Ryan B. MacDonald

An SNP in an ultraconserved regulatory element affects Dlx5/Dlx6 regulation in the forebrain

Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of γ-aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons. Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy, schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade.

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. We find that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI>0 switch is triggered by activation of Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS.

The relationship between dlx and gad1 expression indicates highly conserved genetic pathways in the zebrafish forebrain

The Dlx genes encode a family of transcription factors important for the development of the vertebrate forebrain. These genes have very similar expression domains during the development of the telencephalon in mice and play a role in γ‐aminobutyric acid (GABAergic) interneuron differentiation. We have used triple fluorescent in situ hybridization to study the relative expression domains of the dlx and gad1 genes in the zebrafish telencephalon and diencephalon. We also generated transgenic zebrafish with regulatory elements from the zebrafish dlx1a/2a locus. The zebrafish dlx regulatory elements recapitulated dlx expression in the forebrain and mimicked the relationship between the expression of the dlx genes and gad1. Finally, we show that a putative enhancer located downstream of dlx2b can also activate reporter gene expression in a tissue‐specific manner similar to endogenous dlx2b expression. Our results indicate the dlx genes are regulated by an evolutionarily conserved genetic pathway and may play a role in GABAergic interneuron differentiation in the zebrafish forebrain. Developmental Dynamics 239:2298–2306, 2010.

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.

Cellular Requirements for Building a Retinal Neuropil

Summary How synaptic neuropil is formed within the CNS is poorly understood. The retinal inner plexiform layer (IPL) is positioned between the cell bodies of amacrine cells (ACs) and retinal ganglion cells (RGCs). It consists of bipolar cell (BC) axon terminals that synapse on the dendrites of ACs and RGCs intermingled with projections from Müller glia (MG). We examined whether any of these cellular processes are specifically required for the formation of the IPL. Using genetic and pharmacological strategies, we eliminated RGCs, ACs, and MG individually or in combination. Even in the absence of all of these partner cells, an IPL-like neuropil consisting of only BC axon terminals still forms, complete with presynaptic specializations and sublaminar organization. Previous studies have shown that an IPL can form in the complete absence of BCs; therefore, we conclude that neither presynaptic nor postsynaptic processes are individually essential for the formation of this synaptic neuropil.

Müller glia provide essential tensile strength to the developing retina.

When the formation of Müller glia is inhibited in the zebrafish retina, a major consequence is that the retina begins to rip apart due to a loss of the mechanical resilience that these glial cells provide to the neural tissue.

Reconciling competence and transcriptional hierarchies with stochasticity in retinal lineages.

Highlights • Problems with a strict retinal competence model are explained.• The apparent conflict between transcriptional hierarchies and stochasticity is resolved.• The underlying nature of retinal progenitor cell stochasticity is discussed.• Key issues that can be addressed in the face of stochasticity are enumerated.

Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon.

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.

Activity of dlx5a/dlx6a regulatory elements during zebrafish GABAergic neuron development.

During vertebrate forebrain formation, Dlx homeobox genes play essential roles in the differentiation, migration and survival of subpallial precursor cells that will later give rise to diverse subtypes of γ‐aminobutyric acid (GABA)‐expressing neurons, including inhibitory cortical interneurons in mammals. They also participate in the regulation of the Gad genes encoding the enzymes necessary for GABA synthesis. In mice, at least four cis‐regulatory elements (CREs) control Dlx expression in the telencephalon and diencephalon: URE2 and I12b in the Dlx1/Dlx2 bigene cluster, and I56i and I56ii in the Dlx5/Dlx6 bigene cluster. However, little is known so far with respect to the function of orthologous dlx genes and their regulatory elements during zebrafish GABAergic neuron development. To investigate whether similar dlx‐mediated pathways exist in the early developing zebrafish forebrain, we generated independent lines of transgenic zebrafish carrying two distinct GFP reporter constructs driven by a β‐globin minimal promoter: one containing a ∼1.4 kb dlx5a/dlx6a intergenic sequence (encompassing I56i and I56ii) and one with a ∼1.1 kb fragment containing only the I56i CRE, respectively. The expression patterns of these two transgenes were compared with that obtained with another construct containing the ∼1.4 kb dlx5a/dlx6a intergenic sequence and driven by a ∼3.5 kb dlx6a 5′‐flanking fragment. Our comparative analysis showed that GFP expression of the three transgene is largely overlapping throughout the ventral forebrain. Intriguingly, the dlx6a 5′‐flanking fragment has a major impact on transgene expression in the mesencephalic tectum. Furthermore, comparison of transgene expression between the ∼1.4 kb and ∼1.1 kb intergenic fragments did not show any specific spatial expression conferred by I56ii. Almost all GFP‐expressing cells in the transgenic zebrafish are GABA‐positive and also express various GABAergic interneuron markers. Together, our data suggest that zebrafish dlx5a/dlx6a intergenic CREs may be involved in a conserved genetic pathway necessary for proper dlx expression during zebrafish GABAergic neuron development.