Hypoxia and notch signaling pathway disruptions cause congenital scoliosis in mice


Congenital scoliosis (CS) is the lateral curvature of the spine due to vertebral defects induced during gestation. It occurs in 1 in 1,000 live births of humans. The underlying causes of this abnormality can be caused by genetics; however, other causes have no genetic etiology and are considered to be sporadic congenital scoliosis.

Recent studies have found that sporadic birth defects were caused by a mixture of genes and teratogens in which the mother was exposed. Teratogens cause malformation of the embryo. Examples of teratogens were shown to be infections, dietary deficiencies and drugs. Sparrow et al (2012) reported that genes and the environment explained the etiology of congenital scoliosis after observing heterozygotes for mutations on loci for Hes7 and Mesp2 transcription. Surprisingly, not all individuals with the heterozygous mutations showed a phenotype from CS;  therefore, it was concluded that the mutations were recessive and would result in incomplete penetrance.

Child born with congenital scoliosis due to unlateral unsegmented bar in spine (Letts, R.M., 2014)

Figure 1: Child born with congenital scoliosis due to unlateral unsegmented bar in spine
(Letts, R.M., 2014)

Of the four known scoliosis genes found in humans, haploinsuffiency of Hes7 or Mesp2 was associated with CS in humans according to  (Sparrow et al,2011); however, in mice mutations Hes7 caused a CS like phenotype in nearly 50% (16/30) of the progeny while Mesp2 mutations only showed minor vertebral effects in 10% of progeny (3/30) (Sparrow et al, 2012). Since the Mesp2 mutation known to disrupt somitogenesis in humans caused few disruptions in spine development in the mice,  an extreme environmental factor was hypothesized to possibly trigger CS in the wildtype, Mesp2 +/- and Hes7 +/- mice.

In previous studies, spontaneous and induced mutations in mice have caused deviations in the normal development of the skeleton. Spinal curvature was identified in mice which had mutations in the following Notch signaling genes : DLL3, MESP2, LFNG and HES7 (Sparrow et al, 2011). Since many cases of spondylocostal dysostosis (SCD) and spondylothoraic dysostosis (STD) have been shown to be caused by single mutations, an environmental factor was thought to be causal for congenital scoliosis. Sparrow et al (2012) hypothesized that  CS would result from heterozygous mutations in any of the four causal genes of SCD. After screening families for the heterozygous mutations, the team observed the role of genes and teratogens. Oxygen deprivation has produced CS like phenotypes along with malformations in structural disruptions of the heart, limbs and palate independent from induced and inherited mutations in Mesp2 and Hes7(Geoffr0y Sainte Hellaire, 1820); (Ingalls et al, 1952); therefore, they connected the role of genetics and hypoxia as triggers for congenital scoliosis in mice.

1. Haploinsufficiency of Hes7 or Mesp2 was found to cause CS in humans

First Sparrow et al (2012) screened the previously identified coding region and splice sites of 79 cases of CS in humans and found two probands which were heterozygous for nonsynonymous coding SNPs in Hes7 (family A) and Mesp2 (family B) respectfully. The proband from family A portrayed multiple vertebral defects due to the Hes7 mutation as a result of the amino acid substitution D142Y; however, although the mother of the proband also had the same mutation, no external spinal defects were observed.

The proband from family B portrayed fused posterior elements and rib anomalies due to the mutation of Mesp2 resulting in the amino substitution H102P. The mother, maternal grandparents and three siblings of the proband were screened for the mutation. The mother, two siblings and grandfather all had the same amino acid substitution; however, only the mother had an abnormal vertebral morphology.

These results showed that that the deleterious alleles of Hes7 or Mesp2 caused partial penetrance in CS in humans.

Figure 1 v6 US

Figure 2– Top (A-E): radiographs of two families affected with congenital scoliosis
A: Family A- radiograph of proband A.III.2 at birth
B: Family B- radiograph of proband B.III.4 at age 4
C-D: Family B-MRI images of B.III.4 at age 8
E: lateral radiograph of B.II.1
Bottom: Pedigree of family A (F)
Pedigree of family B (G)
*Probands indicated by the arrows. CS phenotype indicated by gray shading and mutant alleles indicated by black shading. Individuals without known genotype are grey boxes
**Figure 1 (Sparrow et al ,2012)


2. Haploinsufficiency of Hes7 causes CS with incomplete penetrance in Mice

Next, Sparrow et al (2012) determined if heterozygous mutations alone were sufficient to cause the CS phenotype in mice by examining mice lines with null alleles for Hes7 or Mesp2[figure 5]. The embryos were collected at embryonic day 14.5 and examined for vertebral defects. Embryos were classified in the following groups: no defects, mild defects, moderate defects and severe defects. Previous studies had not reported the vertebral phenotype of Mesp2 +/- mice. They found that loss of one allele of Hes7 but not Mesp2 was sufficient to cause vertebral defects in 50% of embryo.

3. Gene-Environment interactions increase frequency and severity of CS in mice

Since the heterozygous mutation of Mesp2 did not cause CS defects in mice , Sparrow et al (2012) hypothesized that intra-uterine hypoxia during early pregnancy could trigger CS in wildtype, Mesp2 +/- or Hes7 +/- genotype embryos[figure 6]. Pregnant mice were exposed to short term hypoxia for eight hours at concentrations of either 5.5%, 6.5%, 7.5%, 8% or 8.5%. Afterwards, each was returned to normoxia and embryos were removed at day 14.5 and analyzed of spine formation. The dose-dependent thresholds of oxygen’s affect on the severity of vertebral defects differed greatly amongst the three genotypes. Wildtype and null allele embryos were collected at embryonic day 14.5.

Basic CMYK

Figure 3: Difference in penetrence and severity of CS phenotype  from three genotypes observed . Top Left: When either mutant experiences normoxia during development, there is low penetrance for CS. Top Middle: Wild-type mice experiencing mild hypoxia have low penetrance for CS. Top Right: Either mutant experiencing mild hypoxia displays higher penetrance and severity for CS. Bottom Left: Normal oxygen levels allow FGF to target the presomitic mesoderm for normal spine development. Bottom Right: Hypoxia disrupts FGF signaling causing spinal defects. *(Sparrow et al ,2012) supplementary image



Although CS in humans has been found by heterozygous mutations in Mesp2 or Hes7, only CS is induced with mice with Hes7 mutations. Sparrow et al(2012) found that short-term gestational hypoxia resulted in increased failure of early development processes during somitogenesis in wildtype and Mesp2 +/- or Hes7 mutants. The addition of hypoxic environments dramatically increased the penetrance of the CS phenotype in Hes7 +/- mutants in comparison to Mesp2 +/- mutants[figure 3].

4. Hypoxia disrupts cyclical Notch1 signaling in presomatic mesoderm

Afterwards, they tested if there was a molecular connection between hypoxia and Noth Signalling by analyzing three Notch pathway genes required for somiteogenesis (Dll3, Dll1 and Notch1). Hypoxic environments were found to temporarily down regulate FGF and Wnt signalling in the presomatic mesoderm which disrupts the cycle of Notch signaling. Notch signaling is necessary during somiteogenesis.

 Hypoxic experiments were conducted on Dll3 +/-, Dll1+/- and Notch1 +/- embryos to determine if vertebral effects would be induced.

Dll3 +/-

These mutants did not display vertebral defects in normoxia. Hypoxia did not induce significant increases in severe cases of verterbal defects compared to control [Figure 5C].

Dll1 +/-

All mutants displayed mild or moderate vertebral defects in normoxia [Figure 6 (E-F); (E’-F’); (E”-F”)] in contrast to Cordes et al (2004) study which showed only 10% of mutants had minor effects. Differences in results were due to different genetic backgrounds of mice used.

The Dll1 +/- mutant in 8% atmospheric oxygen displayed drastic increases in severe vertebral deformities [Figure 5 D] comparable to Hes7  +/- embryos [Figure 5A].


Notch1 +/-

The Notch1 +/- mutant in 8% atmospheric oxygen displayed higher frequencies of  severe vertebral deformities; however, the frequencies observed were less than Hes7 and Dll1 [figure 5].

Since Notch1 is normally activated cyclically in the caudal presomatic mesoderm (PSM)with some expression in the rostral side, they observed how short term hypoxia could affect patterning of presomites and eventually cause CS. They found that embryos in normoxia had cleaved notch1 while embryos in hypoxia displayed Notch1 throughout the PSM [figure 8F]. Dll1 and Notch1 showed higher rates of vertebral defects in mice after hypoxic treatment making them great candidate genes for future CS patient genome sequencing; however, no increased rates of CS were induced in Dll3 mice as found in humans.




Figure 4: Somiteogenesis  in mouse *(Sparrow et al,2011-Figure 1a)

Somiteogenesis is controlled by the interaction of the signalling pathways Notch, Wnt, FGF and Retinoic Acid. The clock and wavefront (determination front) must interact during somitogenesis. Presomatic mesoderm contains gradients of FGF and Wnt signal from the caudal to rostral axis of the embryo. The somites (which later give rise to spinal vertebrae) signals retinoic acid (RA) rostral to caudal. The wavefront  is located based upon the threshold of FGF and Wnt. In the caudal position, undifferentiated cells are maintained while rostral cells form somites.




Hypoxia experiments

Wild Type Embryos

Embryos exposed at 5.5% hypoxia  displayed vertebral defects (n= 20/22) with 15 displays as moderate or severe.

Embryos exposed to 8.5% atmospheric oxygen displayed no vertebral defects  while 8% atmospheric oxygen induced some mild defects (n=4/26).

Embryos exposed to 7.5% atmospheric oxygen displayed few severe defects (n=1/29),  moderate defects (n= 7/29) and mild defects (n=5/29) while 7% atmospheric oxygen induced modern or severe defects (n= 20/24).

Mesp2 +/- embryos

Wild-type embryos progeny produced from crossing Mesp2 +/- males  and wildtype C57BL/6J females exposed to 8% atmospheric oxygen displayed mild vertebral effects (n=6/26) and moderate vertebral effects (n=1/26) [figure 7D ]

Mesp2 +/- embryos displayed higher rates of CS along with increased penetrance of the phenotype [figure 6 (C-D); (C’-D’)]

Hes7 +/- embryos

Hes7 +/- embryos have higher rates and severity for CS with any atmospheric concentration of oxygen compared to all treatment experiments [Figure 5 ]



Basic CMYK

Figure 5: Various hypoxic treatments increase in number and severity in mice embryos with genetic susceptibility to CS *Sparrow et al, 2012 *Figure 3


Figure 2 v2 US

Figure 6: Vertebral phenotypes of control versus hypoxia treated embryos. Dorsal view of Alician blue stained embryos. A-B: Hes7 +/- embryos. C-D: Mesp2 +/0 embryos. E-F: Dll1 +/- embryos. G-H: Notch +/- embryos.
*Figures with apostrophes indicate magnified view of embryo with same letter.
**White arrows show missing pedicle. White arrowheads show fused laminae. Yellow arrows show rib abnormalities. White dots show fusion, split or hemi-vertebral bodies.
***Scales: A-H- 1mm; A’-H’ and E”-F”- 650µm  *Figure 2-(Sparrow et al ,2012)


Figure S2 v3

Figure 7: Hypoxic treatment of wildtype c57BL/ 6J embryos  A- shows no defects
B- mild defects
C- Moderate Defects
D-Severe effects
E- oxygen dose response curve and vertebral morphology in percentages of each category
F-this was used to determine mean age of mouse embryos which underwent the 8 hour 5.5% oxygen treatment *(Sparrow et al, 2012)

v13 title

Figure 8:  Effects of hypoxic treatment of E9.5 mouse embryos on levels of Notch signalling and protein levels of Notch1 receptor, ligands and Hes7
control and treated embryos were compared. A-E control; F-J treated. A & F– cleaved Notch1; B&G– magnified versions of A & F; C & H– Notch1 expression in A & F embryos; D -merged image of A&C; I-merged image of F & H.
K-O control; P-T treated; K & P- Dll1; L & Q-Dll3;M&R- cleave notch1 in Dll1 and Dll3 embryos; N- merged image of K,L & M; S– merged image of P, Q&R ; U-X control; Y-AA treated; U&Y– Hes7; V&Z– cleaved Notch1 of U&Y; W– merged image of U&V; AA– merged  image of Y&Z.  *Arrows indicate cells that lack Notch1 signalling in control “A” and gain signalling after hypoxic treatment
**Figure S4 (Sparrow et al, 2012)



Strengths and Weaknesses of Sparrow et al study

It was very good that the researchers crossed Mesp2 +/- males with wildtype C57BL/6J females to generate sources of wildtype and heterozygous zygotes when observing embryo vertebral differences compared to wildtype to reduce differences in phenotypes induced from genotype.  The authors failed to mention why the 5.5% atmospheric oxygen was the lowest oxygen level that mice could tolerate although it was clearly shown in the supplementary [Figure 7]. It was not clear what percentage of normoxia or normal atmospheric oxygen was in this study although more assumptions can be made from surviving embryos in the wildtypes of Figure 7. It is very difficult to observe the elements of vertebral phenotypes using the arrows and arrow heads in Figure  6 . The dots were very useful for observing abnormalties. It is not clear why the authors of the Sparrow et al (2012) paper decided to use references of previous studies from 1820 and  1952 to decide that hypoxia could induce spinal deformation instead of more recent studies with stronger genetic and molecular approaches.



Geoffr0y Sainte Hellaire, E. (1820). Differents etats de pesanteur des oeufs au

commencement et a la fin d’incubation. Journal complementaire des sciences medicales,7, 271.

Ingalls, T.H. , Curley, F.J. & Prindle, R.A.  (1952). Experimental production of

congenital anomalies; timing and degree of anoexia as factors causing fetal deaths and congenital anomalies in the mouse. New England Journal Medicine, 247, 758-768.

Letts, M.R. (2014). Congenital Spinal Deformity. WebMD. Retreived from


Sparrow, D. B.,  Chapman, G., Dunwoodie, S. L. (2011).

The mouse notches up another success: understanding the causes of human vertebral malformation. Mammalian Genome, 22, 362-372. DOI: 10.1007/s00335-011-9335-5

Sparrow, D.B., Chapman, G., Smith, A. J., Mattar, M. Z., Major, J. A…Dunwoodie, S. L.

(2012). A Mechanism for Gene-Environment Interaction in the Etiology of Congenital Scoliosis. Cell, 149, 295-306. DOI: 10.1016/j.cell.2012.02.054