The role of Sonic hedgehog of neural origin in thalamic differentiation in the mouse.

The specification of the intricate neuronal assemblies that characterize the forebrain is not well understood. The ventral spinal cord is specified through a concentration gradient of Sonic hedgehog (Shh) protein secreted by the notochord. Shh is expressed also in the forebrain neuroepithelium (neural Shh) and the underlying notochord and prechordal plate. Neural Shh is essential for the development of the prethalamus (ventral thalamus), but its effects on the thalamus (dorsal thalamus) are still unclear. We hypothesized that neural Shh would act on a previously regionalized dorsal diencephalic region to promote the emergence of specific thalamic nuclear and histological traits. To find out, we generated a conditional mouse mutant line specifically lacking Shh expression in the diencephalic neuroepithelium. We show that the transcription factor Gbx2, required for thalamic development downstream Shh, is expressed in our mutant in a restricted thalamic region and is necessary and sufficient for the differentiation of the medial and intralaminar thalamic nuclei. In the rest of the thalamus, neural Shh is required to promote neuronal aggregation into nuclei as well as axonal extension. In this way, the individual thalamic nuclei show differential dependence on Shh, Gbx2, or both for their differentiation. Additionally, Gbx2 is required for the survival of thalamic neurons.

Role of neuroepithelial Sonic hedgehog in hypothalamic patterning.

The hypothalamus is a region of the diencephalon with particularly complex patterning. Sonic hedgehog (Shh), encoding a protein with key developmental roles, shows a peculiar and dynamic diencephalic expression pattern. Here, we use transgenic strategies and in vitro experiments to test the hypothesis that Shh expressed in the diencephalic neuroepithelium (neural Shh) coordinates tissue growth and patterning in the hypothalamus. Our results show that neural Shh coordinates anteroposterior and dorsoventral patterning in the hypothalamus and in the diencephalon-telencephalon junction. Neural Shh also coordinates mediolateral hypothalamic patterning, since it is necessary for the lateral hypothalamus to attain proper size and is required for the specification of hypocretin/orexin cells. Finally, neural Shh is necessary to maintain expression of differentiation markers including survival factor Foxb1.

Cadherins mediate sequential roles through a hierarchy of mechanisms in the developing mammillary body.

Expression of intricate combinations of cadherins (a family of adhesive membrane proteins) is common in the developing central nervous system. On this basis, a combinatorial cadherin code has long been proposed to underlie neuronal sorting and to be ultimately responsible for the layers, columns and nuclei of the brain. However, experimental proof of this particular function of cadherins has proven difficult to obtain and the question is still not clear. Alternatively, non-specific, non-combinatorial, purely quantitative adhesive differentials have been proposed to explain neuronal sorting in the brain. Do cadherin combinations underlie brain cytoarchitecture? We approached this question using as model a well-defined forebrain nucleus, the mammillary body (MBO), which shows strong, homogeneous expression of one single cadherin (Cdh11) and patterned, combinatorial expression of Cdh6, -8 and -10. We found that, besides the known combinatorial Cdh pattern, MBO cells are organized into a second, non-overlapping pattern grouping neurons with the same date of neurogenesis. We report that, in the Foxb1 mouse mutant, Cdh11 expression fails to be maintained during MBO development. This disrupted the combination-based as well as the birthdate-based sorting in the mutant MBO. In utero RNA interference (RNAi) experiments knocking down Cdh11 in MBO-fated migrating neurons at one specific age showed that Cdh11 expression is required for chronological entrance in the MBO. Our results suggest that neuronal sorting in the developing MBO is caused by adhesion-based, non-combinatorial mechanisms that keep neurons sorted according to birthdate information (possibly matching them to target neurons chronologically sorted in the same manner). Non-specific adhesion mechanisms would also prevent cadherin combinations from altering the birthdate-based sorting. Cadherin combinations would presumably act later to support specific synaptogenesis through specific axonal fasciculation and final target recognition.

Genetic manipulation of the mouse developing hypothalamus through in utero electroporation.

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Interaction between axons and specific populations of surrounding cells is indispensable for collateral formation in the mammillary system.

BACKGROUND: An essential phenomenon during brain development is the extension of long collateral branches by axons. How the local cellular environment contributes to the initial sprouting of these branches in specific points of an axonal shaft remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: The principal mammillary tract (pm) is a landmark axonal bundle connecting ventral diencephalon to brainstem (through the mammillotegmental tract, mtg). Late in development, the axons of the principal mammillary tract sprout collateral branches at a very specific point forming a large bundle whose target is the thalamus. Inspection of this model showed a number of distinct, identified cell populations originated in the dorsal and the ventral diencephalon and migrating during development to arrange themselves into several discrete groups around the branching point. Further analysis of this system in several mouse lines carrying mutant alleles of genes expressed in defined subpopulations (including Pax6, Foxb1, Lrp6 and Gbx2) together with the use of an unambiguous genetic marker of mammillary axons revealed: 1) a specific group of Pax6-expressing cells in close apposition with the prospective branching point is indispensable to elicit axonal branching in this system; and 2) cooperation of transcription factors Foxb1 and Pax6 to differentially regulate navigation and fasciculation of distinct branches of the principal mammillary tract. CONCLUSIONS/SIGNIFICANCE: Our results define for the first time a model system where interaction of the axonal shaft with a specific group of surrounding cells is essential to promote branching. Additionally, we provide insight on the cooperative transcriptional regulation necessary to promote and organize an intricate axonal tree.