Early head development in the Australian lungfi sh Neoceratodus forsteri (Krefft 1870) has until recently received relatively little attention. In this chapter we summarize recently published studies, mostly from our own laboratories, and some unpublished data that document the early development of the head skeleton and the head musculature in this species. Like in other vertebrates, the cranial neural crest gives rise to a large part of the skull in the Australian lungfi sh. Unlike in related animals, cranial neural crest cells emerge late from the neural tube, but follow the general migratory pattern seen in other vertebrates, forming mandibular, hyoid and branchial streams. Labelling with the lipophilic fl uorescent dye, DiI, allowed us to follow the fate of cranial neural crest cells until Kemp stage 43, when several cranial skeletal elements have started to diff erentiate. Th e cranial neural crest contributes cells to several parts of the head skeleton, including the trabecula cranii and derivatives of the mandibular arch (e.g. Meckel’s cartilage, quadrate), the hyoid arch (e.g. the ceratohyal), and the branchial arches (ceratobranchials I-IV), as well as to the connective tissue surrounding the myofi bers in cranial muscles. Th e myofi bers of cranial muscles are likely to be derived from the head mesoderm, but no proper fate-mapping is availabe to prove this. Later they can be detected using antibodies that mark desmin, and are diff erentiating almost simultaneously in the mandibular and

hyoid arches, but later in the branchial arches. Unlike the Mexican axolotl, which has an origin-to-insert pattern of diff erentiation, no obvious directionality can be observed in the diff erentiating cranial muscles of the Australian lungfi sh. Keywords: head development, morphogenesis, cell migration, fate mapping

Research into head development is an important subject in comparative vertebrate morphology, with a rich classical literature (see e.g. Gegenbaur 1888; de Beer 1937; Goodrich 1930 for reviews). In a modern 3-volume treatise on the vertebrate skull (Hanken and Hall 1993), one entire volume was devoted to development. Most of the recent studies have focused on the mechanisms that determine the early regionalization of the head and give the proper identity to the tissues in the head region (Grammatopoulos et al. 2000; Graham and Smith 2001; Pasqualetti et al. 2000; Rijli et al. 1993, 1998), and have used model species of birds (the quail/chicken system), rodents (mouse, rat), frogs (Xenopus) and salamanders (Mexican axolotl). Less is known about the later processes that regulate morphogenesis and are responsible for shaping the architecture of the developing head (but see, e.g. Depew et al. 2002; Depew and Simpson 2006), and about non-model species. Th e picture emerging from these studies is that, in contrast to the situation in the trunk, most skeletal elements in the head are derived from neural crest cells (Couly and Le Douarin 1990; Couly et al. 1992, 1993; Noden 1991). Th e neural crest is an embryonic cell population that gives rise to a large array of diff erent cell and tissue types (Hall 1999; LeDouarin and Kalcheim 1999). Cranial neural crest cells emerge on top of the neural tube. Th ey migrate in a ventral direction and form streams which feed into the pharyngeal arches. Neural crest cells form most of the head skeleton, but also connective tissue to which the mesoderm-derived muscles attach (Noden 1986a, b; Olsson et al. 2001; Ericsson et al. 2004). Th e muscles of one arch are attached to bones (or cartilages) from the same arch and innervated by cranial nerves emerging from the corresponding rhombomere (Köntges and Lumsden 1996; Olsson et al. 2001). Th us an early pattern of head organisation leaves an imprint even into the adult stages, although the adult vertebrate head does not show a clear segmentation (see Olsson et al. 2005 for review). In the Australian lungfi sh Neoceratodus forsteri (Krefft 1870), the development of the head, and early events in particular, are relatively poorly known (but see Fox 1965; Bartsch 1993, 1994; Bertmar 1966). Despite some recent work on neural (Kemp 2000) and neural crest (Ericsson et al. 2008; Falck et al. 2000; Kemp 1995, 2000) development, most of the literature is old (e.g. Greil 1908, 1913), using the methods then at hand, and material collected in the wild (Olsson et al. 2004). Sometimes they concern other species of lungfi sh (Agar 1906). Only the later stages of development and the larval anatomy have been the subject of thorough

recent study (Bartsch 1993, 1994; Kemp 1999). In this chapter, we summarize our studies on cranial neural crest cells and conclude that they do emerge and migrate in the Australian lungfi sh in the same way as in other vertebrates, and that they form the skeletal derivatives expected from considerations of homology. We also describe the development of cranial muscle in the Australian lungfi sh, in part using our own unpublished work, and compare head muscle development in the Australian lungfi sh with our published work on the Mexican axolotl (Ericsson and Olsson 2004).