Michael Piper

The transcription factor Engrailed-2 guides retinal axons

Engrailed-2 (En-2), a homeodomain transcription factor, is expressed in a caudal-to-rostral gradient in the developing midbrain, where it has an instructive role in patterning the optic tectum—the target of topographic retinal input. In addition to its well-known role in regulating gene expression through its DNA-binding domain, En-2 may also have a role in cell–cell communication, as suggested by the presence of other domains involved in nuclear export, secretion and internalization. Consistent with this possibility, here we report that an external gradient of En-2 protein strongly repels growth cones of Xenopus axons originating from the temporal retina and, conversely, attracts nasal axons. Fluorescently tagged En-2 accumulates inside growth cones within minutes of exposure, and a mutant form of the protein that cannot enter cells fails to elicit axon turning. Once internalized, En-2 stimulates the rapid phosphorylation of proteins involved in translation initiation and triggers the local synthesis of new proteins. Furthermore, the turning responses of both nasal and temporal growth cones in the presence of En-2 are blocked by inhibitors of protein synthesis. The differential guidance of nasal and temporal axons reported here suggests that En-2 may participate directly in topographic map formation in the vertebrate visual system.

Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones.

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation.

It has been proposed that growth cones navigating through gradients adapt to baseline concentrations of guidance cues. This adaptation process is poorly understood. Using the collapse assay, we show that adaptation in Xenopus laevis retinal growth cones to the guidance cues Sema3A or netrin-1 involves two processes: a fast, ligand-specific desensitization that occurs within 2 min of exposure and is dependent on endocytosis, and a slower, ligand-specific resensitization, which occurs within 5 min and is dependent upon protein synthesis. These two phases of adaptation allow retinal axons to adjust their range of sensitivity to specific guidance cues.

The transcription factor Nfix is essential for normal brain development

BackgroundThe Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nfic-deficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice.ResultsHere we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6- and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals.ConclusionThese data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects.

NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1.

The balance between self-renewal and differentiation of neural progenitor cells is an absolute requirement for the correct formation of the nervous system. Much is known about both the pathways involved in progenitor cell self-renewal, such as Notch signaling, and the expression of genes that initiate progenitor differentiation. However, whether these fundamental processes are mechanistically linked, and specifically how repression of progenitor self-renewal pathways occurs, is poorly understood. Nuclear factor I A (Nfia), a gene known to regulate spinal cord and neocortical development, has recently been implicated as acting downstream of Notch to initiate the expression of astrocyte-specific genes within the cortex. Here we demonstrate that, in addition to activating the expression of astrocyte-specific genes, Nfia also downregulates the activity of the Notch signaling pathway via repression of the key Notch effector Hes1. These data provide a significant conceptual advance in our understanding of neural progenitor differentiation, revealing that a single transcription factor can control both the activation of differentiation genes and the repression of the self-renewal genes, thereby acting as a pivotal regulator of the balance between progenitor and differentiated cell states.

A Molecular Mechanism for the Heparan Sulfate Dependence of Slit-Robo Signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Expression of the vertebrate Slit Gene family and their putative receptors, the Robo genes, in the developing murine kidney

The slit (sli) gene, encoding a secreted glycoprotein, has been demonstrated to play a vital role in axonal guidance in Drosophila melanogaster by acting as a signalling ligand for the robo receptor (Rothberg, J.M., Jacobs, J.R., Goodman, C.S., Artavanis-Tsakonas, S., 1990. slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4, 2169-2187; Kidd, T., Bland, K.S., Goodman, C. S., 1999. Slit is the midline repellent for the robo receptor in Drosophila. Cell 96, 785-794). Multiple homologs of both sli and robo have been identified in vertebrates and are thought to play similar roles to their fly counterparts in neural development (Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., Tessier-Lavigne, M., Kidd, T., 1999. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795-806). Slit2 has been shown to bind Robo1, mediating both neuronal and axonal guidance in the developing central nervous system (CNS), (Brose et al., 1999; Hu, H., 1999. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703-711). Importantly, both gene families display distinct expression patterns outside the CNS (Holmes, G.P., Negus, K., Burridge, L., Raman, S., Algar, E., Yamada, T., Little, M.H., 1998. Distinct but overlapping expression patterns of two vertebrate slit homologs implies functional roles in CNS development and organogenesis. Mech. Dev. 79, 57-72; Yuan, W., Zhou, L., Chen, J.H., Wu, J.Y., Rao, Y., Ornitz, D.M., 1999. The mouse SLIT family: secreted ligands for ROBO expressed in patterns that suggest a role in morphogenesis and axon guidance. Dev. Biol. 212, 290-306). Using in situ hybridization on metanephric explant cultures and urogenital tract sections, the expression patterns of Slit1, 2, 3 and Robo1 and 2 were investigated during murine metanephric development. Slit1 was expressed in the metanephric mesenchyme (MM) surrounding the invading ureteric tree (UT). Slit2 was expressed at the tips of the UT and both Slit2 and Slit3 were expressed at the far proximal end of the comma shaped and S-shaped bodies. Expression of Robo1 was initially diffuse throughout the MM, then upregulated in the pretubular aggregates, and maintained at the distal end of the comma and S-shaped bodies. Robo2 was detected in the induced MM surrounding the arborizing UT tips and later in the proximal end of the S-shaped bodies. Coincident expression of Robo1 with Slit1 in the metanephric mesenchyme and Robo2, Slit2 and Slit3 in the far proximal end of the S-shaped bodies was observed during metanephric development.

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of co-expression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface.

Neuropilin 1-Sema Signaling Regulates Crossing of Cingulate Pioneering Axons during Development of the Corpus Callosum

Pioneer axons from the cingulate cortex initiate corpus callosum (CC) development, yet nothing is known about the molecules that regulate their guidance. We demonstrate that neuropilin 1 (Npn1) plays an integral role in the development of the CC. Npn1 is localized to axons of cingulate neurons as they cross the midline, and multiple class 3 semaphorins (Semas) are expressed around the developing CC, implicating these guidance molecules in the regulation of Npn1-expressing axons emanating from the cingulate cortex. Furthermore, axons from the cingulate cortex display guidance errors in Npn1(Sema-) mice, a knockin mouse line in which Npn1 is unable to bind Semas. Analysis of mice deficient in the transcription factor Emx2 demonstrated that the cingulate cortex of these mice was significantly reduced in comparison to wild-type controls at E17 and that the CC was absent in rostral sections. Expression of Npn1 was absent in rostral sections of Emx2 mutants, suggesting that Npn1-expressing cingulate pioneers are required for CC formation. These data highlight a central role for Npn1 in the development of projections from the cingulate cortex and further illustrate the importance of these pioneer axons in the formation of the CC.

Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

The majority of neural stem cells (NSCs) in the adult brain are quiescent, and this fraction increases with aging. Although signaling pathways that promote NSC quiescence have been identified, the transcriptional mechanisms involved are mostly unknown, largely due to lack of a cell culture model. In this study, we first demonstrate that NSC cultures (NS cells) exposed to BMP4 acquire cellular and transcriptional characteristics of quiescent cells. We then use epigenomic profiling to identify enhancers associated with the quiescent NS cell state. Motif enrichment analysis of these enhancers predicts a major role for the nuclear factor one (NFI) family in the gene regulatory network controlling NS cell quiescence. Interestingly, we found that the family member NFIX is robustly induced when NS cells enter quiescence. Using genome-wide location analysis and overexpression and silencing experiments, we demonstrate that NFIX has a major role in the induction of quiescence in cultured NSCs. Transcript profiling of NS cells overexpressing or silenced for Nfix and the phenotypic analysis of the hippocampus of Nfix mutant mice suggest that NFIX controls the quiescent state by regulating the interactions of NSCs with their microenvironment.