Preservation of duplicate genes by complementary, degenerative mutations.

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow (1996, p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh (1995, p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff (1997, p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. (1997, p. 362)

https://www.genetics.org/content/genetics/151/4/1531.full.pdf

Preservation of Duplicate Genes by Complementary, Degenerative Mutations

Vulnerable periods during the development of the nervous system are sensitive to environmental insults because they are dependent on the temporal and regional emergence of critical developmental processes (i.e., proliferation, migration, differentiation, synaptogenesis, myelination, and apoptosis). Evidence from numerous sources demonstrates that neural development extends from the embryonic period through adolescence. In general, the sequence of events is comparable among species, although the time scales are considerably different. Developmental exposure of animals or humans to numerous agents (e.g., X-ray irradiation, methylazoxymethanol, ethanol, lead, methyl mercury, or chlorpyrifos) demonstrates that interference with one or more of these developmental processes can lead to developmental neurotoxicity. Different behavioral domains (e.g., sensory, motor, and various cognitive functions) are subserved by different brain areas. Although there are important differences between the rodent and human brain, analogous structures can be identified. Moreover, the ontogeny of specific behaviors can be used to draw inferences regarding the maturation of specific brain structures or neural circuits in rodents and primates, including humans. Furthermore, various clinical disorders in humans (e.g., schizophrenia, dyslexia, epilepsy, and autism) may also be the result of interference with normal ontogeny of developmental processes in the nervous system. Of critical concern is the possibility that developmental exposure to neurotoxicants may result in an acceleration of age-related decline in function. This concern is compounded by the fact that developmental neurotoxicity that results in small effects can have a profound societal impact when amortized across the entire population and across the life span of humans.

Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models.

Neural circuits are assembled with remarkable precision during embryonic development, and the selectivity inherent in their formation helps to define the behavioural repertoire of the mature organism. In the vertebrate central nervous system, this developmental program begins with the differentiation of distinct classes of neurons from progenitor cells located at defined positions within the neural tube. The mechanisms that specify the identity of neural cells have been examined in many regions of the nervous system and reveal a high degree of conservation in the specification of cell fate by key signalling molecules.

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Neuronal specification in the spinal cord: inductive signals and transcriptional codes

Over the past several decades, significant advances have been made in our understanding of the basic stages and mechanisms of mammalian brain development. Studies elucidating the neurobiology of brain development span the levels of neural organization from the macroanatomic, to the cellular, to the molecular. Together this large body of work provides a picture of brain development as the product of a complex series of dynamic and adaptive processes operating within a highly constrained, genetically organized but constantly changing context. The view of brain development that has emerged from the developmental neurobiology literature presents both challenges and opportunities to psychologists seeking to understand the fundamental processes that underlie social and cognitive development, and the neural systems that mediate them. This chapter is intended to provide an overview of some very basic principles of brain development, drawn from contemporary developmental neurobiology, that may be of use to investigators from a wide range of disciplines.

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The Basics of Brain Development

In the developing vertebrate hindbrain Hoxa1 and Hoxb1 play important roles in patterning segmental units (rhombomeres). In this study, genetic analysis of double mutants demonstrates that both Hoxa1 and Hoxb1 participate in the establishment and maintenance of Hoxb1 expression in rhombomere 4 through auto- and para-regulatory interactions. The generation of a targeted mutation in a Hoxb1 3′ retinoic acid response element (RARE) shows that it is required for establishing early high levels of Hoxb1 expression in neural ectoderm. Double mutant analysis with this Hoxb1(3′RARE) allele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 reveals synergy between these genes. In the absence of both genes, a territory appears in the region of r4, but the earliest r4 marker, the Eph tyrosine kinase receptor EphA2, fails to be activated. This suggests a failure to initiate rather than maintain the specification of r4 identity and defines new roles for both Hoxb1 and Hoxa1 in early patterning events in r4. Our genetic analysis shows that individual members of the vertebrate labial-related genes have multiple roles in different steps governing segmental processes in the developing hindbrain.

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Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning

The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification Here we investigate the possible functional synergy of the two genes The generation of Hoxa1(3'RARE)/Hoxb1(3'RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3'RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background Hoxa1(null)/Hoxb1(3'RARE) and Hoxa1(null)/Hoxb1(null )double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population Strikingly, the second arch of the double mutant embryos involuted by 105 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants

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Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch

Segmentation plays an important role in neuronal diversification and organisation in the developing hindbrain. For instance, cranial nerve branchiomotor nuclei are organised segmentally within the basal plates of successive pairs of rhombomeres. To reach their targets, motor axons follow highly stereotyped pathways exiting the hindbrain only via specific exit points in the even-numbered rhombomeres. Hox genes are good candidates for controlling this pathfinding, since they are segmentally expressed and involved in rhombomeric patterning. Here we report that in Hoxa-2(−/−) embryos, the segmental identities of rhombomere (r) 2 and r3 are molecularly as well as anatomically altered. Cellular analysis by retrograde dye labelling reveals that r2 and r3 trigeminal motor axons turn caudally and exit the hindbrain from the r4 facial nerve exit point and not from their normal exit point in r2. Furthermore, dorsal r2-r3 patterning is affected, with loss of cochlear nuclei and enlargement of the lateral part of the cerebellum. These results point to a novel role for Hoxa-2 in the control of r2-r3 motor axon guidance, and also suggest that its absence may lead to homeotic changes in the alar plates of these rhombomeres.

Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning

Retinoid signalling and hindbrain patterning

Hox genes are instrumental in assigning segmental identity in the
developing hindbrain. Auto-, cross- and para-regulatory interactions help
establish and maintain their expression. To understand to what extent such
regulatory interactions shape neuronal patterning in the hindbrain, we
analysed neurogenesis, neuronal differentiation and motoneuron migration in
 Hoxa1 , Hoxb1 and Hoxb2 mutant mice. This comparison
revealed that neurogenesis and differentiation of specific neuronal
subpopulations in r4 was impaired in a similar fashion in all three mutants,
but with different degrees of severity. In the Hoxb1 mutants, neurons
derived from the presumptive r4 territory were re-specified towards an r2-like
identity. Motoneurons derived from that territory resembled trigeminal
motoneurons in both their migration patterns and the expression of molecular
markers. Both migrating motoneurons and the resident territory underwent
changes consistent with a switch from an r4 to r2 identity. Abnormally
migrating motoneurons initially formed ectopic nuclei that were subsequently
cleared. Their survival could be prolonged through the introduction of a block
in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent
with a partial misspecification of the presumptive r4 territory that results
from partial Hoxb1 activation. The Hoxb2 mutant phenotype is
a hypomorph of the Hoxb1 mutant phenotype, consistent with the
overlapping roles of these genes in facial motoneuron specification.
Therefore, we have delineated the functional requirements in hindbrain
neuronal patterning that follow the establishment of the genetic regulatory
hierarchy between Hoxa1 , Hoxb1 and Hoxb2 .

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Anthony Gavalas

Papers

Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning

Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch

Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning

Retinoid signalling and hindbrain patterning

Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes.