growth-arrested phenotype in culture. We trace this to a mitotic defect, resulting in unstable, binucleated cells in vitro and in vivo. We show that the abnormal mitoses can be ameliorated by inhibiting RhoA, the activity of which is abnormally high. Conversely, we can elicit such mitotic defects in control keratinocytes by elevating RhoA activity. The ability of p120 deficiency to elicit mitotic alterations and chronic inflammatory responses, that together may facilitate the development of genetic instability in vivo, provides insights into why it figures so prominently in skin cancer progression.

Mena and Vasodilator-Stimulated Phosphoprotein Are Required for Multiple Actin-Dependent Processes That Shape the Vertebrate Nervous System

Ena/vasodilator-stimulated phosphoprotein (VASP) proteins regulate the geometry of the actin cytoskeleton, thereby influencing cell morphology and motility. Analysis of invertebrate mutants implicates Ena/VASP function in several actin-dependent processes such as axon and dendritic guidance, cell migration, and dorsal closure. In vertebrates, genetic analysis of Ena/VASP function is hindered by the broad and overlapping expression of the three highly related family members Mena (Mammalian enabled), VASP, and EVL (Ena-VASP like). Mice deficient in either Mena or VASP exhibit subtle defects in forebrain commissure formation and platelet aggregation, respectively. In this study, we investigated the consequence of deleting both Mena and VASP. Mena-/-VASP-/- double mutants die perinatally and display defects in neurulation, craniofacial structures, and the formation of several fiber tracts in the CNS and peripheral nervous system.

Foxd1 is required for proper formation of the optic chiasm.

In animals with binocular vision, retinal ganglion cell (RGC) axons from each eye sort in the developing ventral diencephalon to project to ipsi- or contralateral targets, thereby forming the optic chiasm. Ipsilaterally projecting axons arise from the ventrotemporal (VT) retina and contralaterally projecting axons primarily from the other retinal quadrants. The winged helix transcription factor Foxd1 (previously known as BF-2, Brain Factor 2) is expressed in VT retina, as well as in the ventral diencephalon during the formation of the optic chiasm. We report here that in embryos lacking Foxd1, both retinal development and chiasm morphogenesis are disrupted. In the Foxd1 deficient retina, proteins designating the ipsilateral projection, such as Zic2 and EphB1, are missing, and the domain of Foxg1 (BF-1) expands from nasal retina into the VT crescent. In retina-chiasm co-cultures, VT RGCs from Foxd1 deficient retina are not repulsed by chiasm cells, and in vivo many VT RGCs aberrantly project contralaterally. However, even though the ipsilateral program is lost in the retina, a larger than normal uncrossed component develops in Foxd1 deficient embryos. Chiasm defects include axon stalling in the chiasm and a reduction in the total number of RGCs projecting to the optic tract. In addition, in the Foxd1 deficient ventral diencephalon, Foxg1 invades the Foxd1 domain, Zic2 and Islet1 expression are minimized, and Slit2 prematurely expands, changes that could contribute to axon projection errors. Thus, Foxd1 plays a dual role in the establishment of the binocular visual pathways: first, in specification of the VT retina, acting upstream of proteins directing the ipsilateral pathway; and second, in the patterning of the developing ventral diencephalon where the optic chiasm forms.

The optic chiasm as a midline choice point

The mouse optic chiasm is a model for axon guidance at the midline and for analyzing how binocular vision is patterned. Recent work has identified several molecular players that influence the binary decision that retinal ganglion cells make at the optic chiasm, to either cross or avoid the midline. An ephrin-B localized to the midline, together with an EphB receptor and a zinc-finger transcription factor expressed exclusively in the ventrotemporal retina where ipsilaterally projecting retinal ganglion cells are located, comprise a molecular program for the uncrossed pathway. In addition, the mechanisms for axon divergence in the optic chiasm are discussed in the context of other popular models for midline axon guidance.

A Role for Nr-CAM in the Patterning of Binocular Visual Pathways

Retinal ganglion cell (RGC) axons diverge within the optic chiasm to project to opposite sides of the brain. In mouse, contralateral RGCs are distributed throughout the retina, whereas ipsilateral RGCs are restricted to the ventrotemporal crescent (VTC). While repulsive guidance mechanisms play a major role in the formation of the ipsilateral projection, little is known about the contribution of growth-promoting interactions to the formation of binocular visual projections. Here, we show that the cell adhesion molecule Nr-CAM is expressed by RGCs that project contralaterally and is critical for the guidance of late-born RGCs within the VTC. Blocking Nr-CAM function causes an increase in the size of the ipsilateral projection and reduces neurite outgrowth on chiasm cells in an age- and region-specific manner. Finally, we demonstrate that EphB1/ephrin-B2-mediated repulsion and Nr-CAM-mediated attraction comprise distinct molecular programs that each contributes to the proper formation of binocular visual pathways.