Distinct roles of Tfap2A & Tfap2B in the diversification of GABAergic neuronal subtypes in the cerebellum
Date of Issue2017
School of Biological Sciences
The cerebellum plays a critical role in sensory-motor integration and is important for precise coordination of body, limb and eye movements as well as adaptation and learning of motor skills. The ability of the cerebellum to carry out these tasks relies on the faithful production and assembly of various classes of cerebellar neurons into functional circuits during development. The distinct subtypes of inhibitory neurons are important in providing feedforward and feedback inhibition to the cerebellar circuit. To explore the molecular mechanisms underlying the development of cerebellar inhibitory neurons, we examined the involvement of Tfap2A and Tfap2B in the specification of cerebellar progenitors into distinct inhibitory neuronal subtypes in mice. We show, for the first time, that Tfap2A and Tfap2B are expressed in distinct subsets of cerebellar inhibitory neurons during embryonic development, and that their expression persists into adulthood. The developmental expression pattern of Tfap2A and Tfap2B suggests that these transcription factors might play a role in the specification of GABAergic neuronal subtypes in the developing cerebellum and may continue to regulate the maintenance and refinement of inhibitory circuits in the mature cerebellar cortex. We carried out in utero electroporation and in vivo lentiviral transduction to investigate consequences of manipulating the expression of these transcription factors on specification of GABAergic neuronal subtypes. The identification and characterisation of genes unique to each neuronal subtype is crucial for a better understanding of the function of individual subtypes. To identify genes that are expressed by specific neuronal subtypes, we examined a database generated by using the Ribo-Tag methodology and microarray technology to profile major cerebellar neuronal types. Analysis of this unpublished microarray database revealed that Tfap2A and Tfap2B are exclusively expressed by GABAergic neurons, but not glutamatergic neurons in the cerebellum. We confirmed this specific expression pattern using in situ hybridisation and immunohistochemistry and showed that Tfap2A is expressed in Purkinje projection neurons and Golgi, stellate and basket interneurons while Tfap2B is only expressed in Golgi, stellate and basket interneurons. Unlike transcription factors that are transiently expressed during development, Tfap2A and Tfap2B are expressed early during embryogenesis and remain expressed in adulthood. In order to define the expression of Tfap2A and Tfap2B in different neuronal subtypes during distinct developmental stages, we carried out co-localisation studies using known molecular markers for each neuronal subtype. We observed that both transcription factors are expressed as early as embryonic day (E) 12.5, the onset of cerebellar cortical neuronal production. In addition, Tfap2A+ and Tfap2B+ cells express molecular markers for early GABAergic precursors such as Ptf1a and Olig2 as well as post-mitotic markers such as RORα and Pax2. These results suggest that Tfap2A and Tfap2B may serve as determinants for the specification of GABAergic neuronal subtypes and prompted us to assess the function of these transcription factors in the developing and mature cerebellum. Since Tfap2A and Tfap2B are expressed exclusively in GABAergic neuronal population in the cerebellum, we tested whether manipulation of the expression of these transcription factors individually could alter the molecular phenotypes of these neurons. In utero electroporation and lentiviral transduction were performed to study the consequences of over/misexpression or knockdown of Tfap2A and Tfap2B in the developing and mature cerebellum. Genetic manipulation during early embryogenesis showed that Tfap2B is essential for the specification of Pax2+-+ interneurons and in preventing the specification of Pax6+-+ excitatory neurons. Tfap2A, on the other hand, is capable of inducing Pax2+-+ interneurons when misexpressed in precursor/progenitor neurons. Additionally, we investigated whether Ptf1a, a key determinant of cerebellar GABAergic neuron identity, controls the expression of Tfap2A and Tfap2B. Our results indicate that the expression of Tfap2A and Tfap2B is not dependent on Ptf1a expression and, thus, may represent an independent program in the specification of cerebellar GABAergic neuron diversification. In postnatal experiments, we assessed GABAergic molecular markers such as glutamic acid decarboxylase 67 (Gad67), the enzyme responsible for inhibitory neurotransmitter production. Misexpression studies in vitro using primary cerebellar neuronal culture revealed that neither Tfap2A nor Tfap2B was capable of transforming excitatory neurons into GABAergic inhibitory neurons. In summary, we show that the expression of Tfap2A and Tfap2B molecularly defines GABAergic neuronal subtypes in the cerebellum and, perhaps, other regions of the nervous system and represent an important set of markers for the labelling and monitoring of the development of GABAergic neuronal subpopulations. In addition, we established the role of these transcription factors in the diversification of GABAergic neuronal subtype during embryogenesis.
DRNTU::Science::Biological sciences::Molecular biology