Control of growth and gut maturation by HoxD genes PNAS PLUS and the associated lncRNA Haglr Jozsef Zakanya, Fabrice Darbellayb, Bénédicte Mascreza, Anamaria Necsuleab,1, and Denis Duboulea,b,2 aDepartment of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland; and bSchool of Life Sciences, Federal Institute of Technology, Lausanne (EPFL), 1015 Lausanne, Switzerland Contributed by Denis Duboule, September 21, 2017 (sent for review July 13, 2017; reviewed by Nicoletta Bobola and Natasza Kurpios) During embryonic development, Hox genes participate in the build- side (9), emphasizing a potential link between growth retardation ing of a functional digestive system in metazoans, and genetic con- and alterations in gut development. ditions involving these genes lead to important, sometimes lethal, In this study, we tried to find a common explanatory frame- growth retardation. Recently, this phenotype was obtained after work for the various cases described above. We decided to deletion of Haglr,theHoxd antisense growth-associated long non- complete the allelic series with a few more mutant chromosomes coding RNA (lncRNA) located between Hoxd1 and Hoxd3. In this necessary to unambiguously interpret the previous observations study, we have analyzed the function of Hoxd genes in delayed relating phenotypes to particular genes. By using CRISPR/Cas9 growth trajectories by looking at several nested targeted defi- mutagenesis in embryo, we demonstrate that the growth-retardation – ciencies of the mouse HoxD cluster. Mutant pups were severely phenotype observed in HoxDDel(1 9) mice is caused by the dominant- stunted during the suckling period, but many recovered after wean- negative effect of the HOXD10 protein. We also report comparable ing. After comparing seven distinct HoxD alleles, including CRISPR/ growth deficits in mice lacking the entire region from Hoxd1 to – Cas9 deletions involving Haglr, we identified Hoxd3 as the critical Hoxd4 [HoxDDel(1 4)] (10) and that a mutation in the HOXD3 component for the gut to maintain milk-digestive competence. This DNA-binding domain produces mice with similar growth defi- essential function could be abrogated by the dominant-negative cits, in contrast to mice mutant for either HOXD1 or HOXD4. effect of HOXD10 as shown by a genetic rescue approach, thus We document pathological signs of intestinal failure in three further illustrating the importance of posterior prevalence in Hox independent nested deletions. Together, these results converge BIOLOGY gene function. A role for the lncRNA Haglr in the control of post- toward a central role for Hoxd3 in the growth-retardation phe- DEVELOPMENTAL natal growth could not be corroborated. notypes previously recorded and further explain the strong functional hierarchy at work among HOX proteins. lncRNA | digestive system | Hox regulation | growth control | CRISPR-cas9 To address the potential function of Haglr in this context, we further engineered a pair of alleles carrying either a deficiency or low postnatal weight gain phenotypes can be induced by an inversion of the CpG island containing the bidirectional Smutations in a range of genes important for developmental promoter for both Hoxd1 and the antisense lncRNA. Neither of patterning. However, the molecular and physiological bases of these two alleles affected postnatal growth, even in the resulting such growth retardations are often difficult to pinpoint due to the absence of the reported Haglr RNA, suggesting that the Haglr- high pleiotropy associated with these genes. While none of the associated phenotype may result from an interference with Hoxd3 39 gene members of the Hox family has been included in this broad phenotypic class thus far, we noticed two cases of postnatal Significance growth retardation during the several-years-long studies of our allelic series at the mouse HoxD locus. First, animals homozygous During development, transcription factors are necessary not for a complete HoxD cluster deficiency showed about 30% body- only to pattern the body plan but also to control growth. mass deficit by the time of weaning. However, given that they However, the link between these two developmental compo- suffered severe limb and innervation defects, growth retardation nents has been difficult to establish. Hox genes are involved in seemed compatible with reduced feeding (1). Secondly, a shorter the emergence of a functional digestive system in metazoans, – deficiency removing Hoxd1–Hoxd9 [HoxDDel(1 9)] was associated thus providing a potential impact on growth through nutrition. with postnatal growth retardation in heterozygous mice in the absence Also, genetic conditions involving these genes lead to impor- tant growth retardation. We analyzed several targeted mutant of mobility defects. However, some specimens also displayed facial lines at the HoxD locus and found that stunted phenotypes can defects, again suggesting the existence of a physical problem all be explained by the lack of function of Hoxd3, whose role potentially affecting the feeding behavior (2). seems to be critical in the developing gut of suckling mice, Highly severe postnatal growth retardation in the absence of perhaps as an adaptation to the milk-dependent early post- any obvious malformations was recently reported following a natal period in mammals. targeted deletion of the Hoxd antisense growth-associated long noncoding RNA (lncRNA) Haglr in mice. This midget (Mdgt) Author contributions: J.Z. and D.D. designed research; J.Z., F.D., and B.M. performed phenotype, which was recorded in homozygous-mutant specimens research; J.Z., F.D., B.M., A.N., and D.D. analyzed data; and J.Z. and D.D. wrote the paper. (3), displayed a reduced viability attributed to a function of the Reviewers: N.B., University of Manchester; and N.K., Cornell University. lncRNA distinct from those associated with either Hoxd1 (4, 5) or The authors declare no conflict of interest. Hoxd3 (6, 7), the two genes flanking Haglr in the HoxD cluster. This open access article is distributed under Creative Commons Attribution- Therefore, in these cases, both recessive and dominant phenotypes NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). were recorded, and they were always associated with loci located in Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. the anterior (telomeric) part of the HoxD gene cluster. In addition, GSE103086). other HoxD alleles showed midgut patterning defects, but their 1Present address: Laboratory of Biometry and Evolutionary Biology, CNRS, University detailed analyses were made difficult by lethality at birth (8). Finally, Claude Bernard Lyon 1, 69662 Villeurbanne, France. distant cis-acting regulatory regions necessary for the transcriptional 2To whom correspondence should be addressed. Email: [email protected]. control of these genes in midgut were reported within the large This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. regulatory landscape flanking this gene cluster on its telomeric 1073/pnas.1712511114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1712511114 PNAS Early Edition | 1of10 Downloaded by guest on October 4, 2021 transcription or mRNA stabilization rather than from a direct ef- difference (Table 1). It is noteworthy that, once growth of mutant fect of the lncRNA as previously reported (11). As many of the animals resumed, it reached slopes similar to those of their wild- severely stunted mutant mice largely recovered after weaning, we type siblings weeks before. Therefore, while strong growth re- hypothesize that the suckling period and its particular physiological tardation occurred during milk feeding, growth could catch up requirements may vitally depend on the function of the Hoxd3 after weaning in the surviving specimens. gene in the gut. Previously, we had observed in various alleles carrying partial deletions in the HoxD cluster (13) that the remaining gene located Results next to the breakpoint after the deletion had occurred was likely to Gain of Function-Dependent Growth Retardation. Hoxd deficiencies adopt some of the expression specificities of the deleted genes. have been used extensively to study the complex regulation of this This observation, along with the dominant inheritance pattern of gene cluster (12). One of them involving the DNA interval from this Del(1–9) stock, strongly suggested a gain-of-function mecha- – Hoxd1 to Hoxd9 [HoxDDel(1 9) or Del(1–9), discussed further be- nism, because even a full deficiency of the HoxD cluster did not low] (Fig. S1) resulted in a semilethal slow postnatal weight gain. induce such severe growth retardation in heterozygous-mutant Heterozygous males crossed with wild-type females produced the animals (1). Therefore, we suspected Hoxd10 expression was predicted 50% proportion of wild-type normal and 50% hetero- gained in space and time and thus interfered with more anterior zygous offspring. Soon after birth, growth of half of the progeny Hox gene function following the property of posterior prevalence. lagged. Many growth-retarded individuals dropped below half their Posterior prevalence was defined as the capacity of some pos- normal siblings’ body mass and thus were killed following legal recom- terior HOX proteins to override the function of more anterior mendation. However, the majority of growth-retarded specimens ones when coexpressed in some contexts (14). As this property survived, and they were genotyped along with their killed litter- may rely upon the binding of these proteins on their DNA target mates. All growth-retarded individuals carried a Del(1–9) chro- sites, we induced a small deletion within the Hoxd10 homeobox mosome (Fig. 1A; red labels), whereas all normally growing siblings sequence in cis with the Del(1–9) deficiency by using the CRISPR/ – werewildtype(Fig.1A; green labels). Wild-type individuals showed Cas9 technology in fertilized mouse zygotes. In this HoxDDel(1 9)d10hd a peak of body growth between postnatal days (P)2 and P14, followed allele, while the same Del(1–9) deficiency was present, the adjacent by a few days of slower growth.