Chapter 7 Development and Developmental Disorders of the Brain Stem Hans J.ten Donkelaar,Martin Lammens,Johannes R.M.Cruysberg and Cor W.J.R.Cremers 7.1 Introduction traocular muscles arise from mesomere 2 (the oculo- motor nucleus) and rhombomeres 1 (the trochlear The brain stem is composed of the midbrain (the nucleus) and 5 (the abducens nucleus). The motor mesencephalon) and the hindbrain (the rhomben- nuclei of the cranial nerves,innervating the branchial cephalon), and is, at least during development, seg- arch musculature,arise from the second,fourth,sixth mentally organized. The midbrain is composed of and seventh rhombomeres.The neural crest,flanking two temporarily present segments known as me- the developing rhombencephalon, makes important someres,whereas the hindbrain is composed of eight, contributions to the branchial arches (Chap. 5). A also temporarily present, rhombomeres (Fig. 7.1). great number of genes are involved in the proper de- The cerebellum largely arises from the first rhom- velopment of the brain stem (Cordes 2001; Moens bomere (Chap. 8). The brain stem contains the retic- and Prince 2002). The isthmus organizer regulates ular formation which is involved in the control of res- the early development of the mesencephalon and of piration, circulation, wakefulness and locomotion. the rostral part of the rhombencephalon (Wurst and The brain stem also contributes ten of the 12 pairs of Bally-Cuif 2001; Joyner 2002). Mutations of genes cranial nerves, III–XII. The motor nuclei for the ex- involved such as Otx2,En1 and En2 result in extensive Fig. 7.1 Segmentation of the brain stem (medial views of mus, is isthmus, Lc locus coeruleus, mes mesencephalon, the brain at Carnegie stages 12, 13, 15 and 17). ap alar plate, M1, M2 mesomeres, nIV trochlear nerve, ov otic vesicle, bp basal plate, cbi internal cerebellar bulge, cho chiasma rhl rhombic lip, slH sulcus limitans of His, v4 fourth ventricle, opticum, D1, D2 diencephalic neuromeres, ev eye vesicle, XII hypoglossal nucleus, 1–8 rhombomeres. (After O’Rahilly gV trigeminal ganglion, gVII facial ganglion, hyp hypothala- and Müller 1999) 270 Chapter 7 Brainstem Development and Its Disorders Fig. 7.2 Gene expression pat- terns at the midbrain–hindbrain boundary (MEB) shown in dorsal views of the mouse embryonic neural plate (a–c), and in Otx2 (e) and Gbx2 (f) knockout mice. Otx2 expression is shown in light red, En1/2 expression in medium red and Gbx2 expression in red. Comparison with the nor- mal pattern shown in d indicates the severe effects of the absence of these genes on the formation of the brain stem (see text for further explanation). mes mes- encephalon, met metencepha- lon, pros prosencephalon, r1–r4 rhombomeres, III–VII motor nuclei of cranial nerves. (After Wurst and Bally-Cuif 2001) defects in the midbrain, the cerebellum and the pons. 7.2 Pattern Formation Each rhombomere is characterized by a unique com- and Segmentation bination of Hox genes, its Hox code. In mice, sponta- of the Brain Stem neous and targeted (knockout) mutations in these genes result in specific, rhombomere-restricted dis- 7.2.1 Pattern Formation of the Brain Stem ruptions in the development of motor nuclei of cra- nial nerves. Such ‘rhombomeropathies’ have not been The midbrain–hindbrain boundary organizer (MHB found in humans,although congenital facial paralysis organizer) or isthmic organizer is responsible for and Möbius syndrome may be good candidates. specifying the fate of the midbrain and cerebellum. It In this chapter, patterning of the brain stem, its was first identified through transplantation experi- segmentation and the development and developmen- ments in chick embryos. When MHB tissue was tal disorders of the cranial nerves and their nuclei transplanted into the caudal forebrain of chick will be discussed.Developmental anomalies of one or embryos, the surrounding host tissue adopted an more cranial nerves, with primary or secondary isthmic or midbrain character (Martínez et al. 1991; dysinnervation, may lead to congenital, non-progres- Marín and Puelles 1994; Wassef and Joyner 1997). A sive, sporadic or familial abnormalities of cranial restricted dorsal domain of the isthmic organizer musculature, currently grouped as congenital cranial (the isthmic node) is necessary for the formation and dysinnervation disorders (CCDDs; Gutowski et al. positioning of the roof plate (Alexandre and Wassef 2003).Congenital defects of any component of the ear 2003). Several genes, encoding transcription factors may cause hearing impairment.About 1 in 1,000 chil- such as the Engrailed (En), Pax, Otx and Gbx families dren is born with hearing loss or deafness present at or secreted proteins of the Wnt and Fgf families, are birth or during early childhood (Petit et al. 2001a, b). expressed within the MHB at early embryonic stages Syndromic deafness contributes to about 30% of the (Wassef and Joyner 1997;Acampora et al.2001; Rhinn cases of prelingual deafness.Several hundreds of syn- and Brand 2001; Liu and Joyner 2001; Wurst and dromes including hearing loss have been described Bally-Cuif 2001; Joyner 2002). The isthmus organizer (Toriello et al. 2004). Rapid progress has been made itself is set up by the expression of a complex array of in identifying deafness genes in mice and men. More genes, two of which are central to its development than 100 genes have now been identified that affect (Fig. 7.2). The first, Otx2 (one of the mouse homo- inner ear development or function (Fekete 1999; Petit logues of the Drosophila gene orthodenticle), is ex- et al. 2001a, b; Steel and Kros 2001; Kiernan et al. pressed in the prosencephalon and mesencephalon. 2002; Friedman and Griffith 2003). Its posterior limit of expression marks the anterior 7.2 Pattern Formation and Segmentation of the Brain Stem 271 gradient that decreases anteriorly through the mes- encephalon and posteriorly through the first rhom- bomere. Graded expression of the En genes appears to be regulated by signalling from the isthmus. Muta- tions in these genes cause deletions of mesencephal- ic and cerebellar structures (Millen et al. 1994; Wurst et al. 1994; Kuemerle et al. 1997). En1 knockout mice have complete cerebellar aplasia, whereas En2 dele- tions are less severe and only cause cerebellar hy- poplasia with abnormal foliation (Millen et al. 1994; Kuemerle et al. 1997). Pax2, Pax5 and Pax8 are also required for specification of the isthmus. The isth- mus is deleted in Pax5–/– mice (Urbanek et al. 1994). A possible human homologue of the En2 knockout mice was recently found by Sarnat et al. (2002; Clini- cal Case 7.1). They described two cases of agenesis of the mesencephalon and metencephalon with cerebel- lar hypoplasia, possibly due to mutations in the EN2 Fig. 7.3 Development of catecholaminergic (substantia ni- gene. gra,Sn,locus coeruleus,Lc) and serotonergic (raphe nuclei,Ra) FGF8 is also essential for the formation of the cell groups in the brain stem and the role of various signalling nuclei of the oculomotor and trochlear nerves, the factors (BMP4, FGF4, FGF8, SHH and Wnt1; see text for further dopaminergic neurons in the substantia nigra and explanation).SHH expression is shown in red,FGF8 expression related ventral tegmental area, serotonergic neurons in grey and Wnt1 expression in light red. mes mesencephalon, in the rostral raphe nuclei and noradrenergic neu- p1–p3 prosomeres, r1–r5 rhombomeres; III–V, VII motor nuclei rons in the locus coeruleus (Wurst and Bally-Cuif of cranial nerves. (After Wurst and Bally-Cuif 2001) 2001; Holzschuh et al. 2003; Fig. 7.3). However, a proper interplay with other signalling molecules (SHH, FGF4 and BMP4) is necessary: for the oculo- limit of the MHB.A second gene, Gbx2 (a homologue motor and trochlear neurons and the substantia ni- of the Drosophila gene unplugged), is expressed in gra the interplay of SHH and FGF8, for serotonergic the rostral part of the hindbrain. Its anterior limit neurons FGF4 from the rostral mesoderm and for the marks the posterior limit of the MHB.In Otx2 knock- locus coeruleus BMP4 from the non-neural dorsal out mice, the rostral neuroectoderm is not formed, ectoderm. Pitx3 is required for survival of the subset leading to the absence of the prosencephalon and the of midbrain dopaminergic neurons (van den Munck- rostral part of the brain stem (Acampora et al. 2001; hof et al. 2003). Wurst and Bally-Cuif 2001). Mice with the genotype Otx2–/+; Otx1–/– or Otx2–/+; Otx1–/+ do not form a mesencephalon and show an extension of meten- 7.2.2 Segmentation of the Brain Stem cephalic tissue, leading to a giant cerebellum (Fig. 7.2e). In Gbx2 knockouts, all structures arising The process of segmentation is particularly evident from the first three rhombomeres,including the cere- in the hindbrain. A modular organization of its neu- bellum and pons, are absent (Fig. 7.2f). ronal subtypes and nuclei is set up by its early trans- MHB cells secrete fibroblast growth factors (FGFs) verse subdivision into eight rhombomeres (Lums- and Wnt (mouse homologues of the Drosophila gene den and Keynes 1989; Lumsden 1990; Guthrie 1996; wingless) proteins which are required for the differ- Moens and Prince 2002; Pasini and Wilkinson 2002). entiation and patterning of the midbrain and hind- Rhombomere identity is controlled by Hox genes. brain (Nakamura 2001; Rhinn and Brand 2001; Joyn- Signalling by FGF8 from the isthmus patterns the er 2002; Chi et al. 2003; Fig. 7.3). In Wnt1 knockout anterior hindbrain and establishes the anterior limit mice, the mesencephalon is malformed and a cere- of Hox gene expression (Irving and Mason 2000; bellum is hardly present (McMahon et al. 1992; Moens and Prince 2002). Rhombomere 1 is the only Mastick et al.1996).A number of homeobox-contain- hindbrain segment in which no Hox genes are ex- ing transcription factors are expressed across the pressed.