Evolution of patterning systems and circuit elements for locomotion

Abstract

Evolutionary modifications in nervous systems enabled organisms to adapt to their specific environments and underlie the remarkable diversity of behaviors expressed by animals. Resolving the pathways that shaped and modified neural circuits during evolution remains a significant challenge. Comparative studies have revealed a surprising conservation in the intrinsic signaling systems involved in early patterning of bilaterian nervous systems but also raise the question of how neural circuit compositions and architectures evolved within specific animal lineages. In this review, we discuss the mechanisms that contributed to the emergence and diversity of animal nervous systems, focusing on the circuits governing vertebrate locomotion.

Figure 1. Neural Induction and Early Patterning in Bilateria

(A) Traditional classification of bilateria. Bilaterians are a subgroup of eumetazoan animals characterized by a bilaterally symmetrical body plan and triploblastic development. Bilaterians are subdivided into protostomes (mouth-first) and deuterostomes (mouth-second). Top: The central nervous system (CNS) (in blue) forms ventrally in protostomes and dorsally in deuterostomes. Bottom: A simplified phylogenetic tree, showing the evolutionary relationships amongst bilaterians and other metazoan phyla. (B) Conservation of gene expression patterns along the dorsoventral (DV) axis in protostomes (flies, annelids) and deuterostomes (hemichordates and vertebrates). In both protostomes and deuterostomes expression of neural identity genes is patterned by Bmps along the DV axis of the nerve cord (Esteves et al., 2014). Ventral patterning cues are not portrayed here as they are not homologous in different species (e.g., Dorsal in flies, Shh in vertebrates). As in vertebrates, cholinergic Hb9⁺ MNs derive from pax6⁺nk6⁺ progenitors and directly innervate muscles in annelids (Denes et al., 2007). In flies, there are additional MN populations (not depicted here) in addition to Hb9⁺ MNs. Although Bmp-Chordin signaling is present in hemichordates, many DV patterning genes are not expressed by the neuroectoderm (e.g., nk2.2 in endoderm). The Mnx gene which shares high homology with Hb9 homeodomain is expressed in the hemichordate ventral ectoderm implicating possible conservation in MN specification (Lowe et al., 2006). Homologous genes are color-coded. Schematics on the bottom represent cross-sections of the embryos. (C) Conservation of anteroposterior patterning systems in bilaterians. Although protostomes do not have analogous neuroectodermal signaling centers present in developing vertebrate brains, key genes determining their boundaries are conserved along the anteroposterior axis. The en gene is also expressed at parasegmental boundaries in the epidermis of flies and annelids. In hemichordates, the expression of fezf (not shown here) is not adjacent to that of irx. Homologous genes are color-coded for comparison. pc, protocerebrum; dc, deutocerebrum; tc, tritocerebrum; seg, subesophageal ganglion; vnc, ventral nerve cord; pro, prostomium; peri, peristomium; tr, trunk (both in annelid and hemichordate); pr, proboscis; col, collar; tel, telencephalon; di, diencephalon; mb, midbrain; hb, hindbrain; sc, spinal cord. Comparisons between species represented in panel (A) and (B) do not take into account gene expression differences and therefore do not represent a true cladistics analysis. Furthermore, this model does not fully take into account the development of animals with unsegmented nervous systems such as in molluscs. Panel (A) is modified from (De Robertis, 2008; Philippe et al., 2011) and panel (B) is modified from (Denes et al., 2007; Mizutani and Bier, 2008).

Figure 2. Motor Innervation Programs in Bilaterians

(A) Table showing conservation and divergence of MN cell fate specification programs in invertebrates and vertebrates, emphasizing known conserved transcription factors. Several key transcription factors involved in MN specification are not indicated. NA, not assessed. (B) Comparisons of MN organization and innervation patterns between mouse and zebrafish at trunk levels. Core MN determinants, Isl1/2, Hb9 and Lhx3, are expressed in different combinations in three distinct thoracic columns in mouse. scg, sympathetic chain ganglia. Zebrafish embryos contain four classes of primary MNs, vRoP (ventrally-projecting rostral primary), dRoP (dorsally-projecting RoP), MiP (medial primary) and CaP (caudal primary), and they do not organize into tightly clustered columns (Menelaou and McLean, 2012). They are classified by their specific innervation of axial muscles from dorsal to ventral. The stereotypic innervation patterns of each primary MN are depicted here. Although three Mnx proteins are detected within each primary MN subtype in zebrafish, Mnx proteints are only required in MiP MNs (Seredick et al., 2012). (C) MN organization and specification programs at limb/fin levels in mouse and zebrafish. In zebrafish, pectoral fin innervating MNs are considered to be secondary due to their late development and ventrolateral position relative to primary MNs (Myers, 1985). A GFP reporter under control of an Isl1 enhancer indicates that Isl1⁺ pectoral fin MNs selectively innervate abductor muscles (Uemura et al., 2005). Untested aspects of these models are shown in gray.

Figure 3. Evolutionary Diversity of Spinal Motor Neurons

(A) Evolution of locomotor strategies. Top: A chordate phylogeny showing representative species of tetrapods (dark purple) and vertebrates (light purple). Chondrichthyans represent the most primitive species bearing paired appendages. Bottom: Comparisons of locomotor behaviors in lamprey, salamander and mouse. (B) Altered MN columnar organization in Foxp1 and Hox mutants. In Foxp1 mutants Hox-dependent spinal MN columns (LMC and PGC) are transformed into an HMC-like “ground state”, which may represent a primitive condition. PMC neurons are present in Foxp1 mutants, but not depicted. Loss of LMC neurons at brachial levels is achieved only when HoxA and HoxC gene clusters are mutated. Lumbar LMC neurons are preserved in HoxA/C cluster mutant mice due to Hoxd10 activity. Deletion of the Hoxc9 gene causes global derepression of brachial Hox genes resulting in an extension of the brachial LMC throughout thoracic levels. MMC neurons are considered Hox-independent as their molecular profiles are preserved in each of these mutants. (C) A model showing how MN organization has evolved with changes in body plans. A subset of MNs in agnathan vertebrates (represented by modern lampreys) may have lost Lhx3 activity, permitting the generation of HMC-like neurons. The acquisition of paired-appendages promoted the generation of LMC-like populations, which may have been initially present at most spinal levels. A repressive domain within Hox9 proteins necessary to suppress LMC specification appears to have emerged when the elongate fin split into pectoral and pelvic fins. Studies in zebrafish suggest the pectoral fin MNs were initially positioned in both the hindbrain (HB) and spinal cord (SC) (Ma et al., 2010). Pelvic fin innervating MNs do not align with Hox10 gene expression (Murata et al., 2010). In mammals, PMC neurons are specified by Hox5 proteins and are Foxp1-independent (Philippidou et al., 2012). (D) In snake embryos expansion of Hoxc9 expression blocks LMC generation. The enlarged-finned fish skate, which naturally has lost the HoxC cluster, may have extended LMC population along the anteroposterior axis of the spinal cord.

Figure 4. Central Pattern Generators and Locomotor Behaviors

(A) Genetic mutations in guidance systems that lead to synchronous bilateral activation of limb-level MNs (hopping) in mice. Mutations in EphA4 or ephrinB3 cause multiple classes of excitatory ipsilaterally projecting interneurons (eIINs) to aberrantly cross the ventral midline. Mutation in netrin causes fewer inhibitory commissural interneurons (iCINs) to cross, but preserves some eCINs projections. (B) Examples of fictive locomotor assays in mice. Ventral root recordings from lumbar level L2 showing bursts of MN activation at regular intervals. In control mice bursts recorded from left L2 (lL2) and right L2 (rL2) alternate. In netrin mutants both sides of the spinal cord burst in phase. Images are modified from (Rabe et al., 2009). (C) Intrinsic factors involved in CIN specification. Excitatory and inhibitory CINs are derived from multiple progenitor domains that are defined by transcription factor expression. Factors expressed by postmitotic neurons are indicated. Both V0d and V0v interneurons are derived from progenitors expressing Dbx1. Genetic silencing of V0 populations causes changes in the connections between CINs and target cells on the contralateral side of the spinal cord. (D) Partial list of genetic manipulations that affect left right alternation. Locomotor phenotypes described represent analysis using either fictive locomotor or behavior assays, or the combinations of both.