Differentiation of enteric neurons into their different types.

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HMX2 AND HMX3 IN DEVELOPMENT

Hmx2 and Hmx3 during embryonic development

Hmx2 and Hmx3, together with Hmx1, form the Hmx family of homeobox transcription factors. The literature concerning these genes is complicated by several changes of names and parallel work on different animal models. The first Hmx gene was called H6 at its discovery (now Hmx1), when it was isolated from a cDNA library of the human craniofacial region, screened for homeobox genes (Stadler et al., 1992). Three years later, two novel homeobox genes were cloned in mouse and found to resemble the drosophila gene Nk1 gene and, despite a slight sequence divergence from the NK family (and without recognizing that they formed a separate family), were called Nkx5.1 and Nkx5.2 (now respectively Hmx3 and Hmx2), which are closely linked on the same chromosome (7 in mouse, and 10 in humans) (Bober et al., 1994; W. Wang & Lufkin, 1997). These three genes are likely present in all mammals (Stadler et al., 1995). Their homeodomain is very similar (although, with 5 amino acid differences between Hmx2 and Hmx3, the conservation is less than for other pairs of paralogous homeodomains, such as Phox2a and Phox2b which have identical homeodomains). The developmental expression of Hmx2 and Hmx3 overall has not been studied in great detail and mostly with now outdated techniques such as radioactive in situ hybridization, imprecise ones like wholemount in situ hybridization or indirect ones, like knock-ins of LacZ reporter genes (e.g. (Weidong Wang et al., 2000) or Cre recombinase (Niquille et al., 2018) and some interpretations should be taken with caution. For example Hmx2 was found expressed in sympathetic ganglia at E12.4 (Wang et al., 2000), based on sections though a Hmx2::LacZ embryo, but no sympathetic expression was detected by in situ hybridization at E13.5 (I. Espinosa-Medina et al., 2016a) or by single cell transcriptomics at P5 (unpublished data from my lab). No antibody exists to our knowledge, and the Alllen Brain Atlas or Genepaint database do not provide a clear pattern for either gene. The best studied expression (and the best studied loss-of-function phenotype) is by far in the inner ear where Hmx3 expression starts at E8.5 in otic placode and Hmx2 expression 0.5 day later (Rinkwitz-Brandt et al., 1996; Wang et al., 2001). Mouse lacking Hmx3 give birth to a decreased ratio of homozygous null mutants (Hmx3-/-), display a reduced capacity of pregnancy in the females, and a highly abnormal locomotory behavior: they continuously locomote in circles, stopping only to groom, feed and sleep, suggesting a defect in the inner ear. Indeed, the mutants present a non-separation of the utricle and saccule during development of the inner ear, leading to a reduced sensory epithelial 54 area for both; aside from this, the horizontal semi-circular duct is not properly shaped, hence a non-properly working inner ear and a loss of equilibrium. The expression of Hmx2 and Hmx1 remain unchanged by the lack of Hmx3 (Wang et al., 1998). The case of Hmx2 was examined some years later. Mice lacking Hmx2 also present a circling behavior, in this case combined with head tilting. Unlike Hmx3, the loss of Hmx2 leads to the complete absence of the 3 semi-circular ducts as well as a fused utricle and saccule, all of this entailing a general 60% decrease of the number of neurons in the inner ear at E18.5 (due to a decrease of cell proliferation during development, rather than premature cell death) (Wang et al., 2001). The similar pattern of expression of Hmx2 and Hmx3 in the inner ear, and the fact that the expression of the later gene (Hmx2) does not depend on the earlier one (Hmx3) suggested the possibility of a partial redundancy and inspired the creation of a double mutant. This was not trivial since the two genes are separated by only 8kb (Wang et al., 2004), which in practice prevents recombination between the two loci. Thus, a third knockout line was created, with a 11 kb deletion encompassing the two genes. Animals lacking both Hmx2 and Hmx3 die at around 5 days after birth with a more severe loss of balance than either single knockout: starting at day 3 the Hmx2-/-;Hmx3-/-pups show a fully penetrant loss of balance and are incapable of righting themselves. The inner ear of Hmx2/3 knockouts develops normally until E12.5 but then starts to degenerate and the vestibular part of the inner ear is completely missing at E18.5. At the molecular level, the expression of a number of early markers of the otic vesicle is altered as early as E11.5, among them Bmp4, FoxG1, Pax2, and Dlx5. The double knockout also shows a complete absence of Netrin1 around the otic vesicle, while its expression was not affected in either Hmx2 or Hmx3 simple knockout, showing shared redundant functions, for some aspects at least. At the moment of their death, pups lacking Hmx2/3 present dwarfism which can be explained by a 40% reduction of circulating Growth Hormone, as well as a complete loss of Growth Hormone Releasing Hormone in the arcuate nucleus of the hypothalamus, attributed to the absence of expression of Gsh1, which is regulated by Hmx2/3 only in this specific nucleus (Wang et al., 2004). Hmx genes are well conserved in evolution. Not only the homeodomain of the Hmx genes is very similar to that of the ancestral unique orthologue in Drosophila (Wang et al., 2000), but the latter can rescue a great part of the phenotype of Hmx2/3 double mutants, in particular the lethality and growth deficit, the inner ear phenotype persisting albeit with a variable penetrance (Wang et al., 2004).

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Expression of Hmx2/3 in the Nervous system

In addition to the inner ear and the arcuate nucleus, Hmx2 and Hmx3 are expressed in other regions of the nervous system, without any clear function established yet. As early as E10.5, they are both expressed all along the spinal cord (probably in interneurons), as well as in dorsal root ganglia; later at E11.5 they can be detected in midbrain, hindbrain and trigeminal ganglia (Bober et al., 1994). Hmx3 is also expressed in the preoptic area from where are generated many neurons populating the striatum, olfactory bulb, septum, amygdala or GABAergic interneurons in the cortex (D. Gelman et al., 2011; D. M. Gelman et al., 2009). More precisely, Hmx3 expressing progenitors were shown, by lineage tracing to give rise to the neurogliaform cortical interneurons, an important inhibitory interneuron type in the cortex (Niquille et al., 2018). The function of Hmx3 in these neuronal types is however unexplored. The expression of Hmx2 and Hmx3 was also reported in the peripheral nervous system (Bober et al., 1994) and more recently established as differential marker of parasympathetic ganglia versus sympathetic ones (Espinosa-Medina et al., 2016). These two genes are also found to be expressed in the enteric nervous system (Heanue & Pachnis, 2006), in the same study that uncovered Tbx3:
In situ hybridization of transcription factors in the enteric nervous system at E15.5. Image reproduced from (Heanue & Pachnis, 2006) Common expression of Hmx3 in the ENS and parasympathetic ganglia might be considered as an additional trait shared by these two divisions of the autonomic nervous system. No functional study was made so far.

Table of contents :

Introduction
1 – General Introduction
2 – Development of the enteric nervous system
I – Overview of the development of the ENS
II – Regulation of the neuron numbers in the ENS
A – Embryonic proliferation and death
B – Post Natal proliferation and death
1 – Evolution of the number of enteric neurons after birth
2 – Evidence for ongoing neurogenesis
3 – Neuronal Diversity in the ENS
I – Introduction
III – Types of enteric neurons defined by classical studies
A – Myenteric plexus
2 – Sensory (afferent) neurons
3 – Interneurons.
4 – Intestinofugal neurons
B – Submucosal plexus
1 – Secretomor/Vasodilator neurons
2 – IPANs
3 – Other types
C – Conclusion on the detection of enteric neurons types.
D – Glia.
IV – Types of enteric neurons newly defined by single cell transcriptomics.
V – Differentiation of enteric neurons into their different types.
A – Timing of neuronal diversification.
B – Mechanisms of neuronal differentiation
VI – Transcriptional control of the ENS development
4 – The transcription factor TBX3
I – The T-Box family of TFs.
II – Tbx3 in stem cells
III – Tbx3 in cancer
IV – Developmental roles of Tbx3
V – Tbx3 and the enteric nervous system
5 – Hmx2 and Hmx3 in development
I – Hmx2 and Hmx3 during embryonic development
II – Expression of Hmx2/3 in the nervous system
3 – Unpublished results
I – Genetic interaction between ErbB3 and Ret
II – Hmx2 and Hmx3 knockouts
A – Construction
B – Phenotype of the Hmx knockouts
III – Role of Tbx3 in the development of the ENS
A – Expression of Tbx3 in the ENS
B – Gross phenotype of a Tbx3 conditional KO
C – Histological analysis of the ENS of Phox2b::Cre; Tbx3lox/lox mutants

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