Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions
© Alvarez-Bolado et al.; licensee BioMed Central Ltd 2012
Received: 13 November 2011
Accepted: 20 January 2012
Published: 20 January 2012
The hypothalamus is a brain region with essential functions for homeostasis and energy metabolism, and alterations of its development can contribute to pathological conditions in the adult, like hypertension, diabetes or obesity. However, due to the anatomical complexity of the hypothalamus, its development is not well understood. Sonic hedgehog (Shh) is a key developmental regulator gene expressed in a dynamic pattern in hypothalamic progenitor cells. To obtain insight into hypothalamic organization, we used genetic inducible fate mapping (GIFM) to map the lineages derived from Shh- expressing progenitor domains onto the four rostrocaudally arranged hypothalamic regions: preoptic, anterior, tuberal and mammillary.
Shh- expressing progenitors labeled at an early stage (before embryonic day (E)9.5) contribute neurons and astrocytes to a large caudal area including the mammillary and posterior tuberal regions as well as tanycytes (specialized median eminence glia). Progenitors labeled at later stages (after E9.5) give rise to neurons and astrocytes of the entire tuberal region and in particular the ventromedial nucleus, but not to cells in the mammillary region and median eminence. At this stage, an additional Shh-expressing domain appears in the preoptic area and contributes mostly astrocytes to the hypothalamus. Shh- expressing progenitors do not contribute to the anterior region at any stage. Finally, we show a gradual shift from neurogenesis to gliogenesis, so that progenitors expressing Shh after E12.5 generate almost exclusively hypothalamic astrocytes.
We define a fate map of the hypothalamus, based on the dynamic expression of Shh in the hypothalamic progenitor zones. We provide evidence that the large neurogenic Shh- expressing progenitor domains of the ventral diencephalon are continuous with those of the midbrain. We demonstrate that the four classical transverse zones of the hypothalamus have clearly defined progenitor domains and that there is little or no cell mixing between the tuberal and anterior or the preoptic and anterior hypothalamus. Finally, we show that, in the tuberal hypothalamus, neurons destined for every mediolateral level are produced during a period of days, in conflict with the current 'three-wave' model of hypothalamic neurogenesis. Our work sets the stage for a deeper developmental analysis of this complex and important brain region.
The hypothalamus is essential for the maintenance of homeostasis and the survival of the individual and the species because of its central role in the regulation of eating, drinking, reproductive and parental behavior, as well as sleep-wake rhythms. It fulfills these functions by controlling the autonomic nervous system and hormone secretion; in addition, the hypothalamus integrates input from all other brain regions and the peripheral nervous system [1, 2]. The hypothalamus consists of a wide variety of neuronal clusters with distinct connectivity and function, classically subdivided along the rostral-caudal axis into four regions - preoptic, anterior, tuberal and mammillary - and along the medial-lateral axis into periventricular, medial and lateral zones .
The hypothalamus develops from the rostral diencephalon after induction by the underlying prechordal plate. The secreted morphogen Sonic hedgehog (Shh), initially expressed in the prechordal plate, is essential for this inductive process. Shh null mutant mice lack all hypothalamic structures [4, 5] and Hedgehog protein can induce hypothalamic tissue in forebrain explants [6, 7]. After induction, the presumptive hypothalamus is patterned along the three spatial axes in order to define distinct hypothalamic areas. Shh, which is expressed in a dynamic pattern in the developing ventral hypothalamus, plays a role in these patterning events as well [8, 9]. A recent study in which Shh was specifically inactivated in the neuroepithelium, but not in the prechordal plate, demonstrated that neural Shh plays a role in ventrodorsal and rostrocaudal patterning and in establishing lateral hypothalamic neurons but does not seem to be required for regional hypothalamic specification and eyefield separation, for which non-neural Shh signaling from the prechordal plate appears to be sufficient .
Although numerous studies explore the functional and anatomical complexity of the hypothalamus, relatively little is known about how the diverse array of hypothalamic cell types is generated and organized during development. Even though some of the molecules regulating hypothalamic development have been identified, it remains unclear how these signals are integrated to direct the specification of the large number of distinct hypothalamic cell types. Moreover, the relationship between precursor domains defined by the expression of specific markers and the cell types in the mature hypothalamus remains largely undefined , except for those neuroepithelial regions expressing Pou3 transcription factors . Genetic defects in the induction and specification of distinct hypothalamic cell types might result in metabolic or homeostatic disorders and there is evidence that this is the case in congenital obesity [13–15]. Identifying the developmental origin and lineage relationships of hypothalamic neurons is the first and essential step in shedding light on common pathological processes such as hypertension, diabetes and obesity.
Here, in order to establish a detailed fate map of the hypothalamus, we analyzed the fate of cells derived from Shh- expressing hypothalamic progenitors using genetic inducible fate mapping (GIFM) to permanently mark cells at different embryonic stages (embryonic day (E)7.5 to E12.5) . We show that, before E9.5, Shh- expressing progenitors are primarily located in the ventral midline of the posterior hypothalamic primordium and give rise to neurons and astrocytes throughout the mammillary and posterior tuberal regions, but also to tanycytes, a specialized glial cell type in the median eminence of the tuberal region. After E9.5, Shh- expressing progenitors are still found in the posterior hypothalamic anlage, but become progressively rarer in the most posterior area and ventral midline. This lateral progenitor domain only contributes few cells to the mammillary region and median eminence. However, this domain gives rise to neurons and astrocytes of the tuberal region, in particular the ventromedial nucleus, indicating that the tuberal hypothalamus is largely derived from lateral progenitors. In addition, a progenitor domain appears in the presumptive preoptic region whose contribution to the hypothalamus consists mostly of preoptic astrocytes. At any labeling stage, we observed very few Shh- expressing progenitors in the area between the posterior hypothalamic primordium and the presumptive preoptic region and accordingly only few fate-mapped cells in the anterior hypothalamus in the adult brain. At late labeling time points there is a shift from neurogenesis to gliogenesis, so that by E12.5, Shh- expressing progenitors give rise almost exclusively to hypothalamic astrocytes. Finally, cells derived from progenitors that express Shh at late stages (E10.5 and E11.5) contribute neurons to all mediolateral zones of the tuberal hypothalamus, indicating that the current 'three-wave model', according to which late-born hypothalamic neurons remain in the medial hypothalamus, might have to be revised .
Sonic hedgehog expression in the ventral diencephalon is dynamic
Fate mapping strategy to follow the fate of progenitors in different medial-lateral Shh- expressing hypothalamic progenitor domains
Shh expression defines distinct precursor domains in the developing hypothalamus
To determine the fate of Shh- expressing hypothalamic progenitors, we first assessed whether the progenitor domains that were marked with GIFM corresponded to the dynamic Shh expression pattern observed with RNA in situ hybridization (Figure 1). Fate-mapped cells derived from progenitors expressing Shh at distinct time points (E7.5 to E12.5; Figure 2B) were first analyzed in whole-mount embryos one day after TM administration (Figure 2C). We found that the distribution of the fate-mapped cells at a specific marking time point did indeed correspond to the Shh expression pattern observed at the time of cell marking (Figures 1A1-A3 and 2C; for example, compare TM8.5 with Shh expression at E9.5).
To compare the distribution of the fate-mapped cells between different labeling time points in detail, we also analyzed transverse brain sections at a common embryonic stage (E12.5; or E13.5 for TM12.5; summarized in Figure 2D; Additional files 1 and 2). Shh-GIFM with TM7.5 resulted in the labeling of cells in the notochord and prechordal plate and of a few cells in the ventral midline of the mammillary and tuberal hypothalamic anlage (Figure 2C, D; Additional file 1). Cells derived from Shh- expressing progenitors marked at TM8.5 (hereafter referred to as Shh-derived cells) were found throughout the presumptive mammillary and tuberal hypothalamus. In the anterior hypothalamic primordium, cells were labeled at the ventral midline in more posterior sections, while no marking was found in the anterior aspects of the anterior hypothalamus (Figure 2C, D; Additional file 1). A few labeled cells were found in the future preoptic area. With TM9.5, only few marked cells were found in the presumptive mammillary nucleus. Shh-derived cells were excluded from the ventral midline in posterior sections, but were still located medially in the anterior tuberal hypothalamic primordium and the posterior aspects of the anterior hypothalamic primordium. In addition, a few scattered cells were located in the anterior aspects of the presumptive anterior hypothalamus (Figure 2C, D; Additional file 1). Marked cells were also observed in the preoptic area and the zli. With GIFM with TM10.5, TM11.5 and TM12.5, the distribution of the cells was similar to GIFM with TM9.5, but with less cells located in the presumptive mammillary body and anterior hypothalamus (Figure 2C, D; Additional file 2). We also found that less cells were observed in the mantle layer of the developing hypothalamus with TM11.5 and TM12.5. This is most likely due to the fact that the progenitors were marked only shortly before we analyzed the fate of the labeled progenitor cells at E12.5 or E13.5. The time window of less than 24 hours is too short for the generation of differentiated cells from the marked progenitors.
In summary, our analysis shows that our GIFM approach faithfully marks Shh- expressing progenitor domains. Based on the distribution of fate mapped Shh-derived cells, we can categorize the changes in cell distribution into the following stages: 'very early' (TM7.5) - only very few Shh-derived cells are found in the posterior hypothalamic primordium; 'early' (TM8.5) - Shh-derived cells are distributed in the mammillary and tuberal regions of the hypothalamic primordium; 'late' (TM9.5 to TM11.5) - Shh-derived cells are in the preoptic area and in the posterior hypothalamic anlage, but are largely excluded from the future mammillary nucleus and the ventral midline of the tuberal hypothalamic primordium; 'very late' (TM12.5) - the distribution of the fate-mapped cells is comparable to TM9.5 to TM11.5, but less cells are labeled (Figure 2C, D).
Two discrete transverse domains of Shh-lineage in the hypothalamus
At the very early stage (TM7.5), the few hypothalamic cells derived from Shh- expressing progenitors were found in the mammillary body and posterior hypothalamus (Figure 4C). Shh-GIFM at the early stage (TM8.5) labeled cells that were distributed in the caudal hypothalamic domain that included the mammillary region and the ventral part of the tuberal region (Figure 4D-F). Up to this labeling time point, the Shh-derived hypothalamic cells were restricted to one single caudal territory, which originates first with few cells in the mammillary region (very early pattern; Figure 4C) and then expands rostrally to include the ventral tuberal region (early pattern; Figure 4D-F). This change in the distribution of fate-mapped cells in the adult hypothalamus correlates well with the rostral expansion of Shh- expressing progenitors from the mammillary recess (TM7.5) to a large, ventral and caudal domain including all ventral midline neuroepithelium of the mammillary and tuberal regions (TM8.5) observed during embryogenesis (Figure 2C, D).
Two striking modifications of this pattern appeared at the late stage (TM9.5, TM10.5 or TM11.5). Rostrally, a novel group of fate-mapped cells appears labeled in the preoptic region (ventral and caudal to the anterior commissure) (Figure 4G-J). Caudally, Shh-derived cells no longer contribute to the ventral part of the mammillary or tuberal region (Figure 4G-J). This distribution again correlates well with the corresponding progenitor domains, which show a decrease in labeling in the mammillary neuroepithelium and the medial tuberal progenitor zone from TM9.5 through TM11.5 as well as a large increase in labeled progenitors in the preoptic region (Figure 2C, D). At this point, a rostral and a caudal domain of Shh-derived cells are visible in the hypothalamus. Since the intervening region (anterior hypothalamic region) is almost devoid of labeled cells, hypothalamic Shh-derived cells from the 'late' domain show a remarkable pattern with alternate labeled and non-labeled bands, which are rostrocaudally arranged (Figure 4J). Finally, cells derived from progenitors expressing Shh at E12.5 (very late stage) showed essentially the same distribution pattern, but the total number of marked cells was decreased (Figure 4K).
In summary, on sagittal sections and hemisected brains a remarkable and consistent heterogeneity in the distribution of Shh-derived cells was apparent. Of the four transverse hypothalamic regions, the most anterior (preoptic) and two most caudal (tuberal and mammillary) showed abundant labeled cells (depending on the time point when Shh- expressing progenitors were marked), while the anterior hypothalamic area seemed virtually devoid of them.
Mediolateral extent of the Shh-lineage hypothalamic domains: most tuberal and mammillary nuclei are of Shh descent
Since Shh-GIFM with TM8.0 or TM8.5 marked cells in the ventral midline of the mammillary and tuberal hypothalamic primordium (Figure 2; Additional file 1; data not shown), we could investigate which hypothalamic nuclei are derived from the medial progenitor zone. Consistent with the analysis of sagittal sections, fate-mapped cells were very rare in the preoptic and anterior hypothalamus, but were widely distributed in the tuberal and mammillary regions (Figure 5B, E, H, K, N; Additional file 3). The most rostral level at which fate-mapped cells could be found was the arcuate nucleus, at the level of rostral end of the A13 Th-expressing group (Figure 5H). Caudal to this point, numerous labeled cells were found at all dorsoventral and mediolateral levels of the tuberal and mammillary areas, including the ventromedial and dorsomedial nucleus, the arcuate nucleus, the median eminence (Figure 5K) and the mammillary body (Figure 5N).
Shh-GIFM with TM9.5 to TM11.5 allowed us to assess which nuclei are derived from lateral progenitor domains in the mammillary and tuberal regions, as well as from the rostral domain (Figure 2C, D). The size and position of the Shh- expressing progenitor domains is similar with TM9.5 to TM11.5. Since Shh expression is restricted to the ventricular zone, comparing the distribution of Shh-derived cells labeled with TM9.5, TM10.5 or TM11.5 can help to define the relation between the birth date (the time point when the cells leave the progenitor zone) and the final position of a neuron. With TM9.5, a broad cell population derived from the lateral Shh- expressing progenitor domain should be labeled. Based on their spatial origin, the same population could be labeled with TM10.5 and TM11.5. However, since some neurons have already left the progenitor zone at E10.5 or E11.5, the later GIFM time points will label a more restricted cell population, namely of neurons that have a later birth date.
Consistent with the results from the analysis of sagittal sections, the preoptic region showed numerous labeled cells (rostral Shh-lineage domain; Figure 5C, D) whose morphology in most cases suggested glia (see below). Characteristic anterior hypothalamic nuclei like the paraventricular hypothalamic nucleus or the suprachiasmatic nucleus were virtually devoid of Shh-lineage cells, as was the portion of the lateral hypothalamic zone corresponding to this region (Figure 5F, G). Fate-mapped cells contributed extensively to the tuberal region, but were excluded from the ventromedial tuberal hypothalamus, consistent with their origin in a lateral progenitor domain (Figures 2 and 5I, J, L, M; Additional file 3) . The most rostral cells of the Shh lineage in the caudal domain were again found in the arcuate nucleus (Figure 5I). The two large and characteristic nuclei of the tuberal region, the dorsomedial and the ventromedial nuclei, showed interesting differences in contribution of fate-mapped cells. The ventromedial nucleus (tuberal region) appeared as a large and compact group of Shh-derived cells packed together and showing a clearly recognizable histological pattern (Figure 5I, J, L, M; Additional file 3). In contrast, the dorsomedial nucleus contained Shh-derived cells, which do not seem to aggregate into a histologically recognizable nucleus (Figure 5I, J, L, M). Interestingly, fate-mapped cells were also found in the lateral aspects of the tuberal hypothalamus with GIFM between TM9.5 and TM11.5, indicating that cells that leave the progenitor zone after E10.5 or E11.5 (have a later birth date) can still contribute to the lateral hypothalamus. Finally, very few cells were marked in the mammillary nucleus with Shh-GIFM with TM10.5 and TM11.5 (Figure 5P, data not shown).
Very late stage
Shh-GIFM with TM12.5 labeled numerous cells in the lateral hypothalamus of the tuberal region, in the preoptic region and in the ventromedial nucleus, which was clearly delimited (data not shown). Many of these cells (virtually all in the preoptic region) showed astrocytic morphology (see below). Although the general pattern at this stage was similar as in the previous, the total number of cells was decreased, and cells with glial morphology were most numerous.
In summary, the analysis of sagittal (Figure 4) and transverse (Figure 5; Additional file 3) brain sections demonstrates clearly that the anterior hypothalamic region is not derived from Shh- expressing cells. This set of data suggests that there is little or no cell mixing between the tuberal and anterior or the preoptic and anterior hypothalamus. In other words, the cells produced by a certain progenitor domain tend to remain in the same rostrocaudal level.
Late born hypothalamic neurons settle in medial and lateral aspects of the tuberal hypothalamus
Shh- expressing progenitors give rise to astrocytes in the hypothalamus
Ventral diencephalic and ventral mesencephalic domains
Early in development, the Shh expression domain in the ventral neural tube starts rostrally in the ventral rostral diencephalon (characterized here as the progenitor domain of the tuberal and mammillary regions) and extends caudally uninterrupted through the ventral midbrain, hindbrain and spinal cord. Comparison of our data with recent analyses of Shh-derived cells in the ventral midbrain [19, 42, 43] indicates that the domains we observe in the caudal hypothalamus are indeed continuous (although generated earlier) with the Shh- expressing progenitor domains of the ventral midbrain, suggesting a continuity of rostrocaudal, ventral regions along the neural tube, modulated by local signals. The Shh- expressing progenitor domain appearing in the preoptic region later in development is, however, not part of this continuous domain.
Shh-expressing progenitor domains and restricted gene expression
Progenitor domains are characterized by complex patterns of transcription factor gene expression, and an important yet unresolved question concerns the molecular determinants of the neurons produced in each progenitor domain. An initial mapping of progenitor domains in the hypothalamus and the neuronal nuclei they generate has been made based on the fact that some transcription factors are expressed not only in restricted progenitor domains but also in the neurons they generate  and similar approaches are contributing to unravel the relation between progenitor domain and mantle layer in the hypothalamus . Through recent genomic studies, a glut in resources has been made available that will be key to answer this question . The present study shows that the large Shh-expressing domains are formed by smaller, partially overlapping domains. This corresponds with the ample variety of Shh-derived hypothalamic neuronal nuclei. Intriguingly, the early- and late-expressing domains show differential expression of determinants, pointing at the molecular basis of neurogenetic timing.
Multiple, differential patterns of neurogenesis in the Shh progenitor domains
The neuroepithelial progenitor domains are characterized by restricted gene expression patterns [12, 46] and generate specific neurons according to distinctive rhythms . Some specific hypothalamic neuronal groups are formed in a single neurogenetic peak  while others have distinct subgroups generated at different times [49, 50]. Are Shh- expressing hypothalamic precursors characterized by a specific timing and rate of neurogenesis? The answer is negative. Our data show that the hypothalamic Shh-domains generate many neurons for many nuclei through differential neurogenetic dynamics. As an example, the lateral hypothalamus (in the tuberal and mammillary regions) is generated as a large number of scattered cells over an extended period of time and an extended area of the brain. The two major tuberal nuclei, the ventromedial and the dorsomedial nucleus, generated by opposing strategies, are the best example. While the dorsomedial nucleus seems to receive a steady contribution of scattered cells by the neuroepithelium during TM8.0 through TM12.5 (the same strategy applies to the lateral hypothalamus), the ventromedial nucleus receives massive amounts of neurons, which aggregate forming a histologically recognizable nucleus. In conclusion, the hypothalamic Shh- expressing progenitors do not share common neurogenetic dynamics, suggesting that they respond to proliferation cues differentially, according to specific programs and critical periods.
Hypothalamic patterns of neurogenesis: three waves?
The above paragraph has broader implications. Classically, it has been described that the hypothalamus is formed in three neurogenetic waves producing neurons that accumulate progressively more medial (outside-in) . This has been considered as fundamentally proven, although opposite findings relative to specific hypothalamic neuronal subgroups have been reported [48, 51, 52]. Our data show that since the Shh- expressing progenitor domains remain essentially unchanged (that is, they do not shrink or expand) after E9.5, GIFM with TM9.5, TM10.5 and TM11.5 labels essentially the same progenitor domains, which are generating neurons fated for the same regions. Therefore, by comparing these three stages we can actually make a statement about which cells are born early (before E11.5) or late (after E11.5). If there were distinct waves of neurogenesis generating progressively more medial neuronal zones, they would be obvious in our material (Figures 5 and 6), but they are not.
In conclusion, for a period of several days, the hypothalamic Shh- expressing progenitors generate neurons fated for every mediolateral level, and do not generate neurons for a specific mediolateral level at a specific time. At least for these progenitors, the three wave model  has to be revised.
Shh-derived cells remain within the same region of the progenitor domain
Our results contribute to clarify the controversy about the modes of hypothalamic migration. Analyses of hypothalamic neurogenesis with tritiated thymidine suggested a pattern of migration in the mediolateral plane ('radial' migration) . Clonal analyses of the avian ventral forebrain  and the distribution of specific hypothalamic neurons at different developmental stages in the rat  provided confirmatory evidence, although other diencephalic areas show some widely dispersed clones [54, 55]. In addition, intrahypothalamic tangential migration as well as migration from the olfactory placode, telencephalon and diencephalon into the hypothalamus have been described [44, 56–58]. Here we show that the progenitor domains coincide with the regions where the Shh-derived cells settle, suggesting little or no mixing of cells belonging to different domains. This is clearly confirmed by the fact that neither the preoptic nor the anterior regions are colonized by Shh-derived neurons from caudal regions. Therefore, the Shh-derived hypothalamic cells, that is, most of the tuberal and mammillary regions, migrate mostly in the mediolateral plane (radially) and not in the rostrocaudal plane (tangentially) (with the caveat that short tangential migrations of Shh-derived cells inside the Shh-derived domain would not be detected by our approach).
The retrochiasmatic area
The retrochiasmatic area is a small ventral and medial hypothalamic region between the optic chiasm (rostrally) and the arcuate nucleus (caudally). On the basis of Shh expression [18, 59], this region represents the very rostral tip of the basal plate of the hypothalamus. Therefore, the corresponding neuroepithelium represents also the rostral end of the Shh- expressing progenitor domain (Figure 2), and the retrochiasmatic area is labeled in our material (Additional file 3A). Classically classified as part of the anterior hypothalamic region , this area is anatomically continuous with and functionally related to the arcuate and ventromedial nuclei of the tuberal region [60–62]. We have represented it (Figure 2) as part of the anterior region as is traditional, but this does not modify our conclusion that the anterior hypothalamus is not Shh-derived.
The non-Shh-derived hypothalamus: open questions
The embryonic preoptic region has been considered telencephalic as opposed to hypothalamic based on morphological criteria as well as gene expression patterns [59, 63]. Moreover, since Shh is a key ventral determinant in the neural tube, it follows that in the E12.5 embryo the caudal, Shh- expressing half of the hypothalamus is actually ventral, and the rostral half shares the dorsal portion with the telencephalon [18, 59]. Using GIFM, we demonstrate that Shh progenitor domains of the caudal half of the hypothalamus do not produce cells for the preoptic or anterior regions in the adult hypothalamus. In this sense, the two 'rostral' regions of the adult hypothalamus, preoptic and anterior, derive from the dorsal portion of the neural tube, together with the telencephalon. Obviously, the simple longitudinal versus transverse subdivision that works so well in the spinal cord breaks down in the hypothalamus and telencephalon; therefore, it might be time to redefine a novel dorsal subdivision ventral to the telencephalon. Furthermore, it remains to be investigated which hypothalamic neurons originate in the telencephalon [56, 64, 65] and whether they preserve any anatomical or functional links with their region of origin.
From complex specification to complex adult morphology
The adult hypothalamus is functionally organized into a behavioral control column [2, 62] whose rostrocaudal arrangement obscures the developmental seams between the regions derived from specific progenitor domains as revealed by GIFM. Even so, the developmental heterogeneity we unveil here probably contributes to the staggering functional complexity of the hypothalamus. This is perhaps particularly true of the preoptic and anterior regions, which are the most complex hypothalamic regions and, as has been observed, the ones causing the most confusion [3, 56, 66]. Likewise, finding out the origin of that portion of the lateral hypothalamus corresponding to the non-Shh-derived regions promises to be of great interest.
The Shh-expressing progenitor domains in hypothalamus are continuous (although generated earlier) with the Shh- expressing progenitor domains of the ventral midbrain, and can be seen as the most rostral part of the longitudinal, ventral progenitor regions continuous along the neural tube.
The behavioral control column that constitutes the adult hypothalamus has its origin in a variety of progenitor domains in the ventral midline and in the basal and alar portions of the rostral diencephalon. Of its four transverse subdivisions, the two most caudal (mammillary and tuberal regions, including the retrochiasmatic area) are formed mostly by neurons and glia originated in Shh- expressing progenitor domains of the ventral midline and basal plate. The anterior hypothalamic region is virtually devoid of neurons produced in said domains. The most rostral hypothalamic region (preoptic region) receives mostly glia from an independent, rostral and late-appearing Shh- expressing domain.
There is almost no tangential migration from Shh-derived regions into the non-Shh-derived anterior hypothalamic region. This suggests the existence of molecular mechanisms preventing the mixing of cells generated in the basal and alar plates of the rostral diencephalon.
The hypothalamic Shh- expressing domains are formed by smaller, partially overlapping domains of transcription factor gene expression. The hypothalamic progenitors expressing Shh early are localized to transcription factor expression domains (Nkx2-1/Six3) different from those of progenitors expressing Shh late (Dbx1/Sim1).
The hypothalamic Shh- expressing progenitor domains generate neurons fated for all mediolateral levels during a protracted neurogenetic period of several days. No marked outside-in waves of neurogenesis could be detected in our material.
Materials and methods
Animal studies were approved by the University of Bonn Animal Care and Use Committee. ROSA loxP-STOP-loxP-LacZ (R26 lz ) reporter mice were kindly provided by Dr P Soriano ; ROSA loxP-STOP-loxP-EYFP (R26 EYFP ) were kindly provided by Dr F Costantini ; Shh CreER were kindly provided by Dr C Tabin [19, 20]. Mice heterozygous for the R26 lz or R26 EYFP allele and the CreER alleles were genotyped as previously described [25, 67]. All mice were maintained in an outbred SW or CD1 background. Shh CreER/+ ; R26 lz/lz or Shh CreER/+ ; R26 EYFP/EYFP males were bred with SW or CD1 wild-type females (Taconic, Hudson, NY, USA or Charles River, Wilmington, MA, USA) to generate Shh CreER/+ ; R26 lz/+ or Shh CreER/+ ; R26 EYFP/+ progeny. Noon of the day that a vaginal plug was detected was designated as E0.5. TM (Sigma T-5648, St. Louis, Mo, USA) was dissolved in corn oil (Sigma C-8267) at a final concentration of 20 mg/ml. Pregnant females were given 3 to 4 mg TM through oral gavage with animal feeding needles (Fisher Scientific, Waltham, Ma, USA or Fine Science Tools, Heidelberg, Germany) at 12 pm each day between E7.5 and E12.5 and at midnight of E7.5 (corresponding to E8.0). For the analysis of fate-mapped cells at postnatal stages, the TM solution contained progesterone (Sigma P-0130) at a concentration of 5 mg/ml to reduce the incidence of miscarriages.
Tissue processing, RNA in situ hybridization and immunofluorescent stainings
Embryos or embryonic brains were dissected and fixed in 4% paraformaldehyde for 20 to 90 minutes. Postnatal mice were perfused intercardiacally with 4% paraformaldehyde, and brains were dissected and postfixed in 4% paraformaldehyde overnight. E8.5 to E13.5 embryos or embryonic brains were sectioned on a cryostat at 12 μm, E18.5 brains at 14 μm and postnatal brains at 40 μm (free-floating sections). For RNA in situ hybridization, frozen sections were used or the tissue was manually processed for paraffin embedding and sectioned at 7 μm. RNA in situ hybridization was performed as described . RNA in situ probes were obtained from AL Joyner (Shh, Nkx2.1), JL Michaud (Sim1), K Campell (Dbx1) and G Oliver (Six3).
X-gal and immunofluorescent staining on sections was performed using standard procedures . Primary antibodies were: goat anti-β-gal (1:2,000; Biogenesis Kingston, NH, USA), rabbit or rat anti-green fluorescent protein (1:400, Invitrogen, Carlsbad, CA, USA or 1:2,000, Nacalai, Kyoto Japan) rabbit or mouse anti-tyrosine hydroxylase (1:500; Millipore, Billerica, MA, USA), rabbit anti-calbindin (1:5,000; Swant, Marly, Switzerland), rabbit anti-GFAP (1:500; Millipore), mouse anti-glutamine synthetase (1:500; Millipore). Secondary antibodies were: donkey anti-goat IgG-Alexa 555 and donkey anti-rabbit IgG-Alexa 488 (1:500; Invitrogen); donkey anti-goat Cy3, donkey anti-rabbit Cy3 or FITC, donkey anti-mouse Cy3 or FITC (1:200; Jackson ImmunoResearch, West Grove, PA, USA).
X-gal whole mount stainings
X-gal staining was performed as described . Embryos older than E10.5 were cleared in benzyl-benzoate.
enhanced yellow fluorescent protein
glial fibrillary acidic protein
genetic inducible fate mapping
zona limitans intrathalamica.
We thank Alexandra Joyner and Soline Chanet for contributions to the initial phase of this project. We would like to thank Phil Soriano and Frank Costantini for the R26 reporter mice; Cliff Tabin and Brian Harfe for the Shh CreER mice and Alexandra Joyner, Kenneth Campell, Guillermo Oliver and Jacques Michaud for RNA in situ probes. This study was funded by a Research Award from the Ministry of Science and Research of North Rhine Westphalia (Programm zur Förderung der Rückkehr des wissenschaftlichen Spitzennachwuchses aus dem Ausland, to SB) and by the Deutsche Forschungsgemeinschaft (AL603/2-1 to GAB).
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