Elsevier

Brain Research

Volume 1304, 4 December 2009, Pages 164-186
Brain Research

Research Report
Ongoing expression of Nkx2.1 in the postnatal mouse forebrain: Potential for understanding NKX2.1 haploinsufficiency in humans?

https://doi.org/10.1016/j.brainres.2009.09.050Get rights and content

Abstract

Coordinated movements require the caudate–putamen and the globus pallidus, two nuclei belonging to the basal ganglia, to be intact and functioning properly. Many neurons populating these regions derive from the medial ganglionic eminence, a transient structure that expresses the transcription factor Nkx2.1 during prenatal development. Accordingly, the basal ganglia of Nkx2.1−/− mice are heavily affected and a substantial loss of several types of GABAergic interneurons has been observed. Interestingly, heterozygous mutation of the NKX2.1 gene in humans has been described as causing an unusual disorder from the second year of life onwards, which is mainly characterized by disturbances of motor abilities and delayed speech development. In the present study, we therefore investigated whether Nkx2.1 is still expressed in the young adult and aged mouse forebrain. After birth, the most intense immunolabeling for Nkx2.1 was detected in several components of the hypothalamic region, in the subventricular zone of the ventral tips lining the lateral ventricles, and in neighboring structures including the striatum, the globus pallidus and the various nuclei of the septal complex. Surprisingly, this staining pattern was substantially maintained into adulthood. Double immunocytochemistry for Nkx2.1 and various neuronal markers revealed that mainly parvalbumin-containing GABAergic neurons, but also cholinergic neurons, of the ventral forebrain express this protein. Moreover, in situ hybridization confirmed that these neurons maintain synthesis of Nkx2.1 throughout life. The robust expression of Nkx2.1 by these neurons points to a broad functional spectrum within the adult forebrain.

Introduction

Haploinsufficiency is a rare genetic phenomenon related to semidominant genes. Since these genes only fulfill their normal function in the presence of both wild-type alleles, heterozygous loss of function mutations lead to phenotypic abnormalities (for review, see Nutt and Busslinger, 1999). For instance, haploinsufficiency of the transcription factor NKX2.1 in humans specifically results in dysfunctions of the thyroid gland, the lungs, and the brain (Iwatani et al., 2000, Breedveld et al., 2002a, Breedveld et al., 2002b, Krude et al., 2002, Doyle et al., 2004, Asmus et al., 2005, do Carmo Costa et al., 2005, Moya et al., 2006, Devos et al., 2006, Provenzano et al., 2008; for review, see: Kleiner-Fisman and Lang, 2007). This “brain–thyroid–lung syndrome” emerges in early childhood and displays a variable combination of symptoms including hypothyroidism, respiratory distress, apneic episodes, and choreoathetosis (Krude et al., 2002, Willemsen et al., 2005). The latter is characterized by delayed development of motor abilities, persistent ataxia, dysarthria, muscular hypotonia, hyperextendable knee joints, muscular atrophy of the lower limbs, and rapid choreatic and athetotic movements of the limbs, face and trunk. The severity of the neurological symptoms varies between the patients and mostly depends on the type of mutation affecting the NKX2.1 gene. However, in most cases the symptoms become less severe or even disappear once adulthood is reached. Interestingly, no psychiatric or cognitive abnormalities have been found so far in most of the patients, and only some individuals fail to finish school and/or show mild mental abnormalities (Breedveld et al., 2002a, Willemsen et al., 2005, Moya et al., 2006). Thus, most of the neurological deficits reflect a disturbed development and/or maturation of the motor functions and are most likely associated with malformations of the basal ganglia (Krude et al., 2002, do Carmo Costa et al., 2005).

Nkx2.1 was originally shown to regulate the expression of two thyroid genes, and hence, this protein is also called thyroid transcription factor 1 (TTF1) or thyroid enhancer binding protein (T/EBP; Civitareale et al., 1989, Mizuno et al., 1991). It belongs to the homeobox gene family, and is translated into a protein closely related to members of the Drosophila NK-2 homeodomain protein family (Guazzi et al., 1990). During development of vertebrates, Nkx2.1 is expressed in the Anlagen of the thyroid gland and lungs but also in certain forebrain regions (Lazzaro et al., 1991, Price et al., 1992). In the mouse brain, Nkx2.1 mRNA was first detected at embryonic stage E8.75 (1-somite-stage according to Shimamura et al., 1995). At E10 strong mRNA expression was observed in the hypothalamic primordium, the medial ganglionic eminence (MGE), and ventral parts of the immature telencephalon which give rise to the septal complex, the anterior entopeduncular region and the preoptic area (Sussel et al., 1999, Flames et al., 2007, Garcia-Lopez et al., 2008). Cells produced in each domain (or their smaller subdivisions) migrate either radially to the mantle zone or tangentially to adjacent and distant regions (Marin et al., 2000, Wichterle et al., 2001). The development of the large subcortical compartments formed mainly by projection neurons is thus a result of radial migration. Other precursors deriving from the same proliferative zones tangentially migrate and integrate in these compartments. At their final positions, these cells differentiate into the various types of interneurons which, in general, represent a minor subpopulation. For instance, the MGE gives rise to most of the GABAergic projection neurons in the globus pallidus but also the majority of interneurons in the neighboring caudate–putamen.

The importance of Nkx2.1 for the correct development of these neuronal populations has been demonstrated by the severe phenotype of the Nkx2.1 null mouse. Homozygous Nkx2.1−/− mutants develop only a rudimentary thyroid gland and die directly after birth due to agenesis of the lungs (Kimura et al., 1996). However, severe morphological alterations were already found in the prenatal brain: the globus pallidus is missing while the striatum appears to be enlarged. In addition, certain nuclei of the hypothalamic area are heavily affected or completely missing (Kimura et al., 1996). At cellular level, immunohistochemical analysis of prenatal mutants at E18.5 revealed a 40% reduction in the number of glutamate decarboxylase 67 (Gad67)-positive interneurons in the cerebral cortex. In line with this, neuronal cell numbers for hippocampal GABAergic interneurons coexpressing neuropeptide-Y (NPY), calbindin (CB), or somatostatin (SOM) were also found to be significantly lower at E18.5 (Pleasure et al., 2000). Moreover, in the mutant striatum a subtotal loss of interneurons was observed. Choline acetyltransferase (ChAT)-, parvalbumin (PV)-, SOM-, NPY-, nitric oxide synthase (NOS)-, and calretinin (CR)-positive neurons are either severely reduced or totally absent (Marin et al., 2000). In addition, cholinergic basal forebrain neurons expressing the high affinity nerve growth factor receptor trkA were not detectable (Sussel et al., 1999).

Several studies have shown that the expression of Nkx2.1 extends into the postnatal life of rodents. As demonstrated for mice, Nkx2.1 is not only necessary for the maintenance of the normal architecture and function of the differentiated thyroid gland (Kusakabe et al., 2006), but also for the hypothalamic control of puberty and reproductive function (Mastronardi et al., 2006). Moreover, several reports have indicated that Nkx2.1 synthesis is also maintained in other basal forebrain regions (Marin et al., 2000, Lee et al., 2001, Nakamura et al., 2001, Kim et al., 2006, Xu et al., 2008). However, these studies have only been performed for up to 3 weeks after birth, and they can therefore not explain the sensitivity of motor functions to NKX2.1 haploinsufficiency in humans. In fact, both the delay in speech development and the persisting defects in movement coordination suggest that long-lasting expression of NKX2.1 in the basal ganglia is needed for the sufficient maturation of these abilities. Since a detailed characterization of subcortical Nkx2.1-expressing neurons has not been performed yet for the adult and aged mouse, we used immunocytochemistry and in situ hybridization to identify Nkx2.1-positive cells in the basal forebrain. Here we show that many neurons of both the basal ganglia and septal complex, but also several neighboring regions located more ventrally, maintain synthesis of Nkx2.1 throughout life. In addition, Nkx2.1-expression is not only maintained by subcortical GABAergic interneurons but also by GABAergic and cholinergic projection neurons of several basal forebrain nuclei. These so-called “cholinergic centers” play, for instance, an important role for learning and memory processes (Dutar et al., 1995, Heimer et al., 1997, Berger-Sweeney, 2003, Mufson et al., 2003).

Section snippets

Results

Neurochemical characterization of neurons in rodents reveals remarkable differences not only between rats and mice but also between strains of the same species (e.g., Tunnicliff et al., 1973, Albanese et al., 1985, Naumann et al., 2002, Naumann et al., 2003). We therefore investigated two different mouse lines widely used as laboratory animals: CD1 and BL6 mice. Although the number of Nkx2.1-positive cell profiles in the basal forebrain of CD1 and BL6 mice varied to some extent between P0 and

Discussion

Major divisions of the mammalian subpallium are known to be involved in the regulation of various brain functions such as motor behavior, learning and memory, motivation and hormone homeostasis, and are severely affected in psychiatric and neurological human disorders. In most cases, however, the underlying morphological alterations are not fully understood. Motor functions, for instance, are known to depend on correct neuronal transmission within the basal ganglia (for review, see Gerfen, 2004

Animals and tissue preparation

For the present study postnatal CD1 and C57BL/6 (BL6) mice of either sex were used. In detail, the following age groups were selected for immunocyotchemistry (IC) and in situ hybridization (ISH): P0 (CD1: n = 6; BL6: n = 6), P2 (CD1: n = 4; BL6: n = 3), P5 (CD1: n = 8; BL6: n = 5), P8 (CD1: n = 3; BL6: n = 2), P10 (CD1: n = 5; BL6: n = 5), P11 (CD1: n = 2; BL6: n = 2), P15 (CD1: n = 6; BL6: n = 6), P20 (CD1: n = 2; BL6: n = 2), 4–6 weeks (“young adult mice”; CD1: n = 12; BL6: n = 12), 2–4 months (“adult mice”; CD1: n = 9; BL6: n = 

Acknowledgments

The authors gratefully acknowledge Gudrun Thomascheck for technical assistance.

This work was funded by the German Research Foundation (SFB Berlin 665/A 3).

References (117)

  • DoyleD.A. et al.

    Autosomal dominant transmission of congenital hypothyroidism, neonatal respiratory distress, and ataxia caused by a mutation of NKX2-1

    J. Pediatr.

    (2004)
  • ErlanderM.G. et al.

    Two genes encode distinct glutamate decarboxylases

    Neuron

    (1991)
  • FreundT.F.

    GABAergic septohippocampal neurons contain parvalbumin

    Brain Res.

    (1989)
  • Gonzalo-RuizA. et al.

    Immunohistochemical localization of GABA in the mammillary complex of the rat

    Neuroscience

    (1993)
  • Gonzalo-RuizA. et al.

    Immunohistochemical studies of localization and co-localization of glutamate, aspartate and GABA in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat

    J. Chem. Neuroanat.

    (1996)
  • HeimerL. et al.

    Substantia innominata: a notion which impedes clinical-anatomical correlations in neuropsychiatric disorders

    Neuroscience

    (1997)
  • IwataniN. et al.

    Deletion of NKX2.1 gene encoding thyroid transcription factor-1 in two siblings with hypothyroidism and respiratory failure

    J. Pediatr.

    (2000)
  • KimM.S. et al.

    Regulation of pituitary adenylate cyclase-activating polypeptide gene transcription by TTF-1, a homeodomain-containing transcription factor

    J. Biol. Chem.

    (2002)
  • KimJ.G. et al.

    TTF-1, a homeodomain-containing transcription factor, regulates feeding behavior in the rat hypothalamus

    Biochem. Biophys. Res. Commun.

    (2006)
  • KimJ.G. et al.

    Thyroid transcription factor-1 facilitates cerebrospinal fluid formation by regulating aquaporin-1 synthesis in the brain

    J. Biol. Chem.

    (2007)
  • KitaH.

    Parvalbumin-immunopositive neurons in rat globus pallidus: a light and electron microscopic study

    Brain Res.

    (1994)
  • LeeB.J. et al.

    TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neuroendocrine brain in a developmentally regulated and cell-specific fashion

    Mol. Cell. Neurosci.

    (2001)
  • LledoP.M. et al.

    Origin and function of olfactory bulb interneuron diversity

    Trends Neurosci.

    (2008)
  • MeldgaardM. et al.

    Validation of two reference genes for mRNA level studies of murine disease models in neurobiology

    J. Neurosci. Methods

    (2006)
  • MesulamM.M. et al.

    Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6)

    Neuroscience

    (1983)
  • MufsonE.J. et al.

    Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction

    J. Chem. Neuroanat.

    (2003)
  • NakamuraK. et al.

    Immunohistochemical analyses of thyroid-specific enhancer-binding protein in the fetal and adult rat hypothalami and pituitary glands

    Brain Res. Dev. Brain Res.

    (2001)
  • Nobrega-PereiraS. et al.

    Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors

    Neuron

    (2008)
  • PleasureS.J. et al.

    Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons

    Neuron

    (2000)
  • PriceM. et al.

    Regional expression of the homeobox gene Nkx-2.2 in the developing mammalian forebrain

    Neuron

    (1992)
  • ProvenzanoC. et al.

    Functional characterization of a novel mutation in TITF-1 in a patient with benign hereditary chorea

    J. Neurol. Sci.

    (2008)
  • ReinerA. et al.

    Structural and functional evolution of the basal ganglia in vertebrates

    Brain Res. Brain Res. Rev.

    (1998)
  • RiedelA. et al.

    Principles of rat subcortical forebrain organization: a study using histological techniques and multiple fluorescence labeling

    J. Chem. Neuroanat.

    (2002)
  • RisoldP.Y. et al.

    Chemoarchitecture of the rat lateral septal nucleus

    Brain Res. Brain Res. Rev.

    (1997)
  • RisoldP.Y. et al.

    Connections of the rat lateral septal complex

    Brain Res. Brain Res. Rev.

    (1997)
  • SimerlyR.B.

    Anatomical Substrates of Hypothalamic Integration

  • SofroniewM.V. et al.

    The cholinergic nuclei of the basal forebrain of the rat: normal structure, development and experimentally induced degeneration

    Brain Res.

    (1987)
  • SonY.J. et al.

    TTF-1, a homeodomain-containing transcription factor, participates in the control of body fluid homeostasis by regulating angiotensinogen gene transcription in the rat subfornical organ

    J. Biol. Chem.

    (2003)
  • AdamsJ.C.

    Heavy metal intensification of DAB-based HRP reaction product

    J. Histochem. Cytochem.

    (1981)
  • AllenG.V. et al.

    Mamillary body in the rat: a cytoarchitectonic, Golgi, and ultrastructural study

    J. Comp. Neurol.

    (1988)
  • AlonsoJ.R. et al.

    Hippocampo-septal fibers terminate on identified spiny neurons in the lateral septum: a combined Golgi/electron-microscopic and degeneration study in the rat

    Cell Tissue Res.

    (1989)
  • AlonsoJ.R. et al.

    Organization of the septal region in the rat brain: a Golgi/EM study of lateral septal neurons

    J. Comp. Neurol.

    (1989)
  • AmaralD.G. et al.

    An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat

    J. Comp. Neurol.

    (1985)
  • AndyO.J. et al.

    Septum Development in Primates

    (1976)
  • AsmusF. et al.

    A novel TITF-1 mutation causes benign hereditary chorea with response to levodopa

    Neurology

    (2005)
  • BaimbridgeK.J. et al.

    Calcium binding protein and parvalbumin in the mammillary nuclei in the rat. Abstracts of papers presented at the ninety-ninth meeting

    Anat. Rec.

    (1986)
  • BayerS.A.

    The development of the septal region in the rat: I. Neurogenesis examined with 3H-thymidine autoradiography

    J. Comp. Neurol.

    (1979)
  • BenderR. et al.

    Development of cholinergic and GABAergic neurons in the rat medial septum: different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression

    J. Comp. Neurol.

    (1996)
  • BolamJ.P. et al.

    Glutamate decarboxylase-immunoreactive structures in the rat neostriatum: a correlated light and electron microscopic study including a combination of Golgi impregnation with immunocytochemistry

    J. Comp. Neurol.

    (1985)
  • BreedveldG.J. et al.

    Clinical and genetic heterogeneity in benign hereditary chorea

    Neurology

    (2002)
  • Cited by (24)

    • NKX2-1 Is Required in the Embryonic Septum for Cholinergic System Development, Learning, and Memory

      2017, Cell Reports
      Citation Excerpt :

      At postnatal day (P)30, numerous p75NTR-labeled neurons co-expressed the tomato reporter, confirming that these originated from neuroepithelial cells of the septum that expressed Zic4-CreERT2 at embryonic stages (Figure 1E). Previous work had shown that nearly all cholinergic projection neurons of the basal forebrain and interneurons of the striatum are generated from NKX2-1-expressing precursors (Fragkouli et al., 2009; Magno et al., 2009, 2011; Marin et al., 2000). To identify possible septal-derived cholinergic neurons outside the septum, we examined Zic4-Cre;R26R-YFP transgenic mice.

    • Developmental specification of forebrain cholinergic neurons

      2017, Developmental Biology
      Citation Excerpt :

      Nkx2.1 plays a crucial role in specifying the progenitors of GABAergic and cholinergic neurons (Butt et al., 2008; Sussel et al., 1999). Nearly all ventral forebrain cholinergic neurons arise from an Nkx2.1+ progenitor and maintain its expression into adulthood (Fragkouli et al., 2009; Magno et al., 2011, 2009; Marin et al., 2000; Xu et al., 2008). This distinguishes them from cortical GABAergic interneurons, which rapidly downregulate Nkx2.1 expression as they migrate tangentially into the cortex.

    • Distinct developmental origins manifest in the specialized encoding of movement by adult neurons of the external globus pallidus

      2015, Neuron
      Citation Excerpt :

      Using unbiased, stereological cell counting in adult wild-type mice (see Figure S1), we determined that PPE-expressing (PPE+) GPe neurons also expressed the transcription factor forkhead box protein P2 (FoxP2), and vice versa, and that PPE+/FoxP2+ arkypallidal neurons constitute 20% of all GPe neurons (Figures 1A–1C). The majority of GPe neurons in the rodent and human brain express a different transcription factor, NK2 homeobox 1 (Nkx2-1), and most Nkx2-1+ GPe neurons also express PV (Flandin et al., 2010; Magno et al., 2009, 2011; Nóbrega-Pereira et al., 2010). As such, we reasoned that Nkx2-1 expression in the GPe might be selective for prototypic neurons, irrespective of PV expression.

    • Hypoperfusion in caudate nuclei in patients with brain-lung-thyroid syndrome

      2012, Journal of the Neurological Sciences
      Citation Excerpt :

      We analyzed our patients and found a common, significant reduction in cerebral blood flow in the caudate nuclei. Although most reports describe expression of the NKX2-1 gene in the pallidum [8,21,22], a recent study showed NKX2-1 expression in the postnatal mouse striatum, including the caudate nuclei, in addition to the pallidum [29]. In humans, nuclear image studies indicated a reduction in blood flow [23] and glucose metabolism [15] in the basal ganglia.

    • A Resource of Cre Driver Lines for Genetic Targeting of GABAergic Neurons in Cerebral Cortex

      2011, Neuron
      Citation Excerpt :

      Consistent with previous studies (Miyoshi et al., 2007), E12 induction gave rise to cortical GABA neurons expressing parvalbumin (PV), somatostatin (SST), but not vasoactive intestinal peptide (VIP) (Figures 2J–2L). Recent studies demonstrate that Nkx2.1 expression continues beyond mid-gestation and persists in the ventral ridge of SVZ during late embryonic and postnatal ages (Marin et al., 2000; Magno et al., 2009). Indeed, we found Nkx2.1-Cre activity in ventral SVZ beyond E17 (Figures 2E–2G), when the characteristic eminence of MGE had already fused with the adjacent LGE.

    View all citing articles on Scopus
    View full text