A Hox gene regulatory network for hindbrain segmentation

Abstract

In vertebrates, the hindbrain serves as a highly conserved complex coordination center for regulating many fundamental activities of the central nervous system, such as respiratory rhythms, sleep patterns and equilibrium, and it also plays an important role in craniofacial development. The basic ground plan that underlies the diverse functions of the hindbrain and its neural crest derivatives is established and patterned by a process of segmentation. Through a dynamic series of signaling and regulatory interactions the developing hindbrain is transiently compartmentalized into a set of seven segmental units, termed rhombomeres. The nested expression of the Hox family of transcription factors is tightly coupled to the process of segmentation and provides a molecular code for specifying the unique regional properties of the hindbrain and its neural crest derived craniofacial structures. The high degree of similarity in hindbrain architecture between diverse vertebrates has enabled cross-species regulatory analysis. This has facilitated the experimental assembly of the signaling and regulatory interactions, which underlie the process of segmentation, into a Hox-dependent gene regulatory network (GRN) model. This hindbrain GRN is a key regulatory feature of head patterning, conserved to the base of vertebrate evolution. This regulatory framework also serves as a basis for comparing and contrasting GRNs that govern cranial neural crest formation and axial patterning and provide insight into regulatory mechanisms associated with the evolution of novel vertebrate traits. The purpose of this review is to discuss the majorfeatures of the GRN for hindbrain segmentation and its relationship to the broader functional role of the hindbrain in patterning head development.

Introduction

During vertebrate development, the hindbrain territory undergoes a process of segmentation, forming a transient series of seven swellings along the anterior-posterior (A-P) axis, called rhombomeres (r) (Alexander, Nolte, & Krumlauf, 2009; Krumlauf, 2016; Lumsden, 2004), as shown in Fig. 1A. Rhombomeres represent lineage-restricted compartments of cells, with restricted cell mixing between the adjacent segments (Birgbauer & Fraser, 1994; Fraser, Keynes, & Lumsden, 1990; Guthrie & Lumsden, 1991; Guthrie, Prince, & Lumsden, 1993). This iterated series of functional territories can respond independently to axial patterning signals, creating regional diversity in the developing hindbrain. This is reflected in the patterns of neurogenesis and neuroanatomy of the early hindbrain, as seen in the segmental organization of neurons and cranial nerve roots, establishing a fundamental neuronal connectivity between the hindbrain, other brain centers, and the periphery (Chandrasekhar, 2004; Clarke & Lumsden, 1993; Gilland & Baker, 2005; Lumsden & Keynes, 1989). The hindbrain also contributes more broadly to head development and craniofacial patterning through the generation of the cranial neural crest (cNCC), which delaminates from the neural tube and migrates in a series of discrete streams to populate the pharyngeal arches (Le Douarin & Kalcheim, 1999; Trainor, Bronner-Fraser, & Krumlauf, 2004). An early segmental plan can be seen at the molecular level, as revealed by the restricted expression domains of many developmental genes which encode transcription factors functionally linked with regulating steps in the segmentation process (Alexander et al., 2009; Lumsden & Krumlauf, 1996; Parker & Krumlauf, 2017). This includes the highly conserved Hox genes, which are coupled to hindbrain segmentation, exhibiting nested and striped expression domains that correspond to the boundaries between rhombomeres (Alexander & Krumlauf, 2009; Lumsden & Krumlauf, 1996; Wilkinson, Bhatt, Cook, Boncinelli, & Krumlauf, 1989), as depicted in Fig. 1A.

Hox genes play key roles in specifying segmental identity to rhombomeres and cranial neural crest cells (Alexander et al., 2009; Tumpel, Wiedemann, & Krumlauf, 2009). In mammals, Hox genes reside in four genomic clusters, which arose via ancient duplication events in early vertebrates (Hoegg & Meyer, 2005; Krumlauf, 1994; McGinnis & Krumlauf, 1992). They can be classified into 13 paralogue groups (PG) based on their sequence features, and display temporal and spatial collinearity along their clusters, whereby their timing and spatial domains of expression along the A-P axis correlate with their relative gene order along each chromosomal cluster (Deschamps & Duboule, 2017; Duboule & Dolle, 1989; Graham, Papalopulu, & Krumlauf, 1989; Kmita & Duboule, 2003; Lewis, 1978) (Fig. 1B). The Hox genes also display opposing gradients of responsiveness to retinoic acid (RA) and fibroblast growth factor (FGF) axial signaling pathways along the clusters (Fig. 1B). This gives rise to nested domains of expression, providing combinatorial Hox codes that specify regional properties along the A-P axis in multiple tissues, as shown for the hindbrain and neural crest in Fig. 1A. Hox expression domains are sometimes offset between rhombomeres and pharyngeal arches, in part due to differences between adjacent rhombomeres in their neural crest contributions. Even-numbered rhombomeres provide the major contributions of neural crest, while very few crest cells emerge from odd-numbered rhombomeres (Birgbauer, Sechrist, Bronner-Fraser, & Fraser, 1995; Golding, Trainor, Krumlauf, & Gassmann, 2000). There are also tissue-specific responses to environmental signals that modulate Hox expression in neural crest versus hindbrain domains, such as the repression of Hoxa2 expression in PA1 but not r2 (Trainor, Ariza-McNaughton, & Krumlauf, 2002), yet the regulatory mechanisms underlying this tissue-specificity are not fully understood (Parker, Pushel, & Krumlauf, 2018).

Patterns of segmental Hox gene expression in the developing head are remarkably well conserved across vertebrates (Godsave et al., 1994; Parker et al., 2014, Parker et al., 2019; Prince, Moens, Kimmel, & Ho, 1998), and Hox gene perturbation experiments have revealed roles in rhombomere formation, specification of rhombomere segmental identity and craniofacial patterning (Alexander et al., 2009; Lumsden, 2004; Minoux & Rijli, 2010; Moens & Prince, 2002; Parker & Krumlauf, 2017; Tumpel et al., 2009). Detailed analysis of cis-regulatory elements of Hox genes and other segmental regulators in the developing hindbrain have been reviewed in detail elsewhere (Parker, Bronner, & Krumlauf, 2016; Parker & Krumlauf, 2017; Thierion et al., 2017; Torbey et al., 2018; Tumpel et al., 2009). In this chapter, we provide an outline of the gene regulatory network governing segmental expression of Hox genes in the hindbrain. We discuss recent findings at multiple levels of the network, including how retinoid signaling coordinates maintenance of homogeneous segments, and how segmental border sharpness is subsequently coupled to induction of specialized rhombomere boundary cells during hindbrain tissue patterning (Addison, Xu, Cayuso, & Wilkinson, 2018; Cayuso, Xu, Addison, & Wilkinson, 2019). We then outline key gaps in knowledge that merit further research, including how Hox regulation is integrated between the hindbrain and neural crest.