Bcl11 Transcription Factors Regulate Cortical Development and Function.

Transcription factors regulate multiple processes during brain development and in the adult brain, from brain patterning to differentiation and maturation of highly specialized neurons as well as establishing and maintaining the functional neuronal connectivity. The members of the zinc-finger transcription factor family Bcl11 are mainly expressed in the hematopoietic and central nervous systems regulating the expression of numerous genes involved in a wide range of pathways. In the brain Bcl11 proteins are required to regulate progenitor cell proliferation as well as differentiation, migration, and functional integration of neural cells. Mutations of the human Bcl11 genes lead to anomalies in multiple systems including neurodevelopmental impairments like intellectual disabilities and autism spectrum disorders.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Stability and Function of Hippocampal Mossy Fiber Synapses Depend on Bcl11b/Ctip2.

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.

Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c.

During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.

Ex utero electroporation and organotypic slice culture of mouse hippocampal tissue.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis.

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development.

Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.

The extracellular signal-regulated kinase 3 (mitogen-activated protein kinase 6 [MAPK6])-MAPK-activated protein kinase 5 signaling complex regulates septin function and dendrite morphology.

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 5 (MK5) deficiency is associated with reduced extracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6) levels, hence we utilized the MK5 knockout mouse model to analyze the physiological functions of the ERK3/MK5 signaling module. MK5-deficient mice displayed impaired dendritic spine formation in mouse hippocampal neurons in vivo. We performed large-scale interaction screens to understand the neuronal functions of the ERK3/MK5 pathway and identified septin7 (Sept7) as a novel interacting partner of ERK3. ERK3/MK5/Sept7 form a ternary complex, which can phosphorylate the Sept7 regulators Binders of Rho GTPases (Borgs). In addition, the brain-specific nucleotide exchange factor kalirin-7 (Kal7) was identified as an MK5 interaction partner and substrate protein. In transfected primary neurons, Sept7-dependent dendrite development and spine formation are stimulated by the ERK3/MK5 module. Thus, the regulation of neuronal morphogenesis is proposed as the first physiological function of the ERK3/MK5 signaling module.

Ablation of Sax2 gene expression prevents diet-induced obesity.

Regulation of energy homeostasis is mainly mediated by factors in the hypothalamus and the brainstem. Understanding these regulatory mechanisms is of great clinical relevance in the treatment of obesity and related diseases. The homeobox gene Sax2 is expressed predominantly in the brainstem, in the vicinity of serotonergic neurons, and in the ventral neural tube starting during early development. Previously, we have shown that the loss of function of the Sax2 gene in mouse causes growth retardation starting at birth and a high rate of postnatal lethality, as well as a dramatic metabolic phenotype. To further define the role of Sax2 in energy homeostasis, age-matched adult wild-type, Sax2 heterozygous and null mutant animals were exposed to a high-fat diet. Although food uptake among the different groups was comparable, Sax2 null mutants fed a high-fat diet exhibited a significantly lower weight gain compared to control animals. Unlike their counterparts, Sax2 null mutants did not develop insulin resistance and exhibited significantly lower leptin levels under both standard chow and high-fat diet conditions. Furthermore, neuropeptide Y, an important regulator of energy homeostasis, was significantly decreased in the forebrain of Sax2 null mutants on a high-fat diet. These data strongly suggest a critical role for Sax2 gene expression in diet-induced obesity. Sax2 gene expression may be required to allow the coordinated crosstalk of factors involved in the maintenance of energy homeostasis, possibly regulating the transcription of specific factors involved in energy balance.

Mouse models of Wolf-Hirschhorn syndrome.

Subtelomeric deletion syndromes represent a significant cause of mental retardation and craniofacial disease. However, for most of these syndromes the pathogenic genes have yet to be identified. Currently there is every indication that identification of these genes will be a slow process if we continue to rely strictly upon clinical data. An alternative approach is the use of mouse models to complement the patient studies. Wolf-Hirschhorn syndrome (WHS), caused by deletions in 4p16.3, is the first recognized subtelomeric deletion syndrome. As with other syndromes of this class, WHS has not yet been subjected to an intensive, systematic analysis using mouse models. Nonetheless, a significant number of targeted mutations have been introduced into mouse genomic region, 5B1, which is orthologous to 4p16.3. Included among these mutations are a series of deletions approximating the deletions in some patients. The mouse lines carrying these deletions display a remarkable concordance of phenotypes with the human patient's characteristics, strongly indicating that the mouse models can be used to phenocopy WHS. In this review, we will catalog the currently existing targeted mutations in mice in the regions orthologous to the WHS critical regions. For each mutation we will discuss the resulting phenotype and its potential relevance to the pathogenesis of the syndrome. Further, we will describe how the phenotypes of some of the mutations suggest new directions for the clinical studies. Finally we will outline approaches for the efficient creation of new mouse models of WHS going forward.

Homeobox gene Sax2 deficiency causes an imbalance in energy homeostasis.

The brain, in particular the hypothalamus and the brainstem, plays a critical role in the regulation of energy homeostasis by incorporating signals from the periphery and translating them into feeding behavior. Here we show that the homeobox gene Sax2, which is expressed predominantly in the brainstem, in the vicinity of serotonergic neurons, contributes to this physiological balance. Sax2 deficiency results in a decrease of fat and glycogen storage, reduced blood glucose levels, and raised serotonin levels in the hindbrain. Surprisingly, in the brainstem the expression levels of pro-opiomelanocortin and neuropeptide Y were indicative of a fasting condition, opposed to the observed high serotonin levels implying satiation. Furthermore, Sax2-directed lacZ expression reveals a dramatic change of the distribution of Sax2-expressing cells in the null mutant occurring during perinatal development. These data strongly suggest that Sax2 is required for the coordinated crosstalk of factors involved in the maintenance of energy homeostasis.

Role of Pur alpha in targeting mRNA to sites of translation in hippocampal neuronal dendrites.

Using genetic inactivation in the mouse, PURA, encoding Pur alpha, is demonstrated to be essential for developmentally-timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Pur alpha prompts the hypothesis that Pur alpha functions with non-coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Pur alpha binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Pur alpha binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Pur alpha is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Pur alpha and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Pur alpha dendritic targeting and allow its mislocalization to axons. Using mouse brain, double-RNA immunoprecipitation places Pur alpha together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Pur alpha targets specific mRNA molecules to sites of dendritic translation.

Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction.

Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Here we report that loss of the receptor tyrosine kinase (RTK) antagonist, Sprouty1 (Spry1), causes defects in kidney development in mice. Spry1(-/-) embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. We conclude that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations.

Postnatal lethality in mice lacking the Sax2 homeobox gene homologous to Drosophila S59/slouch: evidence for positive and negative autoregulation.

Homeobox gene transcription factors direct multiple functions during development. They are involved in early patterning of the embryo as well as cell specification, cell differentiation, and organogenesis. Here we describe a previously uncharacterized murine homeobox gene, Sax2, that shows high similarity to the Drosophila S59/slouch and murine Sax1 genes. We show that Sax2 gene expression occurs early during embryogenesis in the midbrain, the midbrain-hindbrain boundary, the ventral neural tube, the developing eye, and the apical ectodermal ridge of the limb. To determine the role of Sax2 during development, we generated a knockout mouse line by replacing part of the Sax2 coding sequences with the lacZ gene. The Sax2 null allele mutants exhibit a strong phenotype indicated by growth retardation starting immediately after birth and leading to premature death within the first 3 weeks postnatal. Intriguingly, our studies also demonstrated a striking autoregulation of the Sax2 gene in both positive- and negative-feedback mechanisms depending on the specific cell type expressing Sax2.