Anthony-Samuel LaMantia

Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain

We have used an in vitro assay to identify sources of retinoic acid (RA) and transgenic mice to identify target domains in the developing forebrain. RA participates in a sequence of events that leads to the establishment of the olfactory pathway. First, the lateral cranial mesoderm activates an RA-inducible transgene in neuroepithelial cells in the olfactory placode and the ventrolateral forebrain. Then, neurons and neurites begin to differentiate in these two regions. Finally, olfactory axons grow specifically into the ventrolateral forebrain and subsequently are limited to the olfactory bulb rudiment. The coordination of these events, perhaps by common signals, implies that retinoid induction and retinoid-activated region-specific transcriptional regulation may help to define a forebrain subdivision and the peripheral neurons that provide its primary innervation.

Diminished dosage of 22q11 genes disrupts neurogenesis and cortical development in a mouse model of 22q11 deletion/DiGeorge syndrome

The 22q11 deletion (or DiGeorge) syndrome (22q11DS), the result of a 1.5- to 3-megabase hemizygous deletion on human chromosome 22, results in dramatically increased susceptibility for “diseases of cortical connectivity” thought to arise during development, including schizophrenia and autism. We show that diminished dosage of the genes deleted in the 1.5-megabase 22q11 minimal critical deleted region in a mouse model of 22q11DS specifically compromises neurogenesis and subsequent differentiation in the cerebral cortex. Proliferation of basal, but not apical, progenitors is disrupted, and subsequently, the frequency of layer 2/3, but not layer 5/6, projection neurons is altered. This change is paralleled by aberrant distribution of parvalbumin-labeled interneurons in upper and lower cortical layers. Deletion of Tbx1 or Prodh (22q11 genes independently associated with 22q11DS phenotypes) does not similarly disrupt basal progenitors. However, expression analysis implicates additional 22q11 genes that are selectively expressed in cortical precursors. Thus, diminished 22q11 gene dosage disrupts cortical neurogenesis and interneuron migration. Such developmental disruption may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.

Disruption of local retinoid-mediated gene expression accompanies abnormal development in the mammalian olfactory pathway

We have evaluated the role of retinoid signaling in the early development of the olfactory epithelium and olfactory bulb. When retinoid‐mediated gene expression is blocked briefly in mouse embryos at midgestation with citral (a general alcohol dehydrogenase antagonist that is thought to interfere with retinoid synthesis), the spectrum of morphogenetic abnormalities includes disruption of olfactory pathway development. It is difficult, however, to assess the specificity of this pharmacological manipulation, insofar as it also compromises several other aspects of central nervous system development. In homozygous Pax6 mutant mice (small eye: Pax6Sey‐Neu), there is a more discrete lesion to the olfactory pathway: The epithelium and bulb cannot be recognized at any time during development, whereas other forebrain subdivisions can still be recognized. This loss of the entire primary olfactory pathway is accompanied by a failure of retinoid‐mediated gene expression limited to the frontonasal region and forebrain. Retinoid receptors are expressed in the forebrain of Pax6Sey‐Neu/Pax6Sey‐Neu embryos, and the mutant forebrain remains responsive to exogenous retinoic acid. However, in Pax6Sey‐Neu/Pax6Sey‐Neu embryos, retinoic acid (RA) is not produced by the frontonasal mesenchyme, which normally provides local retinoid signals to the placode and forebrain. Together, these results suggest that local retinoid signaling is essential for the normal development of the mammalian olfactory pathway. J. Comp. Neurol. 379:171–184, 1997.

Differential adhesion and the initial assembly of the mammalian olfactory nerve

During the initial assembly of the olfactory pathway, the behavior of olfactory axons changes as they grow from the olfactory epithelium toward the telencephalic vesicle. The axons exit the epithelium singly or in small fascicles, and their growth cones are simple and bullet‐shaped. Outside the epithelium, they make a sharp dorsal turn and fasciculate into a single nerve; the growth cones remain simple. Upon entering the ventromedial telencephalon, the axons defasciculate, branch extensively, and end in complex, lamellate growth cones which extend toward the ventrolateral aspect of the telencephalic vesicle. The distribution of laminin, collagen‐IV, and fibronectin varies in register with these changes in olfactory axon and growth cone behavior. Each of these extracellular matrix molecules influences olfactory neurite outgrowth and growth cone morphology in vitro consistent with its distribution in vivo. The distribution of E‐cadherin, L1, neural cell adhesion molecule (NCAM) and the polysialated form of NCAM also varies in register with changes in olfactory axon behavior. In vitro, L1 modulates embryonic olfactory neurite outgrowth and growth cone morphology consistent with its distribution in vivo. Thus, olfactory axon trajectory, fasciculation, and growth cone morphology change within distinct adhesive environments in the nascent olfactory pathway, and some of the molecules that characterize these environments have differential effects upon olfactory neurite growth and growth cone morphology. Consequently, the patterned expression and activity of extracellular matrix and cell surface adhesion molecules may contribute to the initial assembly of the olfactory pathway.

Retinoic Acid Signaling Identifies a Distinct Precursor Population in the Developing and Adult Forebrain

We asked whether retinoic acid (RA), an established transcriptional regulator in regenerating and developing tissues, acts directly on distinct cell classes in the mature or embryonic forebrain. We identified a subset of slowly dividing precursors in the adult subventricular zone (SVZ) that is transcriptionally activated by RA. Most of these cells express glial fibrillary acidic protein, a smaller subset expresses the epidermal growth factor receptor, a few are terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling positive, and they can be mitotically labeled by sustained rather than acute bromodeoxyuridine exposure. RA activation in similar cells in SVZ-derived neurospheres depends on retinoid synthesis from the premetabolite retinol. The apparent influence of RA on precursors in vitro is consistent with key properties of RA activation in the SVZ; in neurospheres, altered retinoid signaling elicits neither cell death nor an acute increase in cell proliferation. There is apparent continuity of RA signaling in the forebrain throughout life. RA-activated, proliferative precursors with radial glial characteristics are found in the dorsal lateral ganglionic eminence and ventrolateral palliumembryonic rudiments of the SVZ. Thus, endogenous RA signaling distinguishes subsets of neural precursors with glial characteristics in a consistent region of the adult and developing forebrain.

Proliferative and transcriptional identity of distinct classes of neural precursors in the mammalian olfactory epithelium

Neural precursors in the developing olfactory epithelium (OE) give rise to three major neuronal classes – olfactory receptor (ORNs), vomeronasal (VRNs) and gonadotropin releasing hormone (GnRH) neurons. Nevertheless, the molecular and proliferative identities of these precursors are largely unknown. We characterized two precursor classes in the olfactory epithelium (OE) shortly after it becomes a distinct tissue at midgestation in the mouse: slowly dividing self-renewing precursors that express Meis1/2 at high levels, and rapidly dividing neurogenic precursors that express high levels of Sox2 and Ascl1. Precursors expressing high levels of Meis genes primarily reside in the lateral OE, whereas precursors expressing high levels of Sox2 and Ascl1 primarily reside in the medial OE. Fgf8 maintains these expression signatures and proliferative identities. Using electroporation in the wild-type embryonic OE in vitro as well as Fgf8, Sox2 and Ascl1 mutant mice in vivo, we found that Sox2 dose and Meis1 – independent of Pbx co-factors – regulate Ascl1 expression and the transition from lateral to medial precursor state. Thus, we have identified proliferative characteristics and a dose-dependent transcriptional network that define distinct OE precursors: medial precursors that are most probably transit amplifying neurogenic progenitors for ORNs, VRNs and GnRH neurons, and lateral precursors that include multi-potent self-renewing OE neural stem cells.

RETINOID SIGNALING DISTINGUISHES A SUBPOPULATION OF OLFACTORY RECEPTOR NEURONS IN THE DEVELOPING AND ADULT MOUSE

We asked whether retinoic acid (RA) influences olfactory receptor neurons (ORNs) in the developing and mature mouse olfactory epithelium (oe). The distribution of retinoid receptors and binding proteins in the oe changes between embryonic days 11.5 and 13.5, the period when ORNs first differentiate and send axons into the nascent olfactory nerve. Coincident with this change, RA, which is produced in the frontonasal mesenchyme at these ages, begins to activate gene expression in a bilaterally symmetric subset of ORNs in the dorsolateral oe, as judged by the expression of an RA‐responsive transgene. Axons from these RA‐activated ORNs are segregated in the olfactory nerve as it extends through the frontonasal mesenchyme toward the forebrain. In vitro, RA potentiates ORN neurite growth on laminin, which, in the embryo, is found in a stripe of frontonasal mesenchyme directly associated with the olfactory nerve. RA does not modify growth on fibronectin, type IV collagen, or L1, which olfactory axons encounter in different regions of the territory between the olfactory epithelium and the brain. The pattern of RA‐mediated transcriptional activation and axon segregation persists in early postnatal mice, and RA signaling can be recognized in a subset of adult ORNs in the dorsolateral oe. Thus, RA‐mediated gene expression distinguishes a subpopulation of ORNs in a distinct region of the oe during the early development of the olfactory pathway, and may influence differentiation and axonal projections of ORNs in this region throughout life. J. Comp. Neurol. 394:445–461, 1998.

P59FYN AND PP60C-SRC MODULATE AXONAL GUIDANCE IN THE DEVELOPING MOUSE OLFACTORY PATHWAY

The Src-family tyrosine kinases p59fyn and pp60c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn gens and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than than src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fever fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59fyn and pp60c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance.

Molecular Specification and Patterning of Progenitor Cells in the Lateral and Medial Ganglionic Eminences

We characterized intrinsic and extrinsic specification of progenitors in the lateral and medial ganglionic eminences (LGE and MGE). We identified seven genes whose expression is enriched or restricted in either the LGE [biregional cell adhesion molecule-related/downregulated by oncogenes binding protein (Boc), Frizzled homolog 8 (Fzd8), Ankrd43 (ankyrin repeat domain-containing protein 43), and Ikzf1 (Ikaros family zinc finger 1)] or MGE [Map3k12 binding inhibitory protein 1 (Mbip); zinc-finger, SWIM domain containing 5 (Zswim5); and Adamts5 [a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5]]. Boc, Fzd8, Mbip, and Zswim5 are apparently expressed in LGE or MGE progenitors, whereas the remaining three are seen in the postmitotic mantle zone. Relative expression levels are altered and regional distinctions are lost for each gene in LGE or MGE cells propagated as neurospheres, indicating that these newly identified molecular characteristics of LGE or MGE progenitors depend on forebrain signals not available in the neurosphere assay. Analyses of Pax6Sey/Sey, Shh−/−, and Gli3XtJ/XtJ mutants suggests that LGE and MGE progenitor identity does not rely exclusively on previously established forebrain-intrinsic patterning mechanisms. Among a limited number of additional potential patterning mechanisms, we found that extrinsic signals from the frontonasal mesenchyme are essential for Shh- and Fgf8-dependent regulation of LGE and MGE genes. Thus, extrinsic and intrinsic forebrain patterning mechanisms cooperate to establish LGE and MGE progenitor identity, and presumably their capacities to generate distinct classes of neuronal progeny.

Age-Dependent Retinoic Acid Regulation of Gene Expression Distinguishes the Cervical, Thoracic, Lumbar, and Sacral Spinal Cord Regions during Development

We evaluated whether differences in the availability of retinoic acid (RA) establish distinct patterns of RA-dependent gene expresion in the embryonic mouse thoracic/sacral versus cervical/lumbar spinal cord regions. Exogenous RA elicits ectopic expression of an RA-activated transgene and the RA receptor beta in the dorsal thoracic and sacral cord in mice at embryonic day (E) 12.5, but not E14.5. This age-dependent regulation is cell autonomous and is not accompanied by changes in expression patterns of several retinoid receptors, binding proteins, or the SMRT nuclear corepressor. Instead, this change apparently reflects the loss of endogenous RA in the dorsal thoracic and sacral cord between E12.5 and E14.5. Thus, chronic exposure to exogenous RA between E11.5 and E13.5 restores ectopic RA-mediated gene expression. These observations suggest that the local availability of RA establishes absolute differences in gene expression that distinguish the thoracic and sacral cord from the cervical and lumbar cord during midgestation.