BMP signaling is required locally to pattern the dorsal telencephalic midline

BMPs have been proposed to pattern the medial-lateral axis of the telencephalon in a concentration-dependent manner, thus helping to subdivide the embryonic telencephalon into distinct forebrain regions. Using a CRE/loxP genetic approach, we tested this hypothesis by disrupting the Bmpr1a gene in the telencephalon. In mutants, BMP signaling was compromised throughout the dorsal telencephalon, but only the most dorsalmedial derivative, the choroid plexus, failed to be specified or differentiate. Choroid plexus precursors remained proliferative and did not adopt the fate of their lateral telencephalic neighbors. These results demonstrate that BMP signaling is required for the formation of the most dorsal telencephalic derivative, the choroid plexus, and that BMP signaling plays an essential role in locally patterning the dorsal midline. Our data fail to support a more global, concentration-dependent role in specifying telencephalic cell fates.     

A Bmp Reporter with Ultrasensitive Characteristics Reveals That High Bmp Signaling Is Not Required for Cortical Hem Fate

Insights into Bone morphogenetic protein (Bmp) functions during forebrain development have been limited by a lack of Bmp signaling readouts. Here we used a novel Bmp signaling reporter (“BRE-gal” mice) to study Bmp signaling in the dorsal telencephalon. At early stages, BRE-gal expression was restricted to the dorsal telencephalic midline. At later stages, strong BRE-gal expression occurred in neurons of the marginal zone and dentate gyrus. Comparisons to nuclear phospho-Smad1/5/8 (pSmad) and Msx1 indicated that BRE-gal expression occurred exclusively in neural cells with high-level Bmp signaling. BRE-gal responsiveness to Bmps was confirmed in reporter-negative cortical cells cultured with Bmp4, and both in vivo and in vitro, BRE-gal expression was switch-like, or ultrasensitive. In the early dorsal telencephalon, BRE-gal expression negatively correlated with the cortical selector gene Lhx2, indicating a BRE-gal expression border that coincides with the cortex-hem boundary. However, in Lhx2 null chimeras, neither BRE-gal nor nuclear pSmad increases were observed in ectopic hem cells. These findings establish BRE-gal as an ultrasensitive reporter of Bmp signaling in the dorsal telencephalon, imply that hem fate can be specified at different Bmp signaling intensities, and suggest that Lhx2 primarily regulates the responses to – rather than the intensity of – Bmp signaling in dorsal telencephalic cells.     

Gli3 is required for Emx gene expression during dorsal telencephalon development.

Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development.     

Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis

Null mutation of the Foxg1 gene causes hypoplasia of the mouse telencephalon and loss of ventral telencephalic structures. We show that a crucial early requirement for Foxg1 is in the induction of ventral cell fate in the telencephalon. To study later proliferative defects, we have adapted an iododeoxyuridine and bromodeoxyuridine double labeling protocol for use in the developing embryo, which allows estimation of cell cycle kinetics in a single specimen. This technique is used to demonstrate that the cell cycle is prematurely lengthened in the Foxg1-null telencephalon. These defects are first apparent at embryonic day 10.5 (E10.5) and are most severe in the rostral telencephalon. We show that apoptosis is also reduced in the same rostral domain. These defects correspond temporally and spatially with a dramatic reduction in expression of the potent signaling molecule Fgf8. We also show that in the absence of Foxg1 an excess of neurons is produced from E11.5, depleting the progenitor pool and limiting the growth of the Foxg1(-/-) telencephalon. The increase in neurogenic division coincides with an increase in BMP signaling, as detected by immunohistochemistry for phosphorylated smad-1, -5, and -8. This study reinforces Foxg1's position as a major regulator of telencephalic neurogenesis and supports the idea that Foxg1 controls precursor proliferation via regulation of Fgf signaling and differentiation via regulation of Bmp signaling.     

Mutations in the BMP pathway in mice support the existence of two molecular classes of holoprosencephaly

Holoprosencephaly (HPE) is a devastating forebrain abnormality with a range of morphological defects characterized by loss of midline tissue. In the telencephalon, the embryonic precursor of the cerebral hemispheres, specialized cell types form a midline that separates the hemispheres. In the present study, deletion of the BMP receptor genes, Bmpr1b and Bmpr1a, in the mouse telencephalon results in a loss of all dorsal midline cell types without affecting the specification of cortical and ventral precursors. In the holoprosencephalic Shh(-/-) mutant, by contrast, ventral patterning is disrupted, whereas the dorsal midline initially forms. This suggests that two separate developmental mechanisms can underlie the ontogeny of HPE. The Bmpr1a;Bmpr1b mutant provides a model for a subclass of HPE in humans: midline inter-hemispheric HPE.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

Generation of Frizzled10-Cre transgenic mouse line: a useful tool for the study of dorsal telencephalic development

Wnt signaling plays an important role in regulating cortical and hippocampal development, but many of the other molecular mechanisms underlying dorsal telencephalic development are largely unknown. We are taking advantage of the highly regionalized expression patterns of signaling components of the Wnt pathway to generate new mouse lines that will be useful for studying forebrain development. Here, we describe a transgenic mouse line where Cre is driven by the promoter of the Wnt receptor, Frizzled10. In these mice, Cre activity is mainly detected in the dorsal telencephalon during development and is confined to the pyramidal cell fields in the adult hippocampus. The Cre recombinase has very high efficiency when assayed by crossing the transgenic line with the ROSA26 reporter line. Thus, this Cre line will be useful for the study of dorsal telencephalic development and conditional inactivation of target genes in the cortex and hippocampus.     

Six3 cooperates with Hedgehog signaling to specify ventral telencephalon by promoting early expression of Foxg1a and repressing Wnt signaling

Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/β-catenin pathway.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelia formation

Choroid plexus (CP) produces the cerebrospinal fluid (CSF) of the central nervous system (CNS), but little is known about the mechanisms underlying development of this important tissue. CP forms in the hindbrain (4th ventricle), diencephalon (3rd ventricle) and dorsomedial telencephalon bilaterally (lateral ventricles). All of these sites lie at or near the embryonic dorsal midline (DM), which acts as a CNS patterning center. We therefore examined DM-CP relationships using normal and Gdf7 (Bmp12) transgenic embryos to fate map or ablate DM cells. These studies revealed a Gdf7 fate map that includes most CP epithelial (CPe) cells of the hindbrain and diencephalon. In the telencephalon, Gdf7 cell lineages were found in the small anterior domain of telencephalic CPe (tCPe), but its large posterior domain was devoid of these lineages. Anterior and posterior tCPe domains, which arise within a contiguous field separate from diencephalic CPe, also exhibited different patterns of apoptosis. Despite lacking Gdf7 cell lineages, the posterior tCPe domain failed to form after ablating Gdf7-expressing DM cells at neural tube stages. The tCPe loss was associated with abrogation of high-level bone morphogenetic protein (Bmp) signaling, which is known to be required for tCPe induction. Taken together, these studies demonstrate intimate DM-CPe relationships throughout the CNS and highlight two distinct tCPe domains, including a posterior domain whose genesis depends on DM cells in a non-cell-autonomous fashion.     

LRP2/megalin is required for patterning of the ventral telencephalon

Megalin is a low-density lipoprotein receptor-related protein (LRP2) expressed in the neuroepithelium and the yolk sac of the early embryo. Absence of megalin expression in knockout mice results in holoprosencephaly, indicating an essential yet unidentified function in forebrain development. We used mice with complete or conditional megalin gene inactivation in the embryo to demonstrate that expression of megalin in the neuroepithelium but not in the yolk sac is crucial for brain development. During early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression and signaling in the rostral dorsal neuroepithelium, and a subsequent loss of sonic hedgehog (Shh) expression in the ventral forebrain. As a consequence of absent SHH activity, ventrally derived oligodendroglial and interneuronal cell populations are lost in the forebrain of megalin-/- embryos. Similar defects are seen in models with enhanced signaling through BMPs, central regulators of neural tube patterning. Because megalin mediates endocytic uptake and degradation of BMP4, these findings indicate a role for megalin in neural tube specification, possibly by acting as BMP4 clearance receptor in the neuroepithelium.     

Pax6 regulates regional development and neuronal migration in the cerebral cortex

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.     

Early embryonic lethality in Bmp5;Bmp7 double mutant mice suggests functional redundancy within the 60A subgroup

Members of the BMP family of signaling molecules display a high conservation of structure and function, and multiple BMPs are often coexpressed in a variety of tissues during development. Moreover, distinct BMP ligands are capable of activating common pathways. Here we describe the coexpression of two members of the 60A subfamily of BMPs, Bmp5 and Bmp7, at a number of different sites in the embryo from gastrulation onwards. Previous studies demonstrate that loss of either Bmp5 or Bmp7 has negligible effects on development, suggesting these molecules functionally compensate for each other at early stages of embryonic development. Here we show this is indeed the case. Thus we find that Bmp5;Bmp7 double mutants die at 10.5 dpc and display striking defects primarily affecting the tissues where these factors are coexpressed. The present analysis also uncovers novel roles for BMP signaling during the development of the allantois, heart, branchial arches, somites and forebrain. Bmp5 and Bmp7 do not appear to be involved in establishing pattern in these tissues, but are instead necessary for the proliferation and maintenance of specific cell populations. These findings are discussed with respect to potential mechanisms underlying cooperative signaling by multiple members of the TGF-beta superfamily.     

The TGF-beta pseudoreceptor gene Bambi is dispensable for mouse embryonic development and postnatal survival

The Bambi (Bmp and activin membrane-bound inhibitor) gene encodes a transmembrane protein highly similar in amino acid sequence to transforming growth factor-beta (TGF-beta receptors, however, the Bambi intracellular domain is short and lacks a serine/threonine-kinase domain that is essential for transducing TGF beta signaling. Previous biochemical assays showed that Bambi interacts directly with BMP receptors and antagonizes BMP signaling. Interestingly, the expression of Bambi largely overlaps, both temporally and spatially, with that of Bmp4 during early embryonic development in Xenopus, zebrafish, and mice, which led to the hypothesis that Bambi may function to regulate BMP signaling during embryogenesis. To directly analyze the roles of Bambi during embryonic development, we generated mice carrying a conditional allele of Bambi, Bambi(flox), with loxP sequences flanking the first exon that encodes the N-terminus and signal peptide region of the Bambi protein. Mice homozygous for this targeted conditional allele appear normal and fertile. We crossed the Bambi(flox)/+ mice to the EIIa-Cre transgenic mice and generated mice carrying deletion of the first exon of the Bambi gene. Surprisingly, mice homozygous for the deleted allele were viable, fertile and did not exhibit any discernible developmental defect. Our data exclude an essential role for Bambi in mouse embryonic development and postnatal survival.     

glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.