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Disease Models & Mechanisms
Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling
Murielle Rallu, Robert Machold, Nicholas Gaiano, Joshua G. Corbin, Andrew P. McMahon, Gord Fishell
Development 2002 129: 4963-4974;
Murielle Rallu
Robert Machold
Nicholas Gaiano
Joshua G. Corbin
Andrew P. McMahon
Gord Fishell
Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, we examined the telencephalon in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persist in Shh-/- mutants, and that these same markers were expanded in Gli3-/- mutants. To understand further the genetic interaction between Shh and Gli3, we examined Shh/Gli3 and Smoothened/Gli3 double homozygous mutants. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon.
Telencephalon
Gli3
The forebrain arises from the most anterior part of the neural plate and is subsequently divided into the telencephalon and the diencephalon (Fishell, 1997; Rubenstein and Beachy, 1998; Shimamura et al., 1995). After neural tube closure (mouse E9), proliferation within the ventral regions of the telencephalon results in the emergence of three eminences: first, the medial ganglionic eminence (MGE) and later, the lateral and caudal ganglionic eminences (LGE and CGE, respectively) (Fishell, 1997) (S. Nery, G. F. and J. G. C., unpublished). Collectively, these eminences make up the anlage of much of the telencephalic basal ganglia. These structures are thought to give rise to the globus pallidus, the striatum, and large populations of oligodendrocytes and cortical interneurons (Wichterle et al., 2001) (S. Nery, G. F. and J. G. C., unpublished). All three eminences appear to be absent in sonic hedgehog (Shh) null mutants (Chiang et al., 1996) (J. G. C., unpublished).
Shh has been shown to play a fundamental role in the establishment of ventral identity throughout the neural tube (reviewed by Ericsson et al., 1995a; Litingtung and Chiang, 2000b; Patten and Placzek, 2000). As a result, much work has been performed to elucidate the mechanisms by which Shh generates cell diversity within the ventral neural tube, especially in the spinal cord. A considerable body of evidence favors the idea that Shh acts as a morphogen, forming a gradient within the ventral neural tube to which cells respond in a concentration-dependent manner (reviewed by Briscoe and Ericsson, 2001; Jessell, 2000). Collectively, in vitro explant studies using recombinant Shh (Ericsson et al., 1996; Ericsson et al., 1997; Roelink et al., 1995) and in vivo electroporation studies with Shh (Agarwala et al., 2001) or a dominant-negative form of patched (Briscoe et al., 2001) strongly support this hypothesis in the midbrain and spinal cord, respectively.
Although Shh is also clearly required in forebrain regions (Chiang et al., 1996; Ericsson et al., 1995b), to date less effort has been focused on its role in patterning the telencephalon. Similar to the spinal cord, embryos that lack Shh fail to form ventral telencephalic structures and to express corresponding markers, while dorsal marker expression is expanded ventrally (Chiang et al., 1996). Furthermore, ectopic expression of Shh is sufficient to induce ventral telencephalic markers in dorsal regions of the telencephalon both in vitro (Ericsson et al., 1995b; Kohtz et al., 1998; Shimamura and Rubenstein, 1997) and in vivo (Barth and Wilson, 1995; Corbin et al., 2000; Gaiano et al., 1999; Hauptmann and Gerster, 1996). However, when and how Shh is required for ventral telencephalic patterning and whether it acts to differentially specify the MGE, the LGE and the CGE is not presently clear. Beginning around E9.0, Shh is expressed within the ventral telencephalon, primarily in the MGE, preoptic area and prospective amygdala (Nery et al., 2001; Sussel et al., 1999). Notably, the specific loss of Shh expression within the telencephalon, which occurs in Nkx2.1 (Titf1 - Mouse Genome Informatics) (Sussel et al., 1999) and BF1 (Foxg1 - Mouse Genome Informatics) (Huh et al., 1999) null mutants, does not recapitulate the phenotype observed in Shh mutants. This suggests that earlier expression of Shh, probably from non-neural tissues such as the dorsal foregut (i.e. prechordal plate) (Shimamura and Rubenstein, 1997) or Hensen's node (Gunhaga et al., 2000), must be responsible for the more extensive telencephalic defects seen in Shh mutants.
The Ci/Gli family of zinc-finger transcription factors has been implicated as required transducers of the hedgehog signaling pathway (reviewed by Aza-Blanc and Kornberg, 1999; Ruiz i Altaba et al., 2002). Gli proteins, like Ci, display both activator and repressor activities that are regulated post-translationally (Aza-Blanc et al., 1997; Ingham and McMahon, 2001). Different regions of the Gli proteins encode distinct functions: N-terminal regions encode a repressor function, whereas C-terminal regions are required for positive activity. Analysis of mice mutants for each of the three murine Gli genes has shown that Gli3 is the only Gli gene that plays a significant role in patterning the telencephalon. Specifically, mice that lack Gli1 and Gli2 show minor defects in telencephalic patterning (Park et al., 2000), whereas Gli3 mutant mice have strong abnormalities in the dorsal telencephalon (Grove et al., 1998; Theil et al., 1999; Tole et al., 2000). Gli3 protein appears to function primarily as a repressor (reviewed by Ingham and McMahon, 2001) and its activity seems to be negatively regulated by Shh (Marigo et al., 1996; Wang et al., 2000). Shh signaling, both in vitro and in the limb, has been shown to inhibit Gli3 processing into an N-terminal fragment that carries a repressor activity. In agreement with this, analysis of mice lacking both Shh and Gli3 revealed that, in the spinal cord, Shh is required to inhibit Gli3 function in order to form motoneurons (Litingtung and Chiang, 2000a).
In the present study, we have used both gain- and loss-of-function methods to study the importance of hedgehog signaling in general and Shh in particular in the establishment of dorsoventral patterning in the telencephalon. We find that virally mediated ectopic sonic hedgehog expression induces different regional markers, depending on the dorsoventral or anteroposterior position of the infection. Conversely, some aspects of ventral telencephalic patterning persist in the absence of Shh. In addition, we observe that dorsoventral patterning in the telencephalon appears to be largely restored in Shh/Gli3 and Smo/Gli3 double homozygous mutants. Our data reveal that Shh and Gli3 antagonize each other's function in patterning the telencephalon. Furthermore, our findings suggest that a hedgehog-independent mechanism, such as possibly the BMP-signaling pathway (Liem et al., 2000), is capable of acting in parallel to Shh in establishing dorsoventral pattern in the telencephalon.
Animals, virus preparation and injections
Mice used in these studies were maintained according to protocols approved by the Institutional Animal Care and Use Committee at New York University School of Medicine. For staging of embryos, noon on the day the vaginal plug was considered as embryonic day 0.5 (E0.5). Virus preparation and ultrasound surgery were both performed as previously described (Gaiano et al., 1999). Stocks of Shh-expressing or ActSmo-expressing viruses were injected at titers of 5×107 and 5×108 cfu/ml respectively. Mouse embryos were injected from E8.5 to E10.5 and sacrificed 4 days later (E12.5 to E14.5). Viral expression usually begins 12 hours after infection (E9.0 at the earliest in our case).
Genotyping of mutant mice
Shh (Chiang et al., 1996) and Gli3 (XtJ from Jackson Laboratory) mutant mice were maintained on a C57/B16 and C3HeB/FeJ background, respectively. Different mutant combinations were generated by intercrossing Shh+/-;Gli3+/- or Smo+/-;Gli3+/- heterozygous animals. Embryos were genotyped by PCR as described previously (Litingtung and Chiang, 2000a; Maynard et al., 2002; Zhang et al., 2001).
Staining of tissue sections
Whole heads or embryos were fixed at 4°C for 1-4 hours in 4% paraformaldehyde, then cryoprotected overnight in 30% sucrose in PBS, embedded in HistoPrep (Fisher Scientific) and frozen. All tissue was sectioned serially at 12 to 20 μm and processed for immunohistochemistry or RNA in situ hybridization.
Infected cells were identified by the expression of the alkaline phosphatase reporter (PLAP) as described previously (Gaiano et al., 1999). Analysis of gene induction was performed by RNA in situ hybridization on adjacent 20 μm sections. Note that the thickness of the sections and the incompatibility of histochemistry and in situ hybridization on the same sections sometimes made it hard to correlate the infected cells with the cells that display ectopic expression (e.g. Fig. 2J-K).
Effects of ectopic Shh signaling on regional patterning in the telencephalon. Embryos were injected with ActSmo-expressing virus at E9.5 and analyzed at E14.5. Histochemical staining for PLAP reveals the location of virally infected cells co-expressing ActSmo (A,D,G,I,K,N). Adjacent sections were assayed for regionally expressed homeodomain genes (A-J) or for the expression of downstream targets of Shh signaling pathway (K-O). (A-C) Confocal immunohistochemistry for PLAP (A) and Pax6 (C) proteins within the lateral neocortex shows a repression of Pax6 expression in clusters of cells infected by ActSmo (asterisks). (G-J) Coronal sections of ActSmo-infected brains stained for ventral genes expression. Ectopic Nkx2.1 expression is restricted to the LGE and lateral neocortex (H, arrows), whereas Gsh2 expression can be induced throughout the neocortex (J, arrows). Dotted bracket illustrates weaker levels of induction within the medial neocortex. Sagittal sections (D-F) reveal a similar restriction along the rostrocaudal extent of the telencephalon. Asterisks in D-J represent the sites of endogenous expression. (K-O) Ectopic expression of ActSmo induces the upregulation of Gli1 and Ptch expression all along the dorsoventral (L,M, arrows and bracket) or anteroposterior (O) axes, showing that the telencephalon has the competency to respond to Shh signaling at this age. The mismatch between areas of viral infection and Ptch or Gli1 induction reflects the fact that these are visualized in adjacent sections. This is unavoidable because of the incompatibility of PLAP histochemistry and in situ hybridization. (P,Q) Schematic representations of a sagittal (P) and coronal (Q) section showing Nkx2.1 and Gsh2 endogenous domains of expression as well as the regions where their expression can be induced by Shh signaling.
Immunofluorescence was performed as described previously (Corbin et al., 2000). The following primary antibodies were used: mouse anti-Pax6 (1:1000, gift of A. Kawakami) and rabbit anti-Gsh2 (1:3500, gift of K. Campbell).
In situ hybridization was performed as described previously (Schaeren-Wiemers and Gerfin-Moser, 1993; Wilkinson and Nieto, 1993) using non-radioactive digoxigenin-labeled probes for Nkx2.1 (Shimamura et al., 1995), Dlx2 (Porteus et al., 1991), Gsh2 (Hsieh-Li et al., 1995), Mash1 (Ascl1 - Mouse Genome Informatics) (Guillemot and Joyner, 1993), Gli1, Gli3 (Hui et al., 1994) and Ptch (Goodrich et al., 1997).
Dlx2 mRNA expression was also determined by X-Gal staining of embryos from a strain of mice in which the taulacZ gene has been targeted to the Dlx2 locus (Corbin et al., 2000) (S. Nery, G. F. and J. G. C., unpublished). X-Gal staining was performed as described previously (Corbin et al., 2000).
The competence of the E9.0 telencephalon to respond to Shh signaling is spatially restricted
To better understand the mechanisms by which Shh patterns the telencephalon, we chose to investigate the effects of ectopic Shh signaling in vivo. We used a gain-of-function approach that combines high-titer retroviral vectors and ultrasound imaging (Gaiano et al., 1999). Retroviruses expressing the full-length Shh protein were injected into the embryonic forebrain at different stages of development, the earliest of which (mouse E8.5) preceded the establishment of overt dorsoventral patterning in the telencephalon. The injection of Shh virus at these early time points had a dramatic effect on the morphology of the brains, with the vesicles being substantially enlarged compared with wild-type embryos (Fig. 1E,F). These morphological defects may be the result of an abnormal proliferation induced by Shh. Indeed, the role of Shh in cell proliferation within the nervous system has been shown in the cerebellum (Dahmane and Ruiz-i-Altaba, 1999; Wechsler-Reya and Scott, 1999), the retina (Jensen and Wallace, 1997) and the dorsal neural tube (Rowitch et al., 1999). The morphological abnormalities in Shh-infected brains made any analysis of dorsoventral patterning in these animals difficult. Thus, we constructed a retrovirus encoding a constitutively active form of the Smoothened protein (ActSmo) (Hynes et al., 2000; Xie et al., 1998). Smoothened is a transmembrane protein that, in the absence of Shh, is repressed by the Shh receptor Patched (Ptch) (reviewed by Ingham and McMahon, 2001; Kalderon, 2000; Murone et al., 1999). This repression is thought to be relieved by the binding of Shh to Patched, allowing Smoothened to activate downstream elements of the Shh signaling pathway cell-autonomously. Activated Smoothened has been shown to mimic the patterning effects of ectopic Shh in the dorsal neural tube (Hynes et al., 2000). Furthermore, the activity of ActSmo is likely to be cell-autonomous (Hynes et al., 2000), allowing us to study the effects of ectopic hedgehog signaling without perturbing the local environment of the infected cells. We found that infections with a virus expressing ActSmo did not result in the gross morphological deformations observed with Shh (Fig. 1C,D) but was indistinguishable from Shh in its ability to alter regional gene expression.
Differential effects of Shh- or ActSmo-expressing retroviruses on brain morphology. Animals were infected in utero with control virus (A,B) and viruses expressing ActSmo (C,D) or Shh (E,F). Injections were performed at E8.5 (A,C,E) or E9.5 (B,D,F) and analyzed 4 days later (E12.5 or E13.5, respectively). Embryos injected with Shh virus showed a dramatic increase in the size of their heads, with substantially enlarged telencephalic vesicles (E,F). Despite the fact that the ActSmo virus was injected at a 10-fold greater titer than Shh virus, heads from these embryos appeared to have largely normal morphology (C,D).
ActSmo- and Shh-expressing viruses were injected at various times ranging from E8.5 to E10.5 and embryos were sacrificed 4 days later for analysis (E12.5 to E14.5). Notably, we observed that the ectopic expression of ActSmo (or Shh itself) resulted in the same modifications of telencephalic patterning regardless of the age of the infection (data not shown). This suggests that the competence of the telencephalic tissue to respond to ectopic induction of the Shh pathway is not temporally regulated within this window of embryonic development.
Ventral patterning by Shh in the spinal cord is in part mediated through the inhibition of dorsally expressed transcription factors (Briscoe et al., 2000; Ericsson et al., 1997). Similarly in the telencephalon, in vitro exposure to Shh can inhibit the expression of dorsal markers (Kohtz et al., 1998). Moreover, dorsal gene expression expands ventrally in the absence of Shh (Chiang et al., 1996). We found that the expression of Pax6, a dorsal telencephalic marker, is indeed repressed in the infected cells (Fig. 2A-C), in accordance with previous results in spinal cord (Ericsson et al., 1996). A similar result was obtained for the dorsally expressed proneural genes such as Ngn2, Math2 and NeuroD (data not shown).
We next sought to determine the extent to which ventral markers characteristic of the MGE (Nkx2.1) and MGE/LGE (pan-ventral genes Gsh2, Dlx2) could be induced ectopically by virally mediated activation of Shh signaling. We observed that the identity of the markers induced, and therefore the cell types, was mainly dependent on the position of the infection. Along the dorsoventral axis, the induction of Nkx2.1 expression in response to ectopic Shh signaling was restricted to the LGE and lateral neocortex (Fig. 2G,H), whereas Gsh2 and Dlx2 could be induced throughout the neocortex (Fig. 2I,J and data not shown). Interestingly, we noted that the induction of pan-ventral gene expression (Gsh2, Dlx2) was weaker in the medial regions of the telencephalon (Fig. 2I,J). Sagittal sections of ActSmo infected brains revealed the rostrocaudal extent of ventral gene induction: while Nkx2.1 induction was restricted to the LGE and septum (Fig. 2D,F and data not shown), pan-ventral gene induction within the cortex was only excluded from the more posterior regions (Fig. 2D,E). These differences could be accounted for by regional variations in the ability of the telencephalon to activate downstream components of the Shh pathway. To address this issue we analyzed patched (Ptch) and Gli1 gene expression, which are believed to be generic downstream response genes in the Shh signaling pathway. We found that the expression of either of these genes was upregulated at all positions along the dorsoventral or anteroposterior axis (Fig. 2K-O). Based on these criteria, this suggests that all regions of the telencephalon are equally competent to respond to Shh signaling.
These gain-of-function experiments show that regional identity can be altered by Shh signaling through the early phases of neurogenesis. However, we found that this plasticity is somewhat limited: the ventral identities that Shh and ActSmo can induce are dependent on the dorsoventral or anteroposterior position of the mis-expression. This suggests that an intrinsic pattern, which is either Shh dependent or independent, has been established within the telencephalon by the time the Shh pathway is activated ectopically (E9.0 at the earliest, i.e. 12 hours after infection).
Pan-ventral gene expression is maintained in Shh mutants
Previous analysis of Shh-null animals revealed that, in addition to the lack of any obvious ventral telencephalic structures, the domain of dorsal gene expression spreads ventrally to include the whole telencephalon (Chiang et al., 1996; Pabst et al., 2000). However, when we investigated the expression of pan-ventral genes in these mutants, we were surprised to observe that some ventral telencephalic patterning remains in the absence of Shh (Fig. 3). Dlx2 expression was observed in the remaining telencephalon of E10.5 to E12.5 Shh mutants as visualized by in situ hybridization or by using a Dlx2taulacZ allele (Fig. 3A-D). Similarly, Gsh2 expression (another pan-ventral gene) persisted in Shh mutants (Fig. 3E,F) (H. Toresson and K. Campbell, personal communication). Notably, in these mutants the domain of expression of both Dlx2 and Gsh2 was restricted to a region adjacent to the ventral midline. As Shh mutants are extremely dysmorphic, it is impossible to know with certainty which ventral telencephalic domains persist in these mutants. Nonetheless, the fact that Nkx2.1 expression was missing in Shh-/- embryos (data not shown) (Pabst et al., 2000) while Dlx2 and Gsh2 expression persisted suggests that the most ventral domain (i.e. MGE) is absent in Shh-/- mutants, while the lateral regions (i.e. LGE) are displaced ventrally. In this regard, this result is likely to be analogous to that in spinal cord, where the laterally positioned V0 and V1 interneurons, which are characteristic of lateral cell fates, persist in Shh-/- embryos but are displaced toward the midline (Pierani et al., 1999). The observation that ventral gene expression can occur in the absence of Shh suggests the existence of a Shh-independent pathway. This prompted us to examine other genes that could play a role in establishing dorsoventral patterning in the telencephalon.
Shh-/- and Gli3-/- mutants show opposite telencephalic phenotypes. Pan-ventral gene expression was analyzed in E10.5 Shh-/- (A-F) and E12.5 Gli3-/- (G-L) mutants. Dlx2 and Gsh2 expression is maintained but displaced ventrally in Shh-/- embryos (B,D,F). By contrast, Dlx2 and Gsh2 expression expands dorsally in Gli3-/- mutants (H,J,L). (K,L): Gsh2 and Pax6 expression domains abut at the corticostriatal boundary in wild-type animals (K, arrow). This boundary is affected in Gli3-/- embryos (L), with Gsh2 expression expanding dorsally. Dlx2 expression was assayed either by X-gal staining on whole-mount embryos (A,B) or by in situ hybridization on coronal sections (C,D,G,H). Gsh2 expression was assayed by immunofluorescence against the Gsh2 protein (E,F,I-L).
Shh has been shown to be required to antagonize Gli3 function in ventral regions of the spinal cord (Litingtung and Chiang, 2000a). In the telencephalon, Gli3 is expressed at high levels in dorsal and lateral regions (cortex and LGE) and at low levels ventrally (in the MGE). Interestingly, extra-toes mice (XtJ), which carry a deletion encompassing the Gli3 gene (Buscher and Ruther, 1998; Hui and Joyner, 1993; Schimmang et al., 1992; Vortkamp et al., 1992), show defects opposite to those observed in Shh mutants. In Gli3 mutants the morphology of the forebrain is perturbed dorsally: the cortex is highly reduced in size, and the hippocampus and the choroid plexus are absent (Grove et al., 1998; Theil et al., 1999; Tole et al., 2000). Furthermore, the expression of a number of pan-ventral genes, such as, Dlx2 (Fig. 3G-H) (Tole et al., 2000), Gsh2 (Fig. 3I-J) and the bHLH gene Mash1 (data not shown), spreads precociously into cortical areas. Even though ventral markers were ectopically expressed, the cortex of Gli3 mutants retained its dorsal character, as shown by the persistence of dorsal gene expression, including Pax6 (Fig. 3K,L) (Tole et al., 2000). Interestingly, Gsh2 and Pax6 proteins appeared to be co-expressed in the cortex of Gli3 mutants, as shown by double immunostaining (Fig. 3L). Gsh2 and Pax6 expression domains generally abut at the boundary between the cortex and the LGE (i.e. the corticostriatal boundary) (Fig. 3K) and these proteins are normally mutually repressive (Toresson et al., 2000; Yun et al., 2001). Our results showed that Gsh2 expression no longer respects this boundary in the telencephalon of Gli3 mutants, suggesting that the repression of Gsh2 by Pax6 is dependent on Gli3.
Partial rescue of dorsoventral patterning in Shh-/-;Gli3+/- mutants
In order to analyze the extent to which dorsoventral patterning can occur in the absence of Shh and Gli3, we analyzed the telencephalic phenotype of Shh/Gli3 compound mutants. Interestingly, Shh-/-;Gli3+/- embryos displayed a remarkable rescue of brain morphology when compared with Shh-/- embryos. Although the telencephalon of Shh-/- embryos at E12.5 was greatly reduced in size and composed of a single vesicle fused at the midline (Fig. 4B,B′) (Chiang et al., 1996), Shh-/-;Gli3+/- embryos had a much bigger telencephalon in which two vesicles were clearly discernable (Fig. 4C,C′, arrows). Moreover, these embryos had a reduced proboscis (arrowheads) and two relatively well-formed eyes, albeit fused together and located at the midline (Fig. 4D-S, arrows).
Dorsoventral patterning of the telencephalon is largely restored in Shh-/-;Gli3+/- mutants. Lateral (A-C) and dorsal (A′-C′) views of the heads from wild-type, Shh-/- and Shh-/-;Gli3+/- E12.5 embryos, showing the extent of the phenotypic rescue. The telencephalon of Shh-/-;Gli3+/- embryos is formed of two paired vesicles (arrows), in sharp contrast to the phenotype of Shh-/- embryos. Note that the eye phenotype is also partially rescued as indicated by the presence of two distinct eyes, albeit fused at the midline and the proboscis observed in Shh-/- animals is reduced in size (asterisks in B,C; arrows in E,G,K,M; arrowheads in B-C′). (D-O) Coronal sections of wild-type and Shh-/-;Gli3+/- E12.5 embryos assayed for various region-specific homeobox gene expression. Dlx2 (D,E), Mash1 (J,K) and Gsh2 (L,M) expression in the mutants closely resembles that in wild-type embryos, showing that ventrolateral patterning is established normally in this context. Double immunohistochemistry for Gsh2 and Pax6 further demonstrate that LGE- and cortex-like structures are properly established in Shh-/-;Gli3+/- animals (N,O). Arrowheads delineate the boundary between the LGE and the cortex. In the ventral midline, a small region of Nkx2.1 expression is observed in Shh-/-;Gli3+/- embryos (G,I, asterisks). Overlay of Nkx2.1 (red) and Dlx2 (green) RNA in situ hybridization from adjacent sections using Adobe Photoshop 4, shows the nested pattern of Nkx2.1 within a broader Dlx2 domain in both wild-type and mutant embryos (H,I). (P-S) Coronal sections of wild-type and Shh-/-;Gli3+/- E12.5 embryos assayed for Gli1 (P,Q) and Ptch (R,S) expression, showing that the Shh pathway is not active. Brackets in P,R indicate the extent of Gli1 (P) or Ptch (R) expression.
As these embryos showed a significant morphological rescue of the Shh-null phenotype, we analyzed in detail the status of dorsoventral patterning in the telencephalon. We observed that Dlx2 expression, which was shifted either ventrally or dorsally in Shh-/- and Gli3-/- mutants, respectively, was restored in Shh-/-;Gli3+/- embryos to a level and distribution which closely resembled the wild-type pattern (Fig. 4D,E). A similar rescue was observed for other pan-ventral genes such as Gsh2 and Mash1 (Fig. 4J-M). The restoration of the expression of pan-ventral genes to their normal regions was further demonstrated by the existence of a sharp boundary between the Gsh2 and Pax6 expression domains, similar to what is observed at the corticostriatal boundary of wild-type brains (Fig. 4N,O). Notably, while the telencephalic morphology of the Shh-/-;Gli3+/- embryos was affected, a thickening in the lateral wall of the telencephalon was observed ventrally to the Pax6 expression domain in a region where pan-ventral genes were strongly expressed (Fig. 4D-S; arrowheads). Thus, it seems that the LGE is properly specified in these mutants.
To further investigate to what extent dorsoventral patterning was restored in Shh-/-;Gli3+/- embryos, we looked at the expression of the homeobox gene Nkx2.1, which is missing in Shh-/- embryos. Surprisingly, we observed that low levels of Nkx2.1 expression were rescued in these embryos (Fig. 4F,G). In these animals Nkx2.1 expression was specifically located in the most ventral part of the telencephalon and was nested within the domains of expression of Dlx2 (Fig. 3H,I), Gsh2 and Mash1. Thus, although not very prominent, it appeared that a small MGE-like structure can also form in these embryos in absence of Shh (Fig. 4D-S, asterisks). The high levels of recovery observed in Shh mutants where only one copy of Gli3 gene is removed prompted us to investigate the extent of recovery in animals lacking both Shh and Gli3 gene function.
Dorsoventral patterning is largely restored in the telencephalon of Shh/Gli3 double mutants
Telencephalic analysis of Shh/Gli3 double homozygous mutants was extremely difficult as a result of a high rate of exencephaly. Most of the Shh/Gli3 double homozygous mutants analyzed were exencephalic (11/12=91%): the neural tube failed to close at various levels of the forebrain or midbrain and a massive overgrowth of the tissue disrupted telencephalic morphology. We found that Shh-/-;Gli3-/- embryos had a higher proportion of exencephaly than did Gli3-/- (2/12=16.6%) or Gli3-/-;Shh+/- (10/32=31%) embryos. Although exencephalic mutants could not be analyzed for dorsal patterning defects, ventral patterning was analyzed in parallel to non-exencephalic mutants. As the exencephalic defects were more severe at E12.5 than at E10.5, we chose to analyze the double homozygous mutants at E10.5.
In a Shh-/-;Gli3-/- double mutant embryo that was not exencephalic, the telencephalon appeared to have a relatively normal morphology, although dorsal midline structures were missing (Fig. 5; arrows). The level and distribution of expression of pan-ventral genes such as Dlx2 and Gsh2 was similar in Shh-/-;Gli3-/- embryos and in their wild-type littermates (Fig. 5A-D). Remarkably, in contrast to Shh-/-;Gli3+/- and Shh-/- embryos, double homozygous mutants had a complete rescue of Nkx2.1 expression (Fig. 5E,F). Similar to wild-type animals, Nkx2.1 expression was observed in an area nested within the wider expression domain of Dlx2 (Fig. 5G,H, asterisk) and Gsh2 (Fig. 5C,D). This nested expression pattern suggested that both the MGE and the LGE were specified in Shh-/-;Gli3-/- mutants. However, in the absence of a more refined analysis of gene expression patterns within the ventral telencephalon, we cannot exclude the possibility that some aspects of dorsoventral patterning (such as the ventral midline) are not restored in these mutants. Nonetheless, our data suggest that a key role of Shh in patterning the ventral telencephalon is to inhibit Gli3 function, thereby indirectly allowing ventral gene expression. Complementary to the restoration of ventral patterning defects observed in Shh-/- mutants, the dorsal telencephalic defects present in Gli3-/- mutants were also rescued in Shh-/-;Gli3-/- double mutants. This argues for a requirement for Gli3 in antagonizing Shh function in the dorsal telencephalon, a role apparently not played by Gli3 in the spinal cord (Litingtung and Chiang, 2001a).
Dorsoventral patterning is established properly in Shh-/-;Gli3-/- and Smo-/-;Gli3-/- double mutants. (A-L) Coronal sections of wild-type and Shh-/-;Gli3-/- E10.5 embryos. The telencephalic morphology appears relatively normal in the mutants, except for the loss of dorsal midline structures (arrows). (A-L) Analysis of various homeodomain genes expression. Dlx2 expression assayed by RNA in situ hybridization (A,B) and Gsh2 expression assayed by immunofluorescence (C,D) show that normal ventrolateral patterning is established in the double homozygous mutants. Arrowheads delineate the boundary between lateral and dorsal regions in both wild-type and mutant embryos. Nkx2.1 expression also appears normal (E,F). Overlay of Nkx2.1 (red) and Dlx2 (green) RNA in situ hybridization on adjacent sections using Adobe Photoshop 4 (G,H) shows a similar nested pattern of expression for these two genes in wild-type and Shh-/-;Gli3-/- double homozygous embryos. This reveals the existence of ventral and lateral structures similar to the MGE and LGE in the wild-type embryos (asterisk and arrowhead, respectively). Gli1 expression is absent in Shh-/-;Gli3-/- double mutants (I,J), showing that the Shh pathway is not active. Brackets in I indicate Gli1 expression. (K,L) Ptch is expressed at low levels in Shh-/-;Gli3-/- mutants, with higher expression in ventral areas (bracket), similar to wild-type embryos. (M,N) Analysis of ventral genes expression in Smo-/-;Gli3-/- E10.5 embryos. As a result of exencephaly, the dorsal telencephalic structures are disrupted. Despite this, the ventral structures can still be analyzed. Nkx2.1 and Dlx2 are expressed in ventral areas of the telencephalon. Despite the exencephalic morphology of the mutants ventral pattern appears unperturbed, as evidenced by the presence of Nkx2.1 expression nested within the broader Dlx2 domain. This suggests that MGE and LGE cell fates are specified properly in Smo-/-;Gli3-/- double mutants.
The persistence of dorsoventral patterning in the absence of Shh and Gli3 gene function could be the result of compensation by other members of the hedgehog family or the actions of a hedgehog-independent pathway. To differentiate between these possibilities, we analyzed ventral telencephalic gene expression in Smoothened/Gli3 double mutants, in which all hedgehog signaling is abrogated (Zhang et al., 2001). Although, as in the Shh/Gli3 mutants, these animals suffer from a high rate of exencephaly, ventral aspects of the Smo/Gli3 telencephalon can still be analyzed. In these mutants both pan-ventral markers such as Dlx2, as well as the MGE-specific marker Nkx2.1 were expressed in their normal distribution (Fig. 5M,N). Furthermore, the fact that Nkx2.1 expression was nested within the Dlx2 expression domain suggested that ventral patterning is largely rescued in these compound mutants. Thus, our results reveal that, with the exception of the dorsal midline, a hedgehog-independent signaling pathway is able to establish at least basic elements of dorsoventral patterning in the telencephalon in the absence of both Gli3 and Shh or Smo gene function.
To determine whether the restoration of dorsoventral patterning in Shh-/- mutants when Gli3 is removed (Shh-/-;Gli3-/-) was due to an activation of downstream elements of the Shh pathway independent of Shh protein, we examined the levels of expression of Gli1 and Ptch, two targets of Shh signaling. Gli1 and Ptch are normally expressed in the ventral telencephalon in close proximity to the sites of Shh expression (Platt et al., 1997). Gli1 expression in the telencephalon is restricted to a small region at the border of the MGE and the LGE (Fig. 4P, Fig. 5I; brackets). As in Shh-/- mutants (data not shown) this expression was absent in Shh-/-;Gli3+/- and Shh-/-;Gli3-/- embryos (Fig. 4Q, Fig. 5J), consistent with the suggestion that Gli1 activation is strictly dependent on Shh. In wild-type animals Ptch is expressed in a broader region than Gli1, encompassing the entire extent of the MGE (Fig. 4R, Fig. 5K; brackets). Whereas Ptch expression is absent in Shh-/- and Shh-/-;Gli3+/- mutants (data not shown and Fig. 4S), low levels of Ptch expression were detected throughout the telencephalon in Shh-/-;Gli3-/- mutants, with higher levels ventrally (Fig. 5L, brackets). Higher levels of expression ventrally were also observed in double homozygous mutants that were exencephalic (data not shown). This suggests that Ptch expression is dependent on the removal of Gli3 repressor activity in ventral areas and that the loss of Ptc1 expression observed in Shh-/- mutants may be due to a lack of inhibition of Gli3 function.
In this paper, we present evidence for the existence of a pathway that can establish elements of dorsoventral patterning in the telencephalon in the absence of both hedgehog signaling and Gli3 gene function. We find that ectopic activation of the Shh pathway at E8.5/E9.0 results in ventralization of the telencephalon. Importantly, it appears that the ventral identity induced upon ectopic activation of the Shh pathway is determined by the dorsoventral position of Shh misexpression. Second, we find that some remnants of ventral gene expression persist in Shh-/- mutant animals and, in a complementary fashion, these same ventral genes are expanded in Gli3-/- mutants. Third, analysis of Shh-/-;Gli3-/- and Smo-/-;Gli3-/- mutants shows that aspects of dorsoventral pattern are established in the telencephalon relatively normally in the absence of hedgehog signaling and Gli3 gene function. Nevertheless, we cannot rule out that a more refined analysis of ventral patterning in the telencephalon in these compound mutants will not reveal further defects. Regardless of this caveat, this analysis suggests that the primary role of Shh in the telencephalon is to repress Gli3 function ventrally, and that the primary requirement for Gli3 is to repress Shh function in dorsal areas. The notable exception to this relationship is in the dorsal midline, which is disrupted in Shh, Gli3 and Shh/Gli3 double homozygous mutants. Indeed, the more penetrant exencephalic phenotype in Shh-/-;Gli3-/- double mutants argues that these genes may actually cooperate in this region.
Telencephalic competence to respond to Shh signaling
In the present study, we undertook an in vivo gain-of-function approach to address the role of Shh in telencephalic patterning. By infecting the mouse telencephalon at different time points (E8.5 to E10.5), we showed that the differential induction of MGE and LGE/MGE markers by Shh signaling was determined by the intrinsic character of the infected tissues, within both the dorsoventral and anteroposterior axes. Although it could be argued that the failure to induce Nkx2.1 in dorsal regions of the telencephalon or Gsh2 in dorsomedial telencephalon was the result of our viral vectors producing insufficient levels of Shh signaling, results from our previous studies argue against this (Kohtz et al., 1998). Indeed, Nkx2.1 expression could not be induced in vitro by Shh in E11.5 rat dorsal telencephalic explants (E9.5 in mouse), even when they were exposed to extremely high levels of recombinant protein (i.e. 300 nM). Furthermore, the differential induction we observed with our viral vectors did not appear to be the result of a differential activation of the Shh pathway in dorsal and lateral regions, as Gli1 and Ptch could be induced ectopically in the telencephalon, regardless of where or when the infection occurred. Thus, our gain-of-function data suggest that by E8.5/E9.0 the telencephalon has been patterned along both the dorsoventral and anteroposterior axis. However, these experiments cannot distinguish whether this apparent prepattern is the result of hedgehog independent signaling or patterning by Shh prior to E9.0. In support of the latter suggestion, work by the Edlund laboratory suggests that the telencephalic expression of Nkx2.1 results from Shh signaling in the node (Gunhaga et al., 2000). Similarly, we found that rat explants at headfold stage (E9.5 in rat, E8.0 in mouse) could uniformly induce Nkx2.1 expression in response to recombinant Shh protein (Kohtz et al., 1998), suggesting that the restriction observed in this paper may not exist in slightly younger embryos (before E9.0).
Even if Shh normally acts to prepattern the telencephalon prior to E9.0, our results show that some ventral pattern is established in the telencephalon in absence of Shh. Specifically, we found that Dlx2 and Gsh2 expression within the ventral telencephalon is reduced but not absent in Shh-/- mutant embryos. This result is reminiscent of the persistence of V0 and V1 cell fates in the spinal cord of Shh-/- mutants (Pierani et al., 1999). Given that in some contexts members of the hedgehog family can partially compensate for the loss of Shh (Pathi et al., 2001), perhaps the persistence of ventrolateral markers indicates the presence of other hedgehog ligands. Indeed, Indian and sonic hedgehog are both expressed in the early foregut (Bitgood and McMahon, 1995; Ingham and McMahon, 2001). Unfortunately, this question cannot be addressed directly as Smo-/- mutants die before any telencephalic patterning is molecularly evident (Zhang et al., 2001). However, based on the analysis of Smo/Gli3 double homozygous mutants (see below), such compensation appears unlikely.
The regulation of ventral and lateral patterning in the telencephalon by Shh and Gli3.
To futher investigate the cross-repressive interaction between Shh and Gli3 in the telencephalon, we analyzed several Shh and Gli3 mutant combinations. The development of the telencephalon is grossly abnormal in Shh-/- (Chiang et al., 1996) and to a lesser extent in Gli3-/- mutants (Grove et al., 1998; Theil et al., 1999). We found that telencephalic morphology is largely restored in Shh-/-;Gli3+/- mutants. Consistent with their rescued morphology, most ventrolateral patterning is established in Shh mutants lacking a single copy of Gli3 (Shh-/-;Gli3+/-). Furthermore, barring the loss of dorsal midline structures, mutants lacking both Shh and Gli3 function possess sets of markers characteristic of MGE, LGE and cortex, showing that all these structures are at least partially specified in the absence of Shh and Gli3. The same result was obtained in Smo-/-;Gli3-/- mutants in which all hedgehog signaling is abolished. Therefore, many aspects of dorsoventral telencephalic patterning can be established in the complete absence of hedgehog provided that Gli3 gene function is also abolished.
Despite this, the interplay between Shh and Gli3 is crucial to the normal positioning of the different domains of ventral telencephalic gene expression. For example, our data suggests that MGE and LGE markers are differentially sensitive to the dose of Gli3 protein or activity. Although the expression of pan-ventral genes characteristic of the LGE is fully restored in Shh-/- animals with one copy of Gli3 removed, the MGE marker Nkx2.1 is only rescued in the complete absence of Gli3 gene function. Consistent with this, unlike more broadly expressed ventral markers, Nkx2.1 cannot be induced by ectopic Shh signaling in the cortex, where Gli3 is highly expressed. Similarly, Nkx2.1 expression does not spread dorsally in Gli3-/- mutants, whereas pan-ventral gene expression does. One explanation for these findings may be that Gli2, which is expressed in a pattern similar to Gli3 in the telencephalon, possesses weaker repressor activity than Gli3 and acts in a complementary fashion. In this scenario, Gli2 prevents the expansion of Nkx2.1 expression in Gli3-/- mutants but is insufficient to repress pan-ventral genes, such as Dlx2 and Mash1. Indeed, the greater sensitivity of MGE markers to repression by Gli repressors may be one mechanism that underlies the differential specification of the MGE and LGE.
In the limb, the processing of Gli3 into an N-terminal repressor form is negatively regulated by Shh (Wang et al., 2000). As a result, Shh and the N-terminal Gli3 repressor are thought to form opposing gradients in the limb, akin to that suggested by our genetic analysis of the telencephalon. Our results also in many respects parallel the phenotype found in the spinal cord of Shh/Gli3 double mutants (Litingtung and Chiang, 2000a). Similar to the telencephalon, ventrolateral cell fates in the spinal cord, including motoneurons and V0-V2 interneurons, were restored to wild-type levels in Shh/Gli3 double homozygous mutants. However, unlike the telencephalon, the ventral-most fates fail to form in the spinal cord of these same animals (as indicated by the loss of Nkx2.2 at the floorplate). Moreover, only partial rescue of ventrolateral genes expression was observed in the spinal cord of these mutants, while ventrolateral telencephalic gene expression appeared normal in Shh-/-;Gli3+/- mutants. Furthermore, in the telencephalon the antagonism between Shh and Gli3 is reciprocal. By contrast, there is no evidence that Gli3 may antagonize Shh function in the spinal cord, as ventral gene expression does not expand dorsally in this tissue in the absence of Gli3 gene function.
Evidence for a hedgehog independent pathway in patterning the ventral telencephalon
In light of the Shh/Gli3 and Smo/Gli3 double mutants phenotypes, it is worth considering how other signaling pathways might contribute to dorsoventral telencephalic patterning. It is possible that hedgehog-dependent and -independent pathways act in parallel and are functionally redundant, each able to specify ventral, lateral and dorsal cell fates in isolation. When hedgehog signaling is missing, as it is in Shh-/-;Gli3-/- and Smo-/-;Gli3-/- mutants, such redundancy would account for the observed rescue of dorsoventral patterning (Fig. 6). The use of two pathways that complement each other is reminiscent of the mechanism of anterior patterning in Drosophila. In flies, opposing anterior and posterior gradients specify the segmented body pattern. Maternal nanos protein acts as a repressor of hunchback in the posterior region, allowing abdominal patterning to occur; and the absence of both genes results in normal embryos (Hulskamp et al., 1989; Irish et al., 1989; Struhl, 1989). In parallel, bicoid specifies anterior patterning and hunchback can substitute for it in thorax and abdomen (Simpson-Brose et al., 1994; Wimmer et al., 2000).
Model of genetic interactions between Shh and Gli3 in patterning the mouse telencephalon. (A) Schematic representation of a coronal section through an E10.5 mouse telencephalon, highlighting different domains along the dorsoventral axis. The prospective regions of the cortex (blue), the LGE (green) and the MGE (red) are shown. On the right, homeodomain genes are indicated that are expressed in these regions and have been shown to play an essential role in patterning the telencephalon (reviewed by Wilson and Rubenstein, 2000). Note that expression of Gsh2 is only restricted to the lateral telencephalon for a short window of embryonic development (E10.0-E10.5) (Corbin et al., 2000). Ultimately Gsh2 is expressed in both the LGE and MGE. (B) The results of the present study demonstrate that Shh is inducing ventral (Nkx2.1) and lateral (Gsh2, Dlx2) telencephalic patterning indirectly, through the inhibition of Gli3 repressor activity. Note that the regulation of Nkx2.1 expression appears to be different from that of pan-ventral genes, as it is not affected in Gli3-/- single mutants (bold blunt arrow). Gsh2 expression and Pax6 expression at the corticostriatal boundary are repressed by each other. However, the repression of Gsh2 expression by Pax6 appears to be regulated by Gli3 (asterisk), as co-expression of these two genes is observed in the cortex of Gli3-/- mutants. Our data reveal the existence of an unknown hedgehog-independent pathway (X).
Alternatively, we cannot formally exclude the possibility that the dorsoventral patterning of the telencephalon is initially established by a hedgehog-independent pathway and that the role of Shh in patterning the telencephalon is to subsequently maintain or refine the organization of the telencephalon. Perhaps Shh and Gli3, through their antagonistic interactions, act to stabilize dorsoventral telencephalic domains during mid-neurogenesis. This might occur by regulating the growth of the progenitor pools, by maintaining a pre-established pattern or by refining the regional boundaries between discrete territories, such as the MGE, LGE and cortex.
The identity of the signal(s) that lead to a normal dorsoventral patterning in the absence of Shh and Gli3 remains unknown. One possibility is that the dorsoventral patterning observed in Shh/Gli3 double mutants is the result of BMP signaling, which in spinal cord acts to induce dorsal and repress ventral gene expression. Recent studies in the spinal cord have also shown that BMPs antagonizes Shh signaling and that this inhibition may be crucial to the establishment of dorsal identities (Liem et al., 2000). It seems unlikely that the late dorsal expression of BMPs contributes to hedgehog-independent patterning as their expression requires Gli3 (Theil et al., 1999). However, earlier BMP7 expression in the prechordal plate or BMP4 expression in presomitic mesoderm might be playing a role (Dale et al., 1997; Dale et al., 1999), as might Nodal signaling (Rohr et al., 2001). Another alternative comes from studies in spinal cord that have implicated the retinoid signaling pathway in the generation of ventrolateral progenitors (Pierani et al., 1999). In the telencephalon, markers of retinoid synthesis are expressed in the LGE and the developing striatum (Li et al., 2000; Toresson et al., 1999) and retinoid signaling regulates striatal neuronal differentiation (Toresson et al., 1999). However, this expression occurs too late to play a role in the initial patterning of the telencephalon. If retinoids do act in the establishment of dorsoventral pattern in the telencephalon, a more likely source is the lateral cranial mesoderm, which expresses high levels of retinoids and is proximal to the telencephalon during the headfold stage ∼E7.5/E8.0 (LaMantia et al., 1993). A third candidate is the Wnt pathway. Recent work has implicated an important role for Wnt inhibitors in the specification of the head (dickkopf) (Niehrs et al., 2001) as well as the telencephalon (masterblind) (Heisenberg et al., 2001; van de Water et al., 2001). Furthermore, a novel secreted Frizzled-related protein expressed in the anterior neural plate at the junction between neural and non-neural ectoderm is involved in promoting telencephalic development within the forebrain territory (Houart et al., 2002). Prior to neural tube closure, the dorsoventral axis of the telencephalon seems to be roughly translated into an anteroposterior axis (Shimamura et al., 1995). Thus, it is worth considering that, in addition to specifying the telencephalon as a whole, graded inhibition of Wnt signaling may also act to establish dorsoventral identity in the telencephalon.
Regardless of the pathway, it is clear that a complete appreciation of regional patterning within the telencephalon will require more than understanding the hedgehog signaling pathway. Determining the nature of these signals and how they act to complement the hedgehog pathway in patterning the telencephalon will no doubt prove interesting.
We thank Susana Nery, Heather Mason, Mike Rutlin, Alex Joyner, Alex Schier and Yorick Gitton for critical reading of this manuscript. We also thank the following people for the gifts of mice, probes or reagents: H. Westphal and C. Chiang (Shh mutant mice), J. Rubenstein (Dlx2), F. Guillemot (Mash1, Ngn2, Math2, NeuroD), F. Savage (actSmo M2), A. Joyner (Nkx2.1, Gli1, Gli3, Ptc1), A. Kawakami (anti-Pax6), and K. Campbell (anti-Gsh2). We are grateful to Steve Wilson and Kenny Campbell for sharing unpublished data, as well as Petra Kraus, who first observed the residual Dlx2 expression in Shh-null mutants. We also acknowledge the contributions of Mike Rutlin and Yuan Yuan Huang for their technical assistance, as well as Jill McMahon for the breeding and genotyping of the Gli3/Smoothened compound mutants. This work was supported by a NIH grant (NS39007), a March of Dimes basic research grant and a Children's Brain Tumor Foundation grant to G. F.; by postdoctoral grants from l'Association pour la Recherche Contre le Cancer to M. R.; by an American Cancer Society grant to N. G. (PF4473); and by a NIH grant to J. C. (NS10962-01) and R. M. (1F32NS42525-01).
Accepted July 23, 2002.
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Meet New York-based Eduardo Leyva-Díaz and Oliver Hobert, authors of a recent Development paper on transcriptional autoregulation in C. elegans.
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Birds In the City w/ The Bronx Zoo: Flamingos, Penguins, and Terns
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What We'll Do
It's 3pm. Do you know what the flamingoes are doing?
Join Atlas Obscura and the Bronx Zoo for a one-of-a-kind sneak peek into the world of curious birds! During this hour-long live online event, you'll get a chance to be digitally up-close and personal with a number of rare and incredible birds. We'll visit the Bronx Zoo's flock of flamingos, and see the Inca terns dive to catch their fish (and show off their mustaches). Our penguins will swim by, and our owl might even come by for a "hoo-llo"! During this virtual experience you will meet one of the Bronx Zoo's Ornithology team (i.e. a bird keeper), and you'll have a chance to ask them questions live in the moment. Find out what it takes to care for these fantastic birds, what working at the Bronx Zoo is like, and the conservation work they do to protect wildlife and wild places. And yes, there will be trivia. Hint: look up what a flock of flamingos is called!
So grab your favorite friends and family and flock together for this unforgettable experience, no matter where you are!
Where We'll Be
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The Bronx Zoo
The Bronx Zoo, the Wildlife Conservation Society's flagship park, opened on November 8, 1899. Since then, the zoo has created a unique window into the natural world for city dwellers in the heart of the Bronx — and serves as a springboard and hub for conservation efforts across the entire planet. The Bronx Zoo is home to over 700 different species of animals and covers 265 acres, making it one of the largest urban zoos.
What Else You Should Know
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redpajama_set_name: RedPajamaCommonCrawl


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Die Ruine Waldenburg ist die bedeutende Ruine einer hochmittelalterlichen Höhenburg bzw. Landvogteischlosses oberhalb des Städtchens Waldenburg im Schweizer Kanton Basel-Landschaft.

Lage
Die Ruine der ehemaligen Burganlage liegt auf  über einer Talenge der Vorderen Frenke auf dem länglichen, schmalen Felsrücken Rehag rund 170 Meter über der Passstrasse über den Oberen Hauenstein. Es sind heute noch bedeutende Mauerreste sichtbar, welche sich etwa 600 Meter östlich des – ehemals ummauerten – Städtchens Waldenburg erheben. Geschützt wird die Burg durch steile Böschungen und Felsen auf der Nord-, West- und Südseite, während die Annäherung an der Ostseite durch einen Halsgraben erschwert ist.

Anlage
Die ganze Anlage der Waldenburg kann in eine langgezogene, ost-west-ausgerichtete Kernburg (50 × 20 Meter) mit neuerem, westlichem und älterem, östlichem Baukörper sowie eine Vorburg gegliedert werden, die die Kernburg auf der nördlichen, westlichen und südlichen Seite umgibt.

Die Kernburg liegt auf dem höchsten Teil des Felsgrates und besteht in ihrem östlichen, älteren Teil aus dem Bergfried (in der nordwestlichen Ecke, wohl ältester Bauteil) sowie mehreren Wohnbauten entlang der rechten Hälfte des südlichen Berings. Im Hof des östlichen Teils befindet sich eine Filterzisterne und Spuren deuten auf einen an den Bergfried angebauten Backofen hin. Der westliche Teil wird durch einen grossen Palas dominiert, der 4 oder 5 Etagen hoch gewesen sein könnte und nur durch den Bergfried überragt wurde. Dieser Palas wurde erst im späten 13. Jahrhundert errichtet und ist auf Abbildungen mit einem Walmdach und Kamin dargestellt.

Die Vorburg beginnt mit den direkt zur Burg gehörenden Oekonomiegebäuden im Norden der Anlage. Von dort führt ein Aufgang zum äusseren Tor in der nordwestlichen Ecke der Burg. Ein Zwinger mit Torhaus führt um die westliche Schmalseite des Burgfelsens herum zu einem Treppenaufgang, der zum inneren Tor etwa in der Mitte des südlichen Berings aufsteigt. Das innere Tor war mit einem kleinen Torgraben geschützt.

Der zur Waldenburg zugehörige Burghof lag im Bereich des heutigen Sennhus 350 Meter nordöstlich der Burg: Das Städtchen Waldenburg ist nicht als Weiterentwicklung des Burghofes zu betrachten, sondern eine eigenständige Gründung, die mit ihren Mauern und Toren die Benutzung der Passstrasse kontrollierte und von wo aus auch die Dienstleistungen (Säumerdienste, Lagerung, Verköstigung, Übernachtung etc.) angeboten wurden.

Geschichte
Die Waldenburg wurde um 1200 von Graf Hermann von Frohburg (1160–1211) auf Eigengut erbaut, das die Frohburger vom Kloster Murbach übernommen hatten. Die ältesten Teile der Burg Waldenburg sind etwas älter als das Städtchen Waldenburg: Hieraus und aus der verkehrs- und wirtschaftspolitischen Aktivität der Frohburger darf geschlossen werden, dass die wesentliche Motivation zur Errichtung der Festung auf der Erhebung von Strassenzöllen und Entlöhnung von Säumerdiensten bei der Benutzung der Passstrasse lag. Die Burg wurde für die Frohburger so wichtig bzw. einträglich, dass sich ein Zweig der Familie nach der Waldenburg benannte.

Die Burg war Ergänzung und mit der Zeit Nachfolger der Schanz (auch Alt-Schloss, aufgegeben um 1300), einer ebenfalls von den Frohburgern errichteten Anlage. Dass die Lage durchaus wirtschaftlich interessant war zeigt auch die Tatsache, dass das Steinhaus in Waldenburg (heute Pfarrhaus) von den Herren von Lampenberg bis zu ihrem Aussterben 1250 benutzt wurde.

Graf Ludwig von Frohburg versuchte in der Mitte des 13. Jahrhunderts die Waldenburg (mitsamt dem Städtchen) an die Habsburger zu verkaufen und als Lehen zurückzuerhalten. Dies entspricht einem damals im Feudalsystem üblichen Vorgehen, um sich des Schutzes eines grösseren Landesherren zu versichern. Da dies misslang, verkaufte er 1265 Burg und Städtchen an den Bischof von Basel (mit Bestätigung 1277) und erhielt alles als Lehen zurück.

Das Erdbeben von Basel hat 1356 weder Burg noch Städtchen Waldenburg stark in Mitleidenschaft gezogen.

Als 1366 Graf Johans von Frohburg starb, zog der Bischof von Basel das Lehen ein und gründete daraus ein Amt (mit Waldenburg, Langenbruck, Oberdorf, Niederdorf, Hölstein, Lampenberg, Liedertswil, Bennwil, Ramlinsburg, Reigoldswil, Lauwil, Ziefen, Bubendorf, Arboldswil, Titterten, Lupsingen), das von einem bischöflichen Vogt auf der Waldenburg verwaltet wurde.

Der Basler Bischof verpfändete das Amt Waldenburg 1374 an Leopold III. (Habsburg) (Herzog von Österreich), da er wegen Streitereien mit Basel nicht die Stadt berücksichtigen wollte. Bereits 1375 gab er aber das Kleinbasel anstelle des Amtes Waldenburg an Leopold III als Pfand: Das Waldenburg verpfändete er sofort an die Grafen von Thierstein und – nach einem Guglerüberfall sowie weiteren Wechseln der Pfandleiher – 1396 an den Markgrafen Rudolf III. (Hachberg-Sausenberg) und 1400 – zusammen mit Liestal und Homburg – an die Stadt Basel. Durch den enormen Geldbedarf des Bischofs von Basel wurde die Pfandsumme auf das Amt Waldenburg weiter erhöht, bis es dem Bischof unmöglich wurde, das Pfand wieder einzulösen: Formell übergab der Bischof aber das Amt Waldenburg erst 1585 an die Stadt Basel.

Seit 1400 sass ein Vogt der Stadt Basel (zuerst Werner Schilling) auf der Waldenburg und verwaltete von dort aus das gleichnamige Amt. Er musste als erstes aber die Festung Waldenburg wieder sanieren, denn der Bischof hatte sie stark vernachlässigt.

Im Rahmen der aggressiven Expansionspolitik von Solothurn nach Norden, versuchten die Solothurner 1444 die Waldenburg – zum Zeitpunkt als die Besatzung mit den Eidgenossen nach St. Jakob gezogen war – und die damit verbundenen Güter und Rechte durch einen Überfall in ihren Besitz zu bekommen, was aber misslang.

Im Umfeld der Revolutionswirren wurde 1798 die Waldenburg vom Basler Vogt verlassen und dann – wie andere Landvogteischlösser (Farnsburg, Homburg) – angezündet.

Die heute sichtbaren Reste der Festung wurden im Wesentlichen 1929 bis 1931 ausgegraben und saniert, wobei allerdings der Schwerpunkt der Arbeiten, entsprechend dem Geist der Zeit und den archäologischen Möglichkeiten, auf der baulichen 'Instandsetzung' und nicht auf der archäologischen Erforschung lagen.

Im Jahre 2002 musste die Ruine durch die Gemeinde Waldenburg und die Kantonsarchäologie Baselland saniert werden, da – auf Grund früherer, unsachgemässer Reparaturen – lose Steine und Mauerstücke die Besucher und die Bausubstanz gefährdeten.

Aussichtsturm
96 Treppenstufen aus Holz und Stein führen zur Aussichtsplattform in ca. 17,5 Meter Höhe. Von dieser hat man einen Ausblick auf die Ortschaften Niderdorf, Oberdorf und Waldenburg sowie auf diverse Täler und Hügel des Juras.

Darstellungen
 Matthäus Merian: Waldenburg von Süden. Stich, 1642.
 Emanuel Büchel: Waldenburg von Norden. Zeichnung, 1754.

Siehe auch
 Liste von Burgen und Schlössern in der Schweiz

Literatur
 Werner Meyer: Burgen von A bis Z. Burgenlexikon der Regio. Herausgegeben von den Burgenfreunden beider Basel aus Anlass ihres 50-jährigen Bestehens. Druckerei Klingental, Basel 1981, S. 130–132.
 Carl Roth: Die Burgen und Schlösser der Kantone Basel-Stadt und Basel-Landschaft. Band 4b, Birkhäuser, Basel 1932, S. 36 ff.

Weblinks

 Burgenwelt: Waldenburg

Einzelnachweise

Waldenburg, Ruine
Kulturgut von regionaler Bedeutung im Kanton Basel-Landschaft
Waldenburg BL

redpajama_set_name: RedPajamaWikipedia


Men's gold-plated and steel wristband watch. Gilt dial with index markers and date aperture at 3 o'clock. Mechanical swiss movement signed Jaeger, calibre 046. Damaged leather strap.
Gross weight : 31.8g (1.12oz).
Condition report : In working order but working condition not guaranteed.


text:
Hep C Risk Highest for Baby Boomers, but Most Haven't Been Tested
Almost three-quarters of those most likely to be living with chronic hepatitis C virus (HCV) infection—baby boomers, notably those born between 1945 and 1965—have never been tested or are unsure if they have been tested for hepatitis C, according to a new survey conducted by the American Gastroenterological Association (AGA).
The survey, released in advance of National Hepatitis Testing Day, also found that 80 percent of polled baby boomers do not consider themselves at any risk for having the disease, despite the fact that 82 percent of the roughly 5 million U.S. residents with the disease are between 47 and 67 years old and 75 percent don't know they have it.
In addition to a lack of knowledge, the survey showed a lack of action: 83 percent of the baby boomers surveyed have never discussed hepatitis C with their health care provider, even though it is diagnosed with a simple blood test and for many people it can be cured.
Because many boomers have been infected for decades, AGA notes, the number of people who die from hepatitis C–related liver problems is expected to increase by 207 percent from 2000 to 2030.
The survey of more than 1,000 baby boomers not previously diagnosed with hepatitis C was conducted online as part of a new Vertex Pharmaceuticals-funded educational campaign called "ID Hep C."
The survey also indicates that 83 percent of baby boomers don't realize their generation is most likely to have hepatitis C. Instead, half (52 percent) believe all age groups have a similar risk, and nearly one quarter (24 percent) think those in Generation X—people between 31 and 46 years old—are more likely to have the disease.
Fifty-five percent of those surveyed also think every ethnic group has the same likelihood of having hepatitis C, even though African Americans and Latinos are affected by hepatitis C at a significantly higher rate than the general population.
Fewer than one in five of those surveyed knew that for many people, hepatitis C can be cured.
"Many baby boomers have a potentially dangerous 'it's not me' mentality about hepatitis C, and this survey underscores how poorly most people in that generation understand that risk factors do apply to them," said Ira M. Jacobson, MD, of Weill Medical College of Cornell University and an advisory to AGA's campaign in a news announcement. "Given the potentially deadly consequences of allowing hepatitis C to go undiagnosed, the AGA urges all baby boomers to talk to their doctors about getting tested."
#baby boomers


We will be making signs, arranging flowers for the winners, creating the Good Luck cards (for people to buy), working on raffle items, making VIP signs and more for the show. All the last minute goodness wrapped up in a party! If you would like to help, let us know. We would love to have you!
We would love help setting up before the pageant. Everyone who helps us set-up can then transition into a "job" when doors open. Below are all of the things we need help setting up when the theater opens to us at 3:00pm.
Post VIP Seating in theater for visiting titleholders, titleholder families (Miss Solano & Miss Napa County) and other important people. Diagram will be provided.


text:
aA Special Report
SOUTH BAY/MID-CITIES: Doldrums Strike After Fleeting Pickup
Special Report: REAL ESTATE QUARTERLY
By Natalie Jarvey
Major events in Los Angeles County commercial and industrial submarkets in the third quarter.
After a mild recovery earlier this year, the South Bay and Mid-Cities industrial markets contracted a case of the summertime blues.
Vacancy rates in the Mid-Cities stayed the highest in the county at 5 percent, while the South Bay saw vacancies rise two-tenths of a percent to 3 percent, according to Grubb & Ellis Co. There also was little change in asking rents; in Mid-Cities they decreased by one cent to 42 cents and in the South Bay they rose by one cent to 53 cents.
"The market is still lethargic. We're fairly flat, kind of rumbling along," said Jim Biondi, senior vice president at Grubb & Ellis Co. "I still don't think we've turned the corner."
Brokers said activity was slow because many businesses took advantage of the market's rebound at the beginning of the year and signed new leases then. Also, decreased container activity at the ports following the pre-holiday surge could explain the quarter's lethargy.
"We saw a dramatic increase in container activity at the ports," said Howard Schwimmer, senior managing partner at Rexford Industrial. "That's tapered off now that people are fully stocked for the holidays."
That also could account for the rise in the vacancy rate in the South Bay, which is traditionally stronger than its Mid-Cities neighbors because of its proximity to the port. But Steve Calhoun, a broker at the Commerce office of Colliers International, said the Mid-Cities market will get just as soft as the South Bay in the coming year.
"The best thing going for the Mid-Cities is that there's no land for development," he said. "It won't be flooded with new inventory."
Industrial Markets At a Glance
Inventory: 331 million square feet
Under Construction: 0
Asking Rents: 42 cents (Mid-Cities); 53 cents (South Bay)
A 5.5-acre parcel in Carson was leased to MV Transportation, a Bay Area passenger transportation services company, in July. MV Transportation paid $5.5 million for a 10-year lease on the property at 21222 S. Wilmington Ave., owned by Hager Pacific Properties and SoCal Industrial Partners LLC. The parcel includes a 22,000-square-foot maintenance and office building and a five-acre paved yard. MV Transportation signed an $86 million contract with the L.A. Metropolitan Transit Authority in June to service its buses.
RPM Transportation, a traffic engineering and consulting firm, renewed its lease for a warehouse in Santa Fe Springs in August. The 174,000-square-foot property is owned by Golden Springs Development Co. RPM, based out of Brentwood, Tenn., will stay in the building in the Golden Springs Business Center at 13225 Marquardt Ave. for another three years. Terms of the deal were not disclosed.
Saber Avalon LLC bought a 71,900-square-foot property in Gardena from Eugene Halperin 2003 Trust in August. The industrial land at 15000 S. Avalon Blvd. has a fenced-in yard and water, electrical and lighting capabilities. The price was unavailable.
Watson Land Co. leased a 103,000-square-foot warehouse in Carson to Coby Electronics. The Lake Success, N.Y., consumer electronics manufacturer leased the space at 989 E. 233rd St. for an unknown price.
Spectrum Bags Inc., a maker of plastic grocery bags, renewed its lease at 12850 Midway Place in Cerritos. Spectrum signed up for five years at the 113,000-square-foot property from Bechler Corp. at 50 cents per square foot.
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MAICO's History
More Than 80 Years of Experience in Making Hearing Screening Easier
At MAICO, we design and build our devices to ease your daily work — and with each new device, we get even better at doing so. Now with more than 80 years of experience in making hearing screening easier, we improve your workflow with fast tests and intuitive operation.
It All Started with a Vision
Our focus on creating easy-to-use hearing test equipment lies in our history and continues to shape our future: our founder Leland A. Watson built MAICO on a vision of creating easy-to-use hearing test equipment. Watson was the son of an ear physician in Minneapolis and worked as a sales clerk for hearing instruments. He recognized early on that the devices he sold to doctors like his father were too complicated and should be easier to use. However, since there were no devices on the market at that time which were easier to use, he decided to take matters into his own hands and founded "the Medical Acoustics Instruments Company" in 1937. The company name was later shortened to MAICO and became known for intuitive "audiometers", a term that Watson coined.
Becoming a Part of Robert Bosch GmbH
MAICO's easy-to-use devices attracted the attention of the Robert Bosch GmbH in Berlin, Germany — and so the audiological division of Robert Bosch GmbH merged with MAICO Minneapolis in 1991. Since then, MAICO has had headquarters in Minneapolis as well as in Berlin.
Shaping the Future of Hearing Screening with Strong Companions
The William Demant Holding A/S — one of the largest hearing healthcare instrument providers in the world — developed an interest for MAICO and its devices as well. In 1995 MAICO joined the Holding and thus has access to a large database of knowledge around hearing testing — a great foundation to develop even better hearing test devices.
Uniting Two Visions to Reach One Goal
Just like MAICO, the William Demant Holding was built on a vision. The history of William Demant starts with the company's founder – Hans Demant — and his passion to help his wife lead a better life — despite her hearing loss. This passion soon evolved into a vision to empower people with hearing loss all around the world and support them in improving their lives. As a member of the William Demant Holding, we still share this goal at MAICO today. The company values of William Demant Holding fit perfectly with our own vision at MAICO: An early detection of hearing loss today for a healthy and enjoyable life tomorrow.
© 2019 | All rights reserved | MAICO Diagnostics may not offer the products, services, or features displayed on this website in all countries.


Nestled on eighteen acres of pasture and wooded areas our animals are raised in their own environment, humanely treated, antibiotic and hormone free. They are fed a well balanced diet of grain and vegetables along with enjoying their choice of pasture grass. We are Certified Disease Free, Pork Quality Assured and USDA inspected. Our smoked products are Nitrate Free and we only use organic based spices, and our sausage links are casing free. Our Duroc and Heritage bred swine provide us with the best tasting pork products that we can provide to our customers. We are a closed herd, we do not let any of our animals leave for breeding purposes, and do not let any animals in to be bred by our boars. We only use our animals that are raised on our farm for purchases of meat, we do however buy local beef from certified farms to supplement our demand of grass or grain fed beef. We invite the public to come see our farm, however during farrowing (baby time) we cannot let people into our barn due to contamination of disease.


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Auto Club Group provides $250,000 in scholarships to 5 HBCUs
DEARBORN, Mich. (AP) — Students at five historically Black colleges and universities will share $250,000 in scholarships from Dearborn, Michigan-based The Auto Club Group.
The AAA Social Justice Innovator Scholarship will be offered to students pursuing careers in science, technology, engineering, math and business at minority serving schools in the 14 states where The Auto Club Group (ACG) operates.
The Auto Club Group says the program is part of its celebration of Black History Month and intended to help reduce barriers for underrepresented students in higher education.
The first round of funding is going to Bethune-Cookman University in Florida, Claflin University in South Carolina, Clark Atlanta University in Georgia, Fisk University in Tennessee and North Carolina Central University.
To read more details on the scholarships, click here.
For more news happening across Tennessee, click here or download the WBBJ 7 Eyewitness News app.
Categories: Tennessee News
'Armed & dangerous': Escaped inmates may be in Tennessee


Chris Murphy Apr 14th 2019 - 11am.
Fargo Force defenseman and University of North Dakota commit Ethan Frisch has called Moorhead home for his entire life. His dad and uncle both played high school hockey for the Spuds. His two aunts were professional figure skating coaches, so he's been skating since he was 2 years old and had a hockey stick in his hand by the age of 3.
This summer he told the Moorhead hockey coaching staff he needed to meet with them. Moorhead varsity hockey coach Jon Ammerman said he pretty much knew what was coming.
"You play with those guys your whole life and know the coaching staff since you were little," Frisch said. "It's really hard. I told my coaches that I was going to continue with Green Bay. I got a lot of support from family and friends and the coaches."
"Obviously, it's a tough decision," Ammerman said. "He had a decision him and his family had to make and one that they thought was best for his career. We're going to cheer for the kid no matter what happens and we hope he has a really long career and does extremely well. He's a great kid from a great family."
"I got a good taste of it," Frisch said. "I loved the coaching staff and area. I liked the lifestyle. It's more hockey, while still taking care of school work."
"You learn to deal with adversity," Frisch said. "Not every season is going to be the best season as far as wins and losses, but you have to battle back and find a way to get better and keep fighting. It was different, but it was cool to see that side of things. I hate losing, but it's good for me to experience and get to know how to deal with those things."
"I had to follow my head in my decision and what's best for me," Frisch said. "It's exactly what I was hoping for. I got to experience two separate sides of the league. It's been a great wealth of knowledge."
"He's our type of player," Eades said. "He's smart, aggressive, a 200-foot defenseman. He can play both sides of the puck. He's all eyes, all ears, one of those players that soaks things in like a sponge."
"I think his defensive side of the game has grown," Eades said. "He was a little bit of a smaller kid, but he's gotten a lot thicker and stronger. That has helped him advance defensively. He always had the offensive side. He's got really good calmness and patience with the puck and, yet, doesn't move it quickly. We preach a lot to get the puck moving north."
Less than three years ago, on Oct. 24, 2016, five days before his 16th birthday, Frisch committed to UND before ever playing a high school hockey game. Since then, he's helped lead the Spuds to a state championship game, played in Hockey Day Minnesota, been part of a documentary following the Spuds hockey season and had to make a decision to move to Green Bay and skip his senior high school hockey season.
All that's in front of him is the USHL playoffs, heading to UND next season and most likely being a late-round draft pick in the NHL draft this June.
He's beginning to learn the business side of hockey, but it's still a game to him. It's a game that's brought him ups and downs, difficult decisions and Moorhead hockey apparel he still wears. It's a game he says a prayer before. It's a game he's learned to step away from to hunt, fish and just sit inside with family and friends.
It's a game he can step away from, but not a game he can imagine living without.
"I wouldn't want to be in any other place," Frisch said. "I've got to experience a lot of things a lot of players will never get to. It's been a fun ride, and I can't complain at all.
"Hockey, to me, I still love it and am having as much fun with it as I ever have. You go into your draft year and you realize it's a business and you have to promote yourself as a player, but if I didn't love the game, I wouldn't want to do it. It's one of my biggest passions."


At Chapelli's Spaghetti Bar and Restaurant we never close!
We are fully licensed 24 hours a day, offering Italian Cuisine and Casual Dining.
Located in busy Chapel Street, Melbourne's very own fashion Mecca. Whether you are shopping or clubbing Chapelli's is always open.