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. Abolition of Cdh11 expression in the entire MBO during development disrupted the combination-based as well as the birthdate-based sorting. In utero 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

Interaction between Axons and Specific Populations of Surrounding Cells Is Indispensable for Collateral Formation in the Mammillary System

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.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.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

Mammillary branching is present in several thalamic mutants.

<p>A, B) <i>Pax6</i> in situ hybridization shows that PTh/VTh and branching point cells are present in the <i>Lrp6</i> mutant (A) and the <i>Gbx2</i> mutant (B) brains at E18.5. C, D) DiI tracing demonstrates presence of a mth in these mutants (C, D). Scale bars 100 micrometers.</p

Axonal fasciculation and cell aggregation impaired in the <i>Foxb1/Pax6</i> double mutant.

<p>A) Slight increase in problem axons but no detectable change in mtc axons in the <i>Pax6</i> mutant. White column, problem axons; black column, mtc axons. Mean +/− SD; (*) P<0.05; n.s. not significant. B, C) Beta galactosidase detection on sagittal section of double <i>Foxb1-tauLacZ/Sey</i> homozygote. The problem axons (red arrow) seem more numerous as in single <i>Sey</i> homozygotes. The dotted line in (B) indicates the approximate plane of section of D, E, F. (C) shows a high magnification detail of the image in (B). D–F) Beta galactosidase detection on sections along the dotted line in (B) (left side is shown) through the branching point of E18.5 brains (genotypes as indicated). In <i>Foxb1</i> single homozygotes (D) there is a mth (branching takes place), the mtg is not subdivided into fascicles and the <i>Foxb1</i> branching point cells are tightly aggregated. In the double mutant (F), the problem axons (red arrow in E, F) are longer and more numerous, the <i>Foxb1</i>-expressing branching point cells (<i>Foxb1</i> BPC in Fig. 9D–F) are less compactly aggregated and the mtg is divided in more fascicles (arrowheads) as in the <i>Foxb1</i> heterozygote/<i>Sey</i> homozygote (E). Scale bars 100 micrometers.</p

<i>Foxb1</i>-expressing cells migrate from the MBO along the pm.

<p>A) <i>Foxb1</i> expression on a transverse section of a wild type E10.5 brain. Arrowheads, column of <i>Foxb1</i>-expressing cells originated in the MBO and migrating dorsally, preceded by a pioneer group (arrow). B, C) <i>Foxb1</i> expression on transverse sections of wild type E14.5 (B) and E16.5 (C) brains. Left side shows Nissl counterstaining, right side shows dark field. Dotted line in C, E, external medullary lamina (zona limitans). D, E) <i>Foxb1</i> expression in a sagittal section of an E14.5 wild type brain. (D) shows Nissl counterstaining, (E) shows dark field. Arrow, pioneer group of <i>Foxb1</i>-expressing cells. Asterisk in C, D: PTh/VTh. Scale bars A, B, C: 50 micrometers; E: 25 micrometers.</p

A complex and specific cell aggregate around the bifurcation point.

<p>A–D) In situ hybridization for <i>Pax6</i> (A, B) and <i>Foxb1</i> (C, D) on sagittal sections of wild type E18.5 brains (rostral to the left). (B) and (D) show high magnification details of (A) and (C). Black arrowheads, specific cell groups around the branching point. ac, anterior commissure; CTX, cortex; PT, pretectum. B) <i>Pax6</i>-expressing cells are also found forming a trail under the mth (black arrow) continuous with the PTh/VTh (asterisk), between the mth axons (white arrowhead), and in the area between mth and mtg (white arrow). E, F) Confocal pictures of antibody detection of Pax6 (red cell nuclei) and beta-galactosidase (green cell bodies; proxy for <i>Foxb1</i> expression) on a sagittal section of an E18.5 <i>Foxb1-tau-lacZ</i> heterozygous brain. Blue labeling, DAPI nuclear staining. E) Double labeling of the branching point shows a compact group of Pax6- and Foxb1-positive cells (arrowhead). F) <i>Foxb1</i>-positive (arrowheads, green cell bodies) and <i>Pax6</i>-positive (arrows, red nuclei) cells are distinct from each other. Asterisk in A, B, C: PTh/VTh. Scale bars 100 micrometers.</p

The mammillary body and its efferents as classically described.

<p>Diagram of MBO efferent connections to diencephalon and brainstem. P, pons; TG, tegmentum. Other abbreviations: see text.</p

<i>Foxb1</i> and <i>Pax6</i>: Mutants and Phenotypes.

<p>The mutants are listed in the order they appear in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020315#s3" target="_blank">Results</a> section. Two different <i>Pax6</i> mutants, with and without reporter were used. The <i>Pax6</i>-driven reporter (<i>Pax6-lacZ</i>) labels the PTh/VTh and <i>Pax6</i>-BPC, but not the mammillary body, axons or <i>Foxb1</i>-BPC. The <i>Pax6 Sey</i> mutant carries no reporter and its phenotype is analyzed by DiI axonal tracing. The <i>Foxb1-tau-lacZ</i> mouse carries a <i>Foxb1</i>-driven reporter labeling the mammillary body and axons and the <i>Foxb1</i>-BPC. The pm and <i>Foxb1</i> BPC have not been examined in the <i>Pax6</i>-<i>lacZ</i> mutant because they express neither <i>Pax6</i> nor the <i>Pax6</i>-driven <i>lacZ</i> reporter.</p>1<p>BPC: Branching Point Cells;</p>2<p>b-gal: beta-galactosidase;</p>3<p>mam: mammillary;</p>4<p>PTh/VTh: Prethalamus/Ventral thalamus.</p