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Holland, ND, Holland LZ, Heimberg A.  2015.  Hybrids between the Florida amphioxus (Branchiostoma floridae) and the Bahamas lancelet (Asymmetron lucayanum): Developmental morphology and chromosome counts. Biological Bulletin. 228:13-24. AbstractWebsite

The cephalochordate genera Branchiostoma and Asymmetron diverged during the Mesozoic Era. In spite of the long separation of the parental clades, eggs of the Florida amphioxus, B. floridae, when fertilized with sperm of the Bahamas lancelet, A. lucayanum (and vice versa), develop through embryonic and larval stages. The larvae reach the chordate phylotypic stage (i.e., the pharyngula), characterized by a dorsal nerve cord, notochord, perforate pharynx, and segmented trunk musculature. After about 2 weeks of larval development, the hybrids die, as do the A. lucayanum purebreds, although all were eating the same algal diet that sustains B. floridae purebreds through adulthood in the laboratory; it is thus unclear whether death of the hybrids results from incompatible parental genomes or an inadequate diet. The diploid chromosome count in A. lucayanum and B. floridae purebreds is, respectively, 34 and 38, whereas it is 36 in hybrids in either direction. The hybrid larvae exhibit several morphological characters intermediate between those of the parents, including the size of the preoral ciliated pit and the angles of deflection of the gill slits and anus from the ventral midline. Based on the time since the two parent clades diverged (120 or 160 million years, respectively, by nuclear and mitochondrial gene analysis), the cross between Branchiostoma and Asymmetron is the most extreme example of hybridization that has ever been unequivocally demonstrated among multicellular animals.

Koop, D, Chen J, Theodosiou M, Carvalho JE, Alvarez S, de Lera AR, Holland LZ, Schubert M.  2014.  Roles of retinoic acid and Tbx1/10 in pharyngeal segmentation: amphioxus and the ancestral chordate condition. Evodevo. 5   10.1186/2041-9139-5-36   AbstractWebsite

Background: Although chordates descend from a segmented ancestor, the evolution of head segmentation has been very controversial for over 150 years. Chordates generally possess a segmented pharynx, but even though anatomical evidence and gene expression analyses suggest homologies between the pharyngeal apparatus of invertebrate chordates, such as the cephalochordate amphioxus, and vertebrates, these homologies remain contested. We, therefore, decided to study the evolution of the chordate head by examining the molecular mechanisms underlying pharyngeal morphogenesis in amphioxus, an animal lacking definitive neural crest. Results: Focusing on the role of retinoic acid ( RA) in post-gastrulation pharyngeal morphogenesis, we found that during gastrulation, RA signaling in the endoderm is required for defining pharyngeal and non-pharyngeal domains and that this process involves active degradation of RA anteriorly in the embryo. Subsequent extension of the pharyngeal territory depends on the creation of a low RA environment and is coupled to body elongation. RA further functions in pharyngeal segmentation in a regulatory network involving the mutual inhibition of RA-and Tbx1/10-dependent signaling. Conclusions: These results indicate that the involvement of RA signaling and its interactions with Tbx1/10 in head segmentation preceded the evolution of neural crest and were thus likely present in the ancestral chordate. Furthermore, developmental comparisons between different deuterostome models suggest that the genetic mechanisms for pharyngeal segmentation are evolutionary ancient and very likely predate the origin of chordates.

Holland, LZ, Carvalho JE, Escriva H, Laudet V, Schubert M, Shimeld SM, Yu JK.  2013.  Evolution of bilaterian central nervous systems: a single origin? Evodevo. 4   10.1186/2041-9139-4-27   AbstractWebsite

The question of whether the ancestral bilaterian had a central nervous system (CNS) or a diffuse ectodermal nervous system has been hotly debated. Considerable evidence supports the theory that a CNS evolved just once. However, an alternative view proposes that the chordate CNS evolved from the ectodermal nerve net of a hemichordate-like ancestral deuterostome, implying independent evolution of the CNS in chordates and protostomes. To specify morphological divisions along the anterior/posterior axis, this ancestor used gene networks homologous to those patterning three organizing centers in the vertebrate brain: the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer, and subsequent evolution of the vertebrate brain involved elaboration of these ancestral signaling centers; however, all or part of these signaling centers were lost from the CNS of invertebrate chordates. The present review analyzes the evidence for and against these theories. The bulk of the evidence indicates that a CNS evolved just once - in the ancestral bilaterian. Importantly, in both protostomes and deuterostomes, the CNS represents a portion of a generally neurogenic ectoderm that is internalized and receives and integrates inputs from sensory cells in the remainder of the ectoderm. The expression patterns of genes involved in medio/lateral (dorso/ventral) patterning of the CNS are similar in protostomes and chordates; however, these genes are not similarly expressed in the ectoderm outside the CNS. Thus, their expression is a better criterion for CNS homologs than the expression of anterior/posterior patterning genes, many of which (for example, Hox genes) are similarly expressed both in the CNS and in the remainder of the ectoderm in many bilaterians. The evidence leaves hemichordates in an ambiguous position - either CNS centralization was lost to some extent at the base of the hemichordates, or even earlier, at the base of the hemichordates + echinoderms, or one of the two hemichordate nerve cords is homologous to the CNS of protostomes and chordates. In any event, the presence of part of the genetic machinery for the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer in invertebrate chordates together with similar morphology indicates that these organizers were present, at least in part, at the base of the chordates and were probably elaborated upon in the vertebrate lineage.

Koop, D, Holland LZ, Setiamarga D, Schubert M, Holland ND.  2011.  Tail regression induced by elevated retinoic acid signaling in amphioxus larvae occurs by tissue remodeling, not cell death. Evolution & Development. 13:427-435.   10.1111/j.1525-142X.2011.00501.x   AbstractWebsite

The vitamin A derived morphogen retinoic acid (RA) is known to function in the regulation of tissue proliferation and differentiation. Here, we show that exogenous RA applied to late larvae of the invertebrate chordate amphioxus can reverse some differentiated states. Although treatment with the RA antagonist BMS009 has no obvious effect on late larvae of amphioxus, administration of excess RA alters the morphology of the posterior end of the body. The anus closes over, and gut contents accumulate in the hindgut. In addition, the larval tail fin regresses, although little apoptosis takes place. This fin normally consists of columnar epidermal cells, each characterized by a ciliary rootlet running all the way from an apical centriole to the base of the cell and likely contributing substantial cytoskeletal support. After a few days of RA treatment, the rootlet becomes disrupted, and the cell shape changes from columnar to cuboidal. Transmission electron microscopy (TEM) shows fragments of the rootlet in the basal cytoplasm of the cuboidal cell. A major component of the ciliary rootlet in amphioxus is the protein Rootletin, which is encoded by a single AmphiRootletin gene. This gene is highly expressed in the tail epithelial cells of control larvae, but becomes downregulated after about a day of RA treatment, and the breakup of the ciliary rootlet soon follows. The effect of excess RA on these epidermal cells of the larval tail in amphioxus is unlike posterior regression in developing zebrafish, where elevated RA signaling alters connective tissues of mesodermal origin. In contrast, however, the RA-induced closure of the amphioxus anus has parallels in the RA-induced caudal regression syndrome of mammals.

Yu, JK, Holland ND, Holland LZ.  2004.  Tissue-specific expression of FoxD reporter constructs in amphioxus embryos. Developmental Biology. 274:452-461.   10.1016/j.ydbio.2004.07.010   AbstractWebsite

Cephalochordates (amphioxus), the closest living invertebrate relatives of the vertebrates, are key to understanding the evolution of developmental mechanisms during the invertebrate-to-vertebrate transition. However, a major impediment to amphioxus as a model organism for developmental biology has been the inability to introduce transgenes or other macromolecules into the embryos. Here, we report the development of a reproducible method for microinjection of amphioxus eggs. Specifically, we show that expression of a LacZ reporter construct including 6.3 kb of AmphiFoxD upstream regulatory DNA recapitulates expression of the endogenous gene in the nerve cord, somites, and notochord. We have also identified the 1.6 kb at the 5' end of this region as essential for expression in the first two of these domains and the 4.7 kb at the 3' end as sufficient for expression in the notochord. This study, which is the first report of a method for introduction of large molecules such as DNA into amphioxus embryos, opens the way for studies of gene regulation and function in amphioxus and for comparative studies with vertebrates to understand the relationship between the extensive gene duplications that occurred within the vertebrate lineage and the evolution of vertebrate innovations such as neural crest. (C) 2004 Elsevier Inc. All rights reserved.

Holland, LZ, Yu JK.  2004.  Cephalochordate (amphioxus) embryos: Procurement, culture, and basic methods. Development of Sea Urchins, Ascidians, and Other Invertebrate Deuterostomes: Experimental Approaches. 74:195-215.Website