Edited by: Fernando Martinez-Garcia, Universidad de Valencia, Spain
Reviewed by: Loreta Medina, Universidad de Lleida, Spain; Isabel Rodríguez-Moldes, University of Santiago de Compostela, Spain
*Correspondence: Nerea Moreno, Faculty of Biology, Department of Cell Biology, University Complutense of Madrid, C/José Antonio Novais 2, Madrid E-28040, Spain. e-mail:
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The expression of the Nkx2.2 gene is involved in the organization of the alar-basal boundary in the forebrain of vertebrates. Its expression in different diencephalic and telencephalic regions, helped to define distinct progenitor domains in mouse and chick. Here we investigated the pattern of Nkx2.2 protein distribution throughout the development of the forebrain of the anuran amphibian,
During the last 10 years our understanding of the organization of the developing forebrain has dramatically changed, in part as a consequence of the impressive number of morphological, chemoarchitectonic, embryological, and, primarily, genoarchitectonic data. In most of the recent studies, the columnar conception of the brain organization (Herrick,
In this scenario, the expressions of key patterning genes involved both in regional and cellular specification processes are currently being analyzed in detail in the main vertebrate models, in order to establish precise traits of the forebrain evolution. Among these developmental regulators is Nkx2.2, a member of the vertebrate homeodomain transcription factor gene family homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene (Kim and Nirenberg,
Most of the data about Nkx2.2 expression in the forebrain were obtained in amniotes, primarily mouse, and chick, and only fragmentary data on Nkx2.2 expression have been reported in anamniotes, especially fishes and amphioxus (Holland et al.,
For the present study, adults, juveniles, and tadpoles of
Adult
A immunohistofluorescence procedure was conducted with the primary antibody on the free-floating sections that, in all cases, was diluted in 5–10% normal serum of the species in which the secondary antibody was raised in PB with 0.1% Triton X-100 (Sigma) and 2% bovine serum albumin (BSA, Sigma).The protocol included two steps, as follows: (1) Incubation for 72 h at 4°C in the dilution of the primary antibody mouse anti-Nkx2.2 (1:500; Developmental Studies Hybridoma Bank, DSHB, Iowa City, USA. Clone: 74.5A5) and (2) the second incubation was conducted for 90 min at room temperature with the labeled secondary antibody Alexa 488-conjugated goat anti-mouse (1:500; Molecular Probes; catalog reference: A21042). After being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA; catalog number: H1000).
The cocktails of primary antibodies were diluted in PB with 0.1% Triton X-100 and used for 60 h at 4°C. They always included mouse anti-Nkx2.2 (1:500; DSHB) in combination with: rabbit anti-Otp (1:1000; produced by “PickCell laboratories” Amsterdam, The Netherlands; according to the protocol described in Lin et al.,
For double histofluorescence labeling experiments, we combined the immunohistochemistry for Nkx2.2 with
For
Hybridization step was done with 3 μl/ml of a DIG-labeled RNA probe, in a 50% formamide containing medium overnight at 55°C. The solution used for prehybridization (at 60°C for 1 h) and hybridization contained 50% deionized formamide (Fluka, Steinheim, Germany), 5× standard saline citrate (Sigma-Aldrich, Steinheim, Germany), 2% blocking reagent (Roche Diagnostics, Mannheim, Germany), 0.1% Tween-20, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich), 1 mg/ml of yeast tRNA (Sigma-Aldrich), 5 mM of ethylenediaminetetraacetic acid (Sigma-Aldrich), and 50 g/ml of heparin (Sigma-Aldrich) in water.
Hybridization was detected using an alkaline phosphatase coupled anti-DIG antibody (Roche Diagnostics, dilution 1:1500). Alkaline phosphatase staining was developed with Fast red tablets (Roche Diagnostics). The
The sections were analyzed with an Olympus BX51 microscope that was equipped for fluorescence with appropriate filter combinations. Selected sections were photographed by using a digital camera (Olympus DP72). Contrast and brightness of the photomicrographs were adjusted in Adobe PhotoShop CS3 (Adobe Systems, San Jose, CA) and figures were mounted in Canvas 11 (ACD Systems, Canada).
The distribution of the Nkx2.2 protein has been analyzed in the prosencephalon of
In
The most anterior expression detected was observed in the hypothalamic territory, helping to the identification of the alar and basal domains (Figures
In order to further characterize the localization of Nkx2.2-ir cells within the forebrain, we carried out double labeling experiments throughout development, using different prosencephalic markers (Figures
To analyze the distribution of the Nkx2.2-ir cells in the alar hypothalamus we combined Nkx2.2 with the transcription factor Orthopedia (Otp; Figures
At SC levels, the double staining for Nkx2.2/MST (Figure
From early premetamorphic stages, the double staining for Nkx2.2/Nkx2.1 (Figure
Caudally in the diencephalon, the banded staining pattern obtained for the pair Nkx2.2/Tbr1 confirmed the lack of Nkx2.2 expressing cells in the prethalamic eminence (PThE), rich in Tbr1 expression (Figure
Finally, Nkx2.2 has been functionally related to the morphogen Shh implied in the alar-basal boundary establishment (for review see Fuccillo et al.,
All the results obtained from the double labeling analysis confirmed the localization of the Nkx2.2-ir cells in the regions described above and summarized in Figure
Nkx2.2 was the first gene of the NK2 homeobox class to be demonstrated in all the deuterostomes, showing homology in all the models analyzed. Even possible homologies were suggested in the nervous system between invertebrates and vertebrates given the high conservations in the expression and function of this gene (Holland et al.,
In general terms, in the vertebrate central nervous system Nkx2.2 has been implicated in the ventral neuronal patterning, at early developmental stages (Briscoe and Ericson,
In all vertebrates analyzed Nkx2.2 is expressed in the prosencephalon where it is known to be involved in the specification of progenitor domains and in establishing regionalization patterns. Moreover, it contributes to the differentiation of distinct areas and their compartmentalization, as well as to the acquisition of cellular identity and the regulation of the distribution of the earliest neurons in the brain (Wilson et al.,
The expression of Nkx2.1 has been deeply analyzed in the prosencephalon of many vertebrates and it has served to identify the preoptic (PO), SC, and tuberal territories (Tub) during development (Rohr et al.,
In mammals, the most anterior Nkx2.2 expression was observed in the medial ganglionic eminence (MGE), also called pallidal domain, which consists of, at least, five progenitor domains globally defined by the strong expression of Nkx2.1, weak expression of Nkx2.2, and lack of Pax6 and Shh-expressions (Flames et al.,
Also in the telencephalon, the PO region in mammals contains at least two distinct progenitor domains (Flames et al.,
The boundary between the telencephalon and the hypothalamus has been recently defined, first in chicken (Bardet et al.,
The hypothalamus constitutes a very complex structure, as regards its functionality, cell complexity, hodology, development, and morphology. Therefore, it is not surprising that the elucidation of its genoarchitecture is particularly important to understand how is generated and maintained the diversity in the hypothalamus. That is one of the reasons that makes fascinating the hypothalamus and, thereby, the focus of analysis in many comparative studies. This has as its counterparts the numerous studies in which the nomenclature and interpretation of the nuclei and boundaries are constantly under debate (Szabó et al.,
On the basis of combined expression analysis, at least two different longitudinal alar domains have been proposed in the chick hypothalamus: the Dlx- and Shh-negative SPV area, which lies under the border of the FoxG1-positive telencephalic field, and the subparaventricular area, which lies under it and is adjacent to the Shh-positive basal plate, and expresses Dlx5 and Nkx2.2 (Bardet et al.,
The SC region is complex in terms of nuclei organization that most likely reflects a complex genetic specification. In general, the SC is defined as a Dlx-expressing zone in all vertebrates analyzed (Bachy et al.,
The SC of
Finally in the BH, like in the rest of the forebrain regions, the elucidation of genetic combinatorial expression patterns has served to characterize and define regions, and their comparative analysis in different vertebrates has been used as a useful tool to establish their grade of conservation. Thus, it was defined that Nkx2.1 expression in the BH is required to maintain molecular characteristics of the developing hypothalamus (Kimura et al.,
In the last 10 years an impressive number of morphological, chemo- and genoarchitectural data has helped to the interpretation in the diencephalon of the boundaries, extent of the areas and identification of distinct nuclei and subnuclei. These data have often been gathered not only for mammals but also in a number of non-mammalian species and the evolutionary perspective shows the impressive degree of conservation of this forebrain region, not only regarding the expression of different genes but also their functions. In all vertebrates analyzed, the alar diencephalon early develops into three major neuroepithelial domains along the anterior–posterior (A/P) axis, known as the prethalamus (P3), the thalamus (P2), and the pretectum (P1), and it is limited by two boundaries, the most anterior one that lies along the supraopto-mammillary region and the posterior one that lies between the pretectum and the mesencephalon (Puelles and Rubenstein,
In mammals, from early embryonic stages Nkx2.2 is expressed in a distinct pattern in all three diencephalic prosomeres (Price et al.,
In the diencephalon of mouse and chicken, the gene Nkx2.2 is expressed during development in a rostroventral band of the thalamus (Puelles and Rubenstein,
In the chick, the combined expression of Gbx2, Nkx2.2, and Pax6 (Martínez-de-la-Torre et al.,
In
Interestingly, also in
As regards the Zli, it expresses Shh in all vertebrates analyzed and is involved in the correct acquisition of the P2 and P3 gene expression and regionalization pattern (Braun et al.,
Finally, the Nkx2.2 expression observed in the thalamus of the mouse and chicken continues into P1 (Puelles and Rubenstein,
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work supported by grants from Spanish MICINN and the UCM (Grant numbers: BFU2009-12315 and BSCH-UCM GR58/08). We are grateful to Dr. Ruth Morona for the fruitful discussions about the diencephalic regionalization and to Dr. Jesús M. López for the critical reading of the manuscript.