Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins

Abstract

The {Pcdh-γ} gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons {(MNs),} do not undergo excessive apoptosis in {Pcdh-γ(del/del)} null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in {Pcdh-γ} mutants appear clumped, and fill the space between individual {MNs;} quantitative analysis shows a {\textasciitilde}2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons {(vINs),} many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in {Pcdh-γ} null mice by introduction of a Bax null allele. This indicates that loss of {vINs,} which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the {Pcdh-γ} cluster using conditional mutants and multiple Cre transgenic lines {(Wnt1-Cre} for sensory neurons; {Pax2-Cre} for {vINs;} {Hb9-Cre} for {MNs)} also revealed a direct requirement for the {γ-Pcdhs} in Ia neurons and {vINs,} but not in {MNs} themselves. Together, these genetic manipulations indicate that the {γ-Pcdhs} are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of {vINs} that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target {vINs.}